Ceftrioxne Injection 250 Dossior

HYGEIA PHARMACEUTICALS:
Plot No. 295, Industrial Triangle, Kahuta Road, Islamabad,

Pakistan
HYTREX 250mg Injection (IM)
“FORM 5”
[See rule 26(1)]
APPLICATION FOR REGISTRATION OF A DRUG FOR LOCAL MANUFACTURE.
HAVING THE SAME ACTIVE INGREDIENT OR SALT THEREOF, THERAPEUTIC USE, DOSAGE
FORM AND ROUTE OF ADMINISTRATION THAT HAS ALREADY BEEN APPROVED BY THE
DRUG REGULATORY AUTHORITY OF PAKISTAN, ALREADY ON SALE IN LOCAL AND/OR
INTERNATIONAL MARKET.
I M/s Hygeia Pharmaceuticals of Plot No. 295 Industrial Triangle, Kahuta Road, Islamabad hereby
apply for registration of the drug, namely HYTREX 250mg Injection (IM)details of which are enclosed.
Date ……………… Signed:

Place: ___________ Chief Executive
Quality Control Manager Production Manager
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FORM 5
[See rule 26(1)]
ENCLOSURES OF THE APPLICATION FOR REGISTRATION OF A DRUG FOR

LOCAL MANUFACTURE
Sr.No.
Dosage Form Solution
1. Name and address of the manufacturer (applicant): Plot No. 16 A, Road SS4,
National Industrial Zone,
Rawat Islamabad
2. Brand (Proprietary) name of Drug. Tincture Benzoin co
3. The chemical name(s) and, as appropriate and available the Tincture Benzoin co
established (generic) names and synonyms of the drug.
4. Strength of active ingredient(s) per unit, e.g. each tablet or 5 ml, Each 100 ml contains
etc. contains. Benzoin = 10 gm
Storex = 10 gm
Aloes = 2 gm
5. Pharmacological group. Antiseptic plus
Expectorant
6. Recommended clinical use. Annexure-A
7. Proposed route of administration. Topical
8. Proposed dosage. Three times daily
9. Proposed shelf life of the drug. 3 years
10. Proposed storage conditions of finished product. Store in cool & dry place
11. Unit price of the drug, e.g. per tablet, per capsule, per 5ml, etc. Annexure-B
12. In case of international availability, provide the following Annexure-C
information, namely:-
a. name of the drug;
b. country where sold / registered; and
c. name of company selling the drug or having registration to
manufacture (include supporting documents/proof of
International registration.
13. Brand name(s) of drug available in Pakistan. Tr.Benzoinco
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14. Name(s) of company(s) manufacturing in Pakistan. Rehmat pharma, Lahore

15. Composition (actives & excepients) including statement of the Annexure -D
quantitative
composition, giving the weight or measure for each active
substance used in the
manufacture of the dosage form.
16. Outline of method of manufacture. Annexure -E
17. Persons under whose direct supervision and control the drug is
manufactured with
the following details, namely:- Total Number =12
a. total number of technical staff; and Waqar Bin Ahmad
b. name, qualification and designation of the persons directly B.Pharm
supervising the Production Manager
manufacture of the drug applied for registration, and any change
shall be
properly documented and record maintained by the
manufacturer.
18. Name of equipments Annexure -F
that will be used in the manufacture
of the drug applied for registration:
cGMP Compliant
19. Full descriptions of the specifications and analytical methods Annexure -G

necessary to assure the identity, strength, quality, purity and
homogeneity through out the shelf life of the drug product.
20. Name, qualification and designation of the persons who will be Bilal Masood
responsible for the quality control of the drug. M.Sc Chemistry
Quality Control Manager
21. Description of the equipment to be used for the quality control of Annexure -H
the active raw
material and the finished products.
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22. Labeling and prescribing information ( to be mentioned on the N/A

pack/leaflet) specimen
or draft shall be submitted for the following class as of drugs,
namely:-
a. C.N.S. stimulants;
b. drugs affecting uterine motility;
c. drugs inhibiting hormonal production;
d. hormones and other steroidal preparation excluding
preparations for
external and topical use;
e. narcotic drugs as per Single Convention on Narcotic Drugs
1961; and
f. psychotropic substances mentioned as per convention on
psychotropic
Substances, 1971.
(Specimen of label to be submitted as soon as production starts)
23. Facility of water processing with specifications. Annexure –I

24. Environment control processing with details. Annexure -J
25. Type of container/packaging. Pet Bottle
26. A copy of last Inspection Report conducted by the Ministry of Attached
Health.
UNDERTAKING
We hereby undertake that the above given information is true and correct to the best of our knowledge
and belief.
Production Manager Quality Control Manager
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LIST OF EQUIPMENTS IN QUALITY CONTROL LAB:

S.No. MACHINE NAME: QUANTITY:
1 HPLC (Shimadzu) and HPLC wufeng 01+01
2 UV/Vis Spectrophotometer 02
3 Hot Air Oven 01
4 Melting Point Apparatus (Shimadzu) 01
5 Refractometer 01
6 Hot Plate Stirrer 02
7 Distillation apparatus 01
8 Ultrasonic Bath 01
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9 Analytical Weighing Balance 02

10 Vacuum Pump 01
11 Heating Mental 01
12 D.T Apparatus 01
13 pH Meter 01
14 Conductivity meter 01
15 Friabiliator 01
16 Hardness Tester 01
17 Helipath Stand 01
18 Moisture analyzer 01
19 Centrifuge Machine 01
20 Blister Test Apparatus 01
21 Water Bath 01
22 FTIR 01
23 Automatic Polarimeter 01
24 TOC analyzer 01
25 Thin layer chromatography 01
Annex-D
The label will be submitted at the time of Manufacturing.
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1. GLASS VIALS:
Tests Admissibility conditions
Description Clear,colorless, USP Type (1,II) glass vials.
Outer Diameter, mm 22mm±2mm
Inner Diameter, mm 20mm±2mm
Length, mm 52mm±2mm
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2. PATIENT INFORMATION LEAFLETS:
Parameters Specifications
Description. Printing quality /Color scheme:

As per Approved shade card
Text matter As per Approved Art Work.
Dimensions. Length NLT 160mm
Breadth: NLT 103mm
3. UNIT CARTON:

Text matter. As per Approved Art Work.
Mfg.License No. As per drug manufacturing License
Registration No. As per registration letter.
Dimensions. LxBxH
Grammage. NLT 250 g/m2
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Annexure E
Water Processing Facility
Schematic Representation
Of Double Pass R.O Plant
2nd Pass R.O Water

U.V Lamp with 0.2
Micron Filter
R.
Ist Pass
O
Storage Tank
Pu
Raw mp
Ist Pass R.O Water
Water U.V Lamp With 0.45
feeding Micron Filter
Pump Column Water

Colum 20” R.
n1 2 Softener Hosing O
Sand Activate 5 micron Filter De-ionized
Pu
Filter d Filter water
mp air
Quality Control Manager Carbon Production Manager
tight Storage
Filter
Tank
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Treated
Water
Specification
Plot No. 295, Industrial Triangle,
s Kahuta Road, Islamabad,
Pakistan
PH:5-7
Conductivity: 2
HYTREX 250mg
µS cm Injection (IM)
TDS: Nil
-1
Chloride Free
Multiple Effects Still
Environmental Control processing

REPROCESS OF REJECTED PACKAGING MATERIAL
1. Unit cartons/shippers will be shredded by using shredding machine and sold to the scrap.
2. Aluminum foils rejected during the stripping process will be treated as above.
3. Defective bottles, spoons, measuring cups, flip off seals, vials and rubber stoppers will be sent
back to the supplier for reprocessing.
4. Polythene bags: all discarded polybags (used for granules, powder, raw material, capsules
etc.) will be collected in a container labeled as “USED POLYBAGS”. Theses polybags are to be
collected by housekeeping person and are sent to scrap yard.
DESTRUCTION OF MATERIALS:
Any quantity of non-complying bulk granules and/or capsules, dry suspension are destroyed
under the supervision of the production and quality control as per following procedure.
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1. Obtain the destruction authorization from production, Quality Control, Quality Assurance and
management.
2. Soak granules/Capsules etc. in a suitable container full of water for 2 hours.
3. Then mix with a rod.
4. Check the pH with pH meter.
5. Neutralize with Acid/Base. (pH 7.0)
6. Drain in ‘Industrial waste sewerage line.
QUALITY CONTROL WASTES:
1. There is no chemical of extreme hazard used in objectionable quantities.

2. The most frequently used chemicals are dilute acids and alkali solution, which will be drained
after neutralization.
TEMPERATURE AND HUMIDITY CONTROL:
1. All the raw materials will be kept under controlled conditions of temperature, humidity and light.
For this purpose, air conditions and HVAC system is provided.
2. Mixing and filling process of capsules and powder for oral suspension are carried out under
controlled condition of temperature and humidity through HVAC.
3. The temperature and humidity control system has also been provided in the Dry powder
injectable section through HVAC.
PEST CONTROL AND DISINFECTION PLAN

1. Flying insects are controlled by using insect killer.
2. For rodents like rats etc. trap cages will be used.
Following is the pest control and disinfection plan:

i. The service for disinfection may be arranged on contractual agreement with professional
organizations.
ii. This service covers disinfection of all pests, cockroaches, rodents, white ants, silver fish etc.
iii. Administration and co-ordination of this service is done by the engineering department.
iv. The required documentation is carried out by the contractor. These are counter signed by a
responsible person from engineering department after verification.
v. In case of any complaint after servicing, the contractor sends a responsible person from his office to
attend the same and render additional service for effective control. Method and procedure of pest
control to various insects are done as mentioned below:
TERMITE CONTROL:
1. Regular inspection during the month is carried out by contractor all around the factory premises
and inside the building for termite. After identification, the source of termites is located and patent
chemicals are sprayed.
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2. After the completion of the job, the activity is documented and an authorized person initials the
same.
Note: During treatment, precaution is taken to avoid contamination of these chemicals with product
of raw materials and packing materials. For this utmost care is exercise that all production
containers are closed and no material is left exposed during this operation? A production officer
supervises this operation.
DIS-INFESTATION:
1. Treatment for controlling the pest is given once in a month as per schedule in specified areas.
2. The areas for pest control treatment are sprayed with a desired pesticide, as follows:
For Pesticide – Malathion

For Rodents – Zinc Phosphide
3. The whole operation is checked by a company’s responsible person and the same is measure to
avoid the contamination to the products and process.
Note: Utmost care is taken to close all the areas while treatment is given as precautionary
measures to avoid the contamination to the products and process.
RODENT CONTROL:
1. Treatment for controlling the rodent is given in the specified areas as per schedule.
2. For rodent control zinc phosphide is used as rodenticide.
3. The whole operation is checked by an authorized person and the same is documented.
After the pest control treatment is given and before restarting the work, whole area where
treatment is given, is cleaned up to remove any debris of pests or residue of chemicals used.
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Author Reviewed By: Approved By:

Validation Officer. Validation Manager. Management Representative.
Signature Signature Signature
Anex-H
1.0 Purpose
To describe the procedure for carrying out the validation of manufacturing processes & other in
processes thus standardizing the process sequence & parameters. This procedure will eventually ensure
the maintenance of validation state & the compliance to validation requirements
2.0 Scope
This procedure deals with the validation of the critical manufacturing steps i.e. uniformity of actives in
3.0 Terms & Definitions
Tablets, Capsules, dry Suspension manufacturing to consistently produce quality products.
Terms Definitions
Establishing documented evidence which provides high degree of
Validation confidence that a specific system will consistently produce a
product meeting its predetermined specifications and quality.
To demonstrate that the critical processes involved in the
manufacturing of a pharmaceutical/biological products e.g. mixing
Process Validation
of active and inactive ingredient, formulation, filling, sampling and
testing performed are reliable, repeatable and reproducible.
Process validation which includes these considerations that should
be made before an entirely new product is introduced or when there
Prospective Validation is a change in the manufacturing process which may affect the
product characteristics such as uniformity and identity of the
product.
Concurrent Validation Process validation of a product that is already in routine production.
A comprehensive and well planned documented procedure of the
Validation Protocol
validation activities to be performed.
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4.0 Responsibilities
a. Production Manager
i. To supervise and ensure the execution of process validation according to schedule.
ii. To approve the validation protocol and validation report.
b. Quality Control Manager

i. To manage the analysis for the process validation.
c. Validation Manager
i. To manage the process validation
ii. To monitor the critical manufacturing processes.
iii. To arrange samples to Quality Control for testing purpose.

iv. To review the process validation protocol and report and submits to Production Manager for
approval.
v.To analyze the results received from quality control and submits the results to Production
Manager for approval.
vi. To take corrective action with the consent of Production Manager if any deviation is
observed during process validation activities.
d. Officer Validation
i. To prepare process validation protocol.
ii. To perform process validation according to procedure.
iii. To provide samples to quality control for test analysis.
iv. To compile process validation report.
5.0 Environment, Safety and Health Precautions
All the environment, safety and health precautions that are implemented in the relevant process are
also applicable in validation activities
a. During process, the person responsible for validation should use all personal protective equipment.
b. Do not insert finger in any machine during operation.
c. Note any abnormal sound from machines and take remedial action to avoid the noise pollution.
d. Draw samples according to procedure by using masks and gloves.
e. During analysis all the chemical, reagents and dilutions should properly be identified.
f. The analysts during analysis should wear masks and gloves.
g. Pipette filler should be used by the analyst for filling the pipettes.
h. Dispose of the sample hygienically according to procedure for the disposal of solid waste and
liquid effluents.
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1.0 6.0 Procedure
Validation Manager coordinates (Through Inter office Memo) with the production department and
quality control department for proper arrangement before commencing of process (concurrent/
prospective). The following are the prerequisite of the process (concurrent/ prospective) validation.
Process validation is to be performed as mentioned in schedule of validation Master plan (will be

Developed by Validation Department) in coordination with relevant departments head in accordance
with Process validation protocol SOP and results are recorded in Process validation Report.
Process Validation protocol comprises into:
Abstract:
Brief description of the validation protocol.
Unit Operation:
Activity to be performed.
Objective
Change Control
Responsibilities
Acceptance Criteria
Statistical Technique
Observation/Deviation encountered during validation
Impact of deviation activity.
Corrective action
Corrective action taken in response of any deviation and finally
Conclusion
a. Validation Manager prepares and reviews the Process (concurrent/ prospective)
validation protocol and get these approved by the Production manager. (All the Validation
Protocol/ Reports will not be official without signatures)
b. All the equipment, material, test method, acceptance criteria, specification and the
personnel must be controlled and defined in relevant validation protocol form.
c. Explain the manufacturing procedure as mentioned in the relevant BMR/SMP with the
help of Flow Diagram.
d. Validation Manager monitors all the activities at the stage of mixing, and sampling processes to
check the applicability of the procedure as mentioned in the relevant Batch Processing Record,
and note down any deviation observed during manufacturing process.
e. Each process to be validated must be a specific and controlled procedure, clearly
defined in the relevant documents.
f. At the end of each stage, samples will be provided to Quality Control for testing purpose
as mentioned in the relevant Protocol. Quality Control analyzes the validation samples in
accordance with the relevant SOP (Testing Procedure), and submits the results to Validation
Manager, who reviews the results, and hands over the results to validation Department.
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g. Validation Manager analyzes the analytical results with the help of Statistical Techniques as
mentioned in the relevant validation protocol, and presents the results in form of graphs if
applicable.
h. After the completion of the manufacturing process, Validation Manager prepares the
validation report based on the validation study of at least three consecutive batches.
i. Finally the validation Report will be furnished. The validation report should cover but not
limited to use of validation equipment i.e. qualified equipment through I.Q/OQ/PQ.,
The validation report comprises into following:
i. Product Name, Batch No’s, Mfg. & Exp. Date

ii. Protocol No.
iii. Results of Dose Monitoring (Weight Variation)
iv. Results of assay of 10 samples of all stages
v. Calculation of all the Analytical Results with the help of Statistical Techniques (if required)
vi. Graphical presentation of the results (if applicable)
vii. Observation
7.0 Change Control Procedure
Once the Manufacturing Process is validated, it is expected that it remains under control, provided
no changes are made. The Process needs to be revalidated if modifications to the Manufacturing
Process are made, or a problem occurs, or the equipment or systems involved in the
Manufacturing Process are changed or the vender has been changed
Any modification or change in the procedure of the validated process as mentioned in the relevant
Batch Manufacturing record should be controlled in accordance with Change Control Procedure
SOP.
8.0 References
Document No. Description

Change Control Procedure.
Testing SOPs (Chemical)
Testing SOPs (Microbiological)
9.0 Distribution List
Copy No. Designation Issued To

Original Master Document
1. Management Representative
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2. Quality Control Manager

Note: When a revision of the SOP is issued, the obsolete version should be returned back to
Management Representative for disposal.
Annexure-I
Stability Study protocol
PURPOSE
The purpose of this study is to monitor the stability of finished product (Tablets, capsules,
liquid, dry suspension, & powder) over a period of time when storage conditions of 40ºC /
75%RH, and 25ºC / 60%RH.
PRODUCT INFORMATION
Name of product: ---------------------Batch#:------------------Batch size: ----------------

Mfg date: --------------------------------Exp date: -----------------------------------------------
PACKING INFORMATION
STORAGE CONDITIONS
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The products will be stored at 2 different storage conditions as outline below:

 40 degrees C±2 degrees / 75 % Relative Humidity ±5%
 25 degrees C±2 degrees / 60 % Relative Humidity ±5%
TEMPERATURE AND HUMIDITY WILL BE MONITORED AS PER THE FOLLOWING:

Stability chamber /room temperature and humidity will be at the level specified in the stability
protocol and will be maintained within ± 2 degree C for temperature and ± 5 % for humidity of
the setting. The temperature and relative humidity readings will be recorded after every 3
hours.
TOLERANCES FOR PULL DATES AND STABILITY TESTING

To tolerance for dates around which samples must be pulled from their storage condition is
plus or minus one week .All testing for routine and accelerated stability must be initiated
within 2 weeks of removal from test conditions.
STABILITY TESTS
The following test will be performed on the actives:
 Physical appearance
 Water/LOD
 Endotoxin
 PH
 Assay
STABILITY TESTING SCHEDULE

 Samples stored at 40ºC/75/% RH will be pulled and tested at time points 0,1,2,3 and 6
months.
 Samples stored at 25ºC/60/% RH will be pulled and tested at time points
0,3,6,9,12,18,24,36 and 48 months.
STABILITY ANYALYTICAL REPORT
Sample Name : ____________________________________
Packing Configuration : ____________________________________
Sample ID : ____________________________________
Date Manufactured : ____________________________________
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Date Placed on stability : ____________________________________
Storage Condition : _________Temp: _________ RH________
Date pulled from stability : ____________________________________
Time point : _____________________________________
Notebo Chemist
Acceptance Reviewed
Test Name Method Results ok (Test
Criteria by
Ref Date)
Physical
Appearance
Water/LOD
Endotoxin
 PH
 Assay
Stability Study Reports

Annexure-J
Validation of Cleaning Method
SCOPE:
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This document on cleaning validation is intended to address special consideration and issues
pertaining to validation of cleaning procedures for machine / equipment or area used in manufacturing
of Pharmaceutical.
This document is also intended to establish inspection consistency and uniformity with respect to
equipment cleaning procedures.
This document is intended to cover validation of equipment cleaning for the removal of contaminants
associated to the previous product, residues of cleaning agents as well as the control of potential
microbial contaminants.
PRINCIPLE:
1- The objective of Cleaning Validation includes the prevention of possible contamination and cross-
contamination of pharmaceutical starting material and product.
2- Pharmaceutical products can be contaminated by a variety of substances such as contaminants

associated with microbes, previous product (both active pharmaceutical ingredients (API) and
excipient residues), residues of cleaning agents, airborne matter, such as dust and particulate
matter, lubricants and ancillary material, such as disinfectants, and decomposition residues which
include:
 Product residue breakdown occasioned by, e.g. use of strong acids and alkalis during the
cleaning process, and
 Breakdown product of the detergents, acids and alkalis that may be part of the cleaning
process.
3- Adequate cleaning procedures play an important role in preventing contamination and cross-
contamination. Validation of cleaning method provides documented evidence that an approved
cleaning procedure will provide clean equipment, suitable for use.
4- The objective of cleaning validation is to prove that the equipment is consistently cleaned from
product, detergent and microbial residues to an acceptable level, to prevent possible
contamination and cross-contamination.
5- Cleaning validation is not necessarily required for non-critical cleaning such as between batches of
the same product (or different lots of the same intermediate in bulk process), floor, walls, outside,
of vessels, and some intermediate steps.
Note: There should be written SOPs detailing the cleaning process for equipment.At least three
consecutive applications of the cleaning procedure should be performed and shown to be
successful in order to prove that the method is validated
VALIDATION PROGRAM:
A validation program generally encompasses three consecutive successful replicate to establish
that the procedure is reproducibly effective.
If the equipment of the similar size, design and construction is cleaned by the same procedure,
studies need not to be conducted on each units as long as a total of three successful replicates
are done on similar piece of equipment, this concept is known as equipment grouping.
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CHANGE CONTROL:
Any of the following proposed changes are evaluated fully for their impact on the validated state of
the procedure. Changes may be,
i. Processing Equipment changes.

ii. Cleaning Equipment changes.
iii. Change in cleaning agents used.
iv. Change in cleaning procedure.
All the above-cited changes should be performed in accordance with the change control procedure.
SAMPLING PROCEDURE:
There are following two general types of sampling procedures that are considered to be
acceptable.
i. DIRECT SURFACE SAMPLING (SWAB METHOD):
A. Area difficult to clean and which are reasonably clean can be evaluated by direct surface sampling
method (Swab Method), leading to establish a level of residue per given area i.e. 60-100 in 2. The
residue that are dried out or are insoluble can be sampled by Swab Method.
B. The suitability of the method to be used for sampling method. it is important to assure the sampling
medium and solvent (used for extraction from the medium) are satisfactory and can be readily
used.
C. For determination of the Microbiological Contamination on surfaces is to use sterile cotton swabs
moistened with sterile peptone water, WFl, or Phosphate Buffer. Using sterile forceps and aseptic
technique, an area of predetermine size, e.g. 60-100 in 2 is wiped with a sterile Swab is then
aseptically transferred to a sterile tube containing a suitable diluents. The tube is then agitated to
suspend any viable microorganisms and aliquots are placed in a semisolid medium to obtain
quantitative results.
ii. RINSE SAMPLING:
Rinse Samples; allow sampling of large surface area and of inaccessible system or parts that can
not be routinely disassembled. However consideration should be given to the fact that the residue
or contaminant may be soluble or may be physically occluded (Hidden some where)in the
machine.
iii. Placebo:
Placebo sampling method provide the best simulation of the production of the subsequent batch of
product, to use this techniques a suitable placebo formulation must be chosen, solubility of the
compounds being studied and accurate simulation of the actual production conditions.
For liquid production either sterile, or non-sterile, water is often the best placebo formulation. For
sterile liquids WFI is usually selected, where purified water is generally best for non-sterile liquids.
If a single placebo batch can be processed through most or all of the processing steps, it will have
been exposed sequentially to all possible source of contamination of the residuals.
If the placebo method is used to validate the cleaning process it should be used in conjugation with
rinse or swab method.
iv. Along with taking any type of the samples, it is important to use visual inspection as wel to ensure
the process acceptability.
TESTING METHOD:
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i. Physical Testing: Along with taking any type of the samples, it is important to use visual
inspection as well to ensure the process acceptability.
ii. Chemical Testing: specificity and sensitivity of the analytical methods should be determined. If
levels of contamination or residue are not detected, it does not mean that there is no residual
contaminant is lower than the sensitivity or detection limit of the analytical method.
Test the samples i.e. swab sample, detection of active or inactive impurity according to relevant
testing procedure of the ingredient to be detective and provide quantitative results.
iii. Microbiological Testing:
a. PREPARATION OF MEDIA FILLED PLATES:-
 Tryptic Soy Agar Plates (TSA Settle Plates)

i. Weigh the required quantity of the media as per manufacturer direction on Digital Balance (as
per SOP).
ii. Dissolve it into Distill Water.
iii. Dispense the required quantities in conical flask.
iv. Sterilize in an Autoclave (as per SOP) at 121ºC for 20 minutes.
v. Cool the media to 45 ºC and pour into sterilized 100x20mm (Glass) and 90x20mm (disposable)
plates.
vi. Allow them to solidify under laminar flow.
vii. Preincubate all plates for 48 hours before using them for environmental monitoring.
 Tryptic Soy Broth:

i. Weigh the required quantity of TSB powder medium on digital balance as per SOP and
dissolve it into distill water.
ii. Dispense 250ml in conical special flask and pre-incubate at 32.5 ±2.5ºC for 3-5 days.
b. SETTLE PLATES (PLATE SEDIMENTATION):-

i. Disinfect the media filled (TSA) plates externally with an approved disinfectant .Allow to dry in a
clean area for 10-15 minutes.
ii. Transfer the plates to the desired location in a clean sealed container.
iii. Expose the media filled plates by taking off the lid aseptically and place it besides the open plate
facing downwards. Do not reach over the exposed plate.
iv. Allow the plate to remain open for one hour.
v. After completion of exposure time, place the lid on the plate taking care such that the plate does not
get contaminated.
vi. Label the plate with site, date and area.
vii. Transfer the plates into the plate box and return to Q.C. Micro Lab. For incubation.
viii. After receiving the plates from production, incubate them in the incubator as per SOP at 32.2 ±2.5ºC
for 48-72 hours.
ix. After incubation count the number of CFU on each plate and report as No. of
CFU/Plate/hour/location.
x. Incubate one media filled plate along with the exposed plates to serve as negative media control.
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c. FINGER DABS :-
i. Disinfect the plates with an approved disinfectant and allow to dry for 10-15 minutes.
ii. Transfer the plates in a sealed container from Q.C. Micro Lab. to the production area.
iii. Deride the plate into two halves on the backside of media filed plate using a Marker; label it as
right and left.
iv. Ask the operator to press his finger tips on to the agar surface. Close the plate.
v. Label with the name of the operator, date and area.
vi. Transfer the plates Q.C. Micro Lab for incubation.
vii. Incubate the plates at 32.5ºC±2.5ºC for 48 hours in an incubator as per SOP.
viii. After incubation count the colonies on the colony counter as per SOP and report as No of CFU
per hand.
d. SWAB TEST:-
i. The sterilized Cotton Swab prepared on S.S sticks.
ii. Prepare Phosphate buffer pH 7.2 and dispense 5 ml in each screw capped test tubes to be
sterilized.
iii. Material is transferred into sterile area for swab test after disinfection.
iv. Remove the sterilized, soak in phosphate buffer and touch to the surface to be checked.
v. 2.5x2.5 inch2 area is touched against each soaked swab.
vi. Place back the swab into the tube containing phosphate buffer and label the tube with the
testing part and date.
vii. Transfer the tested swab to micro lab for microbiological test.
viii. Pour plate method is used to check the contaminants.
ix. Plates are incubated for 48 hours, the result is declared as number of CFU per part or surface.
INSPECTION CRIERIA:-
i. After receiving intimation for cleaning of machine / equipment or area, with the cleaning according
to the predetermined cleaning procedure.
ii. During cleaning, check and note down the following points.
 Description of machine/ equipment/ area:____________________________________
 Major product contact components: ____________________________________
 Product contact area: ____________________________________
 Previous product: ____________________________________
 Batch No: ____________________________________
 Previous batch completed on: ____________________________________
 Equipment used on: ____________________________________
 After cleaning the equipment used on: ____________________________________
 Subsequent product: ____________________________________
 Name of API: ____________________________________
 Batch size of the subsequent product: ____________________________________
 Maximum daily dose of the subsequent product:__________________________
 Detergent / solvent used:____________________________________
 Composition of the detergent used: ____________________________________
 Cleaning tools: ____________________________________
 Ancillary Utilities: ____________________________________
 Cleaning Cycles: ____________________________________
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 Cleaned by: ____________________________________

 Supervised by: ____________________________________

iii. After cleaning, take sample either by direct surface sample (swab Method) or rinse sample
depending upon the nature of machine/equipment and product.
iv. Send the sample to Quality Control department along with technical information sheet for
analysis.
v. Quality Control department analyzes the sample according to written procedure and gives the
results.
vi. Quality Control department analyzes the results, if the results comply with the specified limit,
then the machine/equipment is considered as cleaned and is allowed for further process.
vii. If the results do not comply with the specified limit, then test until it is cleaned. This concept
involves cleaning, sampling& testing until an acceptable residue limit is obtained.
viii. Also record the following.
a. Sampling Method followed: _________________________________________
b. Analytical Method followed: _________________________________________
Annexure-X
PERFORMANCE QUALIFICATION PROTOCOL

PERFORMANCE QUALIFICATION PROTOCOL (PQ)
Of
EXECUTED QUALIFICATION PROTOCOL REVIEW AND ACCEPTANCE:-
All executed test procedures in this qualification protocol have been reviewed and found to
have been executed according to the approved procedures.
Reviewed By:
____________________________ _________________
Date
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All executed test procedures in this qualification protocol have been reviewed and found to
have been executed according to the approved procedures. The signatures below indicate
acceptance of the results.
Approved By:
____________________________ _________________
Date
SIGNATURE IDENTIFICATION PAGE:-

This page is a record of each individual who signs or initials any page included in this
qualification protocol. Each person shall be identified by typed or printed name, full signature
and written initials, and department represented (Quality Assurance / Control, Production,
Validation, Research and Development, Engineering, Contractor, etc.)
Department
or
Name
Signature Initials Contractor
(Type or Print)
(as
Applicable)
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TABLE OF CONTENTS
Description Page No.

Approval Page 1
Executed Qualification Protocol Approval 2
Signature Identification Page 3
Table of Contents 4
Objective 5
Scope 5
References 5
Equipments / System Description 6
Qualification Test Procedures 7
Appendices:-
Appendix A - Qualification Notes
Appendix B – Instrument Calibration Certificate
Appendix C – Deviations
Appendix D – Test Data Documentation
OBJECTIVE:-
To assure that the equipment used for analysis, is operated, and qualified according to the
specifications mentioned in the equipment catalog and complies with company and regulatory
standard and guidance.
Scope:-
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Reference:-
EQUIPMENT / SYSTEM DESCRIPTION
Equipments Name
Model
Manufacturer
Vender
Description
Utilities
Software
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QUALIFICATION TEST PROCEDURE

Performance of the equipment will be qualified when minimum five trade batches of different
products will be analyzed / tested on different days. Purpose of using running different
products, is to check most of the parameters of the equipment.
PRODUCT NO. 1
Product Specification:-
Test Parameters:-
Parameter Settings Parameter Settings
Test Results:-
Analyst /
Test / Assay Test Limits Results Status
Microbiologist
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PRODUCT NO. 2
Test Parameters:-
Test Results:-
Analyst /
Microbiologist
Analyst: ___________________________ Date: ____________________
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PRODUCT NO. 3
Test Parameters:-
Test Results:-
Analyst /
Microbiologist
Analyst: ___________________________ Date: _________________
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PRODUCT NO. 4
Test Parameters:-
Test Results:-
Analyst /
Microbiologist
Analyst : ___________________________ Date: _________________
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PRODUCT NO. 5
Test Parameters:-
Test Results:-
Analyst/
Microbiologist
Analyst : ___________________________ Date: _________________
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Acceptance Criteria Met?
Yes: ______________________________
NO: ________________________________
Initials:______________________________
Date: ________________________________
If “NO”, Explain in Comment:-
Comments:-
Reviewed By: ____________________ Date: _________________
Appendices:-
Appendix A - Qualification Notes

Appendix B – Qualification Test Calibration Certificate
Appendix C – Deviations
Appendix D – Test Data Documentation
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Appendix - A, Page ________
QUALIFICATION NOTES
Prepared By: ________________________ Date: _________________
Reviewed By: ________________________Date: _________________
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Appendix - B, Page ________
QUALIFICATION TEST EQUIPMENT CALIBRATION CERTIFICATES
Instrument
Description
Manufacture
Model No.
Serial No.
Protocol No.
Instrument Used For
Calibration No.
Calibration Date
Calibration Due Date
Verified By / Date
Comments:-
Reviewed By: ___________________________Date: _________________
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Appendix - C, Page ________
DEVIATION LISTING
Deviation Deviation Page Addendum No. (if

Deviation No
Description No. Applicable)
Reviewed By: ___________________________Date: _________________
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QUALIFICATION PROTOCOL DEVIATION FORM
Deviation No: Test No. : Page No. :

Deviation Description
Prepared By: ___________________________ Date:

_________________
Corrective Action Response
Prepared By: ___________________________ Date:

_________________
Approved By: ___________________________ Date:

_________________
Summary of Corrective Action Results
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Prepared By: ___________________________Date: _________________
Approved By: __________________________Date: _________________
QUALIFICATION PROTOCOL ADDENDUM FORM
Deviation No: Test No. : Page No. :

Modification Description / Comments:
Completed By: _________________________Date: _________________
Approved By: __________________________Date: _________________
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Appendix - D, Page ________
TEST DOCUMENTATION
(Executed)
Title / Document ID Document Rev. Verified By/

Item No.
Description No. Date No. Date
Comments:-
Reviewed By: ___________________________Date: _________________
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UNDERTAKING
SUBJECT: SUBMISSION OF STABILITY STUDY DATA FOR PROPOSED SHELF LIFE AND
STORAGE CONDITIONS OF DRUGS.
We, both production manager and quality control manager on behalf of ms Hygeia
Pharmaceuticals, Plot no 295 –Industrial Triangle Kahuta Road Islamabad. Hereby declare
and undertake: - that, Before marketing of drug i.e. (HYTREX 250mg Injection (IM) ), study
data of six months accelerated stability study and one year real-time study of 3 batches (one
lab and 02 pilot scales) for verification of 2 years of shelf life will be conducted.
Recommended storage condition as per ICH and WHO guidelines will be maintained. We also
undertake to continue these stability studies up to complete shelf life.
We will be responsible for stability, integrity, efficacy and genuineness of Stability data that will
be submitted to DRAP.
Thanks and regards.
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FINISHED PRODUCT:
HYTREX 250 MG INJECTIONS (Im)
Composition:
Each vial contains
Ceftriaxone sodium equ. To ceftriaxone….250mg
Identification: Ceftriaxone sodium is positive.
Ceftriaxone Injection is a sterile powder of Ceftriaxone Sodium for injection. It contains the
equivalent of not less than 90.0 percent and not more than 115.0 percent of the labeled amount of
Ceftriaxone (C18H18N8O7S3).
Identification:
A: The chromatogram of the Assay preparation obtained as directed in the Assay exhibits a major
peak for Ceftriaxone, the retention time of which corresponds to that exhibited in the chromatogram
of the Standard preparation obtained as directed in the Assay.
C: It responds to the tests for sodium.
Weight variation: out of 20 vials checked weight of 18 vials should be in the range of + 5% of the
average weight. Only 2 vials can vary up to + 10%.
Bacterial endotoxins: It contains not more than 0.20 USP Endotoxin Unit per mg of Ceftriaxone.
Sterility: It meets the requirements when tested as directed for Membrane Filtration under Test for
Sterility of the Product to be Examined.
Particulate matter: meets the requirements for small-volume injections.
Crystallinity: meets the requirements.
pH: between 6.0 and 8.0 in a solution (1 in 10).
Water: between 8.0% and 11.0%.
Assay by HPLC:
PH 7.0 Buffer: Dissolve 13.6 g of dibasic potassium phosphate and 4.0 g of monobasic potassium
phosphate in water to obtain 1000 mL of solution. Adjust this solution with phosphoric acid or 10 N
potassium hydroxide to a pH of 7.0 ± 0.1.
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PH 5.0 Buffer: Dissolve 25.8 g of sodium citrate in 500 mL of water, adjust with citric acid solution
(1 in 5) to a pH of 5.0 ± 0.1, and dilute with water to a volume of 1000 mL.
Mobile phase: Dissolve 3.2 g of tetraheptylammonium bromide in 400 mL of acetonitrile, add 44 mL
of pH 7.0 Buffer and 4 mL of pH 5.0 Buffer, and add water to make 1000 mL. Filter through a
membrane filter of 0.5 µm or finer porosity, and degas. Make adjustments if necessary.
Chromatographic system: The liquid chromatography is equipped with a 270-nm detector and a
4.0-mm × 15-cm column that contains 5-µm packing L1. The flow rate is about 2 mL per minute.
Chromatograph the Resolution solution, and record the peak responses as directed
under Procedure: the resolution, R, between the Ceftriaxone E-isomer and Ceftriaxone peaks is not
less than 3. Chromatograph the Standard preparation, and record the peak responses as directed
under Procedure: the column efficiency determined from the analyte peak is not less than 1500
theoretical plates; the tailing factor for the analyte peak is not more than 2; and the relative standard
deviation for replicate injections is not more than 2%.
Standard preparation: Dissolve an accurately weighed quantity of USP Ceftriaxone Sodium
RS in Mobile phase, to obtain a solution having a known concentration of about 0.2 mg per mL. Use
this solution promptly after preparation.
Resolution solution: Dissolve a suitable quantity of USP Ceftriaxone Sodium E-Isomer
RS in Standard preparation, and dilute with Mobile phase to obtain a solution containing about 160
µg of USP Ceftriaxone Sodium E-Isomer RS per mL and 160 µg of USP Ceftriaxone Sodium RS per
mL. Use this solution promptly after preparation.
Assays preparation:
Assay preparation 1: Transfer about 40 mg of Ceftriaxone for Injection, accurately weighed, to a
200-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix. Use this
solution promptly after preparation.
Assay preparation 2: (where it is represented as being in a single-dose container).Constitute
Ceftriaxone for Injection in a volume of water, accurately measured, corresponding to the volume of
solvent specified in the labeling. Withdraw all of the withdrawable contents, using a suitable
hypodermic needle and syringe, and dilute quantitatively with Mobile phase to obtain a solution
containing about 180 µg of Ceftriaxone per mL. Use this solution promptly after preparation.
Assay preparation 3: (where the label states the quantity of Ceftriaxone in a given volume of
constituted solution)—Constitute Ceftriaxone for Injection in a volume of water, accurately measured,
corresponding to the volume of solvent specified in the labeling. Dilute an accurately measured
volume of the constituted solution quantitatively with Mobile phase to obtain a solution containing
about 180 µg of Ceftriaxone per mL. Use this solution promptly after preparation.
Procedure: Separately inject equal volumes (about 20 µL) of the Standard preparation and
the Assay preparation into the chromatograph, record the chromatograms, and measure the
responses for the major peaks. Calculate the quantity, in µg, of Ceftriaxone (C18H18N8O7S3) per mg of
the Ceftriaxone for Injection taken by the formula:
200(CP / W)(rU / rS)
in which C is the concentration, in mg per mL, of USP Ceftriaxone Sodium RS in the Standard
preparation; P is the designated potency, in µg, of Ceftriaxone per mg of USP Ceftriaxone Sodium
RS; W is the quantity, in mg, of Ceftriaxone for Injection taken to prepare Assay preparation
1; and rU and rS are the Ceftriaxone peak responses obtained from Assay preparation 1 and
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the Standard preparation, respectively. Calculate the quantity, in mg, of Ceftriaxone (C18H18N8O7S3)

withdrawn from the container, or in the portion of constituted solution taken by the formula:
(L / D)(CP)(rU / rS)
in which L is the labeled quantity, in mg, of Ceftriaxone (C18H18 N8O7S3) in the container, or in the
volume of constituted solution taken; D is the concentration, in µg per mL, of Ceftriaxone in Assay
preparation 2 or Assay preparation 3, based on the labeled quantity in the container or in the portion
of constituted solution taken, respectively, and the extent of dilution; C is the concentration, in mg
per mL, of USP Ceftriaxone Sodium RS in the Standard preparation; P is the designated potency, in
µg, of Ceftriaxone per mg of USP Ceftriaxone Sodium RS; and rU and rS are the Ceftriaxone peak
responses obtained from the Assay preparation and the Standard preparation, respectively.
REFERENCES: AS PER USP-35 SPECIFICATIONS.
MICROBIOLOGICAL METHODS
Sterility test:
All Officers carrying out sterile work must have received training in aseptic techniques.
Section A
Media & apparatus
All Medias, clothing and testing apparatus must be sterile for each item thermolag's strip and
autoclave tape must be checked properly & recorded.
-- Thioglycollate medium (100 ml for each test)

-- Tryptone Soya broth (100 ml for each test)
-- Rinsing solution (100ml for each test)
-- Filter units and filter manifold.
-- Vacuum source
-- Filter of 0.2 micron
-- Forcep and pair of scissors
-- Sterile containers for sample collection
-- 1% Savlon solution
Procedure
1- Transfer samples (washed with 70% alcohol), medias (labelled with B.No. Portion and test
date) and equipment to sterile area as described in FF/SOP/QC/020
2- Enter the sterile area following FF/SOP/QC/020.
3- Remove the inner paper of each item in UV Hatch.
4- All flasks after removing the paper must be kept in L.A.F. cabinet
5- Connect up filtration system & switch on the pump.
6- Assemble the filter units putting sterile 0.2 micron filter in place.
7- Turn on the manifold
8- Add sterile purified water in each vial using a sterile syringe and collect the sample in sterile
container.
9- Filter 20ml solution of each portion.
10- Rinse with sterile Diluent.
11- Switch off the manifold.
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12- Cut the filter in 2 equal pieces transfer half piece in to Thioglycollate medium and half in to
Tryptone soya broth.
13- Place completed test flasks in UV hatch.
14- The testing session is concluded as described in FF/SOP/QC/039.
15- See FF/SOP/QC/008 for flask cleaning details and FF/SOP/QC/013 for Formaldehyde
gassing.
For sterility testing the following points must be noted;

1- L.F.C. and workbench must be swabbed before use.
2- Env. Of sterile room must be monitored before starting the test.
3- Room temperature must be checked and recorded before test.
4- Medias and sample must ONLY be opened within the LFC.
5- Any container with loose taps and equipment or clothing with torn paper must be discarded.
6- All Medias and rinsing solution must come from one batch for which there is avalid growth
control test either completed or ongoing
7- Gloves must be frequently swabbed with 1% savlon solution during working & to be changed
IVmediately if torn, become damaged or the wearer carries out potentially dirty job.
8- Care must be taken to prevent the membrane filter becoming contaminated during transfer to
and from the filter unit.
9- Any item dropped during the testing session must be left until testing has been completed on
no account should it be used.
10- Any major spillage should be cleaned up after the testing session NOT during it.
Session B: Incubation details refer to SOP

Session C: Interpretation of results refers to SOP
Limit: Sterility must conform
BACTERIAL ENDOTOXIN TEST

01 Reagents and Materials
-- Water, distilled and pyrogen free
-- LAL - Reagent (Lysate)
-- Endotoxin (e.g. NP-1, highly purified Lipopolysachride from Salmonella abortus)
-- Stopwatch
-- Test tube stirrer (Vortex)
-- Dry block incubator
-- Heating oven for depyrogenisation
-- 5ml pipettes (graduated) & 1ml
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-- Universal containers for dilutions

-- Test tubes 10 x 75 mm for incubation
-- Eppendorf micropipettes (1000, 10 microliter) with blue and yellow tips
-- Test tube holders
-- Flasks (100ml)
-- Vortex Type mixer
1.1 Water, distilled and pyrogen-free
Water is freshly distilled transferred to previously depyrogenised 100ml flasks and
IVmediately sterilized for 15 minutes at 121oC. The water should be stored below 8
oCbefore its first use it is to be tested for suitability.
1.2 LAL - Reagent
The Lysate is reconstituted by dissolving in 5ml water, distilled and pyrogen-free and rotating
gentle the vial at least 30 seconds taking care that no foam is formed the daily required
amount of Lysate may be proportioned into several tubes which should be covered by
parafilm.
Storage The reconstituted Lysate can be kept for 1 day at temperature below 8OC protected
from light or it can be kept maxIVum 4 weeks at temperature below -10oC.
1.3. Endotoxin NP-1

Adopt the vial to room temperature and agitate it for 60 seconds on a Vortex.
At first three tenfold dilutions are carried out using pyrogen free water to get 10 EU /ml,1
EU / ml and 0.1 EU/ml
02 Handling Description for LAL Test

Preparation of Dilutions
2.1 Endotoxin Standard
To prepare a set of working standard then two fold dilutions are made from the 0.1 EU /ml
Dilution.The final dilution should usually contain no more than one-half the labelled LAL-
Sensitivity (0.125, 0.06, 0.03 EU /ml) the standard will be tested with.Whenever the NP-1
Standard is used the vessel has to be shaken on the Vortex for 15 seconds.
Storage. The standard solution with 1 EU /ml can be stored at 4oC or below 8oC for 4 weeks.
2.2 The water is directly transferred to a previously depyrogenised 100ml flask .Normally the
water has not to be diluted with pyrogen free water, only if the first test was positive. Also the
dilution is to be agitated for 15 seconds.
2.3 Test Procedure
- Mark the test tubes (10 x 75 mm) -
Negative control
+ 0.025 EU/ml positive controls
+ 0.0125 EU/ml positive controls + 0.06 EU/ml positive controls
+ 0.03 EU /ml Positive control
T/2 Water for Injection diluted 1:1 with pyrogen-free water.
- Pipetteing:
Positive control
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Before dosing out of every dilution containing Endotoxin agitates for 15 seconds and transfer
IV mediately 100 microliter into the test tube by individuated the tip 2cm under the marked
side of the tube. The dosing is started from the lower concentration to the higher ones.
Negative control
Dose 100 microliter water, pyrogen free
Dosing Lysate
Agitate the Lysate brought to room temperature by 5 gentle horizontal rotations and dose
carefully (without spoiling the tip with Endotoxin or test material) on the top of the tube
opposite to the mark, from now the tubes have to be moved very consciously.
Dilution Method
0.1 ml ampoule content + 0.8 ml pyrogen free water.
Dilution ratio on which passes = 1:8
MVD around = 1 : 64
2.4 Incubation
Every test tube is taken in constant intervals 4 tIVes rotated horizontally and put into the
heating block without circulation taken so far 60 +/- 2 minutes at a temperature of 37 +/- 1oC
03 Reading
When the tIVe has passed exactly the tubes are taken in the same sequence from the
heating block taking care that the glass tube does not touch the tube holder or anywhere
else. The tube is now inverted at 180o. When in this position a firm holding gel is formed the
result is regarded as positive, the contrary, a liquid or a slipping clot is observed, the result is
negative.
3.1 Negative control
Must be negative. A positive result indicates either the water or the vessels or the lysate are
contaminated with endotoxins. Then reason for this has to be searched for.
3.2 Positive control - Sensitivity of the test

The lowest test dilution which resulted in gel forming indicates the sensitivity of the test.
04 Evaluation
If the original gives a negative result its Endotoxin content is expressed being lower than the
sensitivity of the LAL - Test carried out at that day.
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Annexure-L
HVAC System Performance Qualification Protocol
Performance Qualification Protocol of:
Performance Qualification Protocol for : ( )

(Not Valid without Signature)
Personnel involved in
Signature Designation
Qualification Study
Officer Validation/Calibration
Prepared By:
Manager Validation
Reviewed By:
Production Manager/MR
Approved By:
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1.0 Introduction:
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Several different utility systems are necessary to operate a manufacturing plant, but only a few have
a direct or incidental product contact and therefore require qualification. The most important of these
are Heating Ventilation and Air Conditioning (HVAC), Water and compressed gases. Other utilities,
such as electricity, plant steam and lighting are obvious requirements but not considered process
critical.
A conventional Air Handling System has 4 sub-systems:
1. Air handling of the incoming (fresh) air: elimination of coarse contaminants and protection from
frost if necessary. In the case of air re-circulation, the fresh air is also called make-up air.
2. Central air handling unit (AHU), where the air will be conditioned (heated, cooled, humidified or
de-humidified and filtered), and where fresh air and re-circulated air, if any, (indicated here by the
dotted line) will be mixed.
3. Air handling in the rooms: under consideration (pressure differential system, additional filtration,
air distribution).
4. Air exhausts system (filtration).
The style and approach to facility qualification may differ from firm to firm, but the principles of a
well-designed qualification program are similar. The supply of filtered air under positive pressure is
the single most important means of maintaining control of environment in an aseptic manufacturing
environment. An air handling system introduces pre-treated air, in order to provide a manufacturing
environment with specified cleanliness, temperature and humidity in order to prevent product
contamination and degradation. Air is then exhausted from the manufacturing environment
Features of the HVAC system that affects product quality and the acceptance criteria for PQ has
been established and the data of the following monitoring will be complied.
i. Room Conditions
ii. HEPA Filter integrity.
iii. Airborne particle control
iv. Air Flow direction.
v. Differential pressure balancing.
vi. Air changes and Air velocity.
The primary purpose of an air conditioning, heating and ventilation systems to provide a specific set
of environmental conditions required for the non-sterile operations.
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2.0 Objective:
The objective of this validation: the primary element in the HVAC system for the purpose of controlling air
cleanliness in the aseptic environment is the HEPA filters integrity and to certify that cleaned air,
differential pressure, Temperature and Humidity provided by HVAC system within the aseptic production
facility meets the established standards (URS).
3.0 Scope:
The scope of this protocol is to outline the types of parameters that affect the quality of products in HVAC
system. The testing and acceptance criteria of HEPA filter integrity, airborne particle control, differential
pressure in rooms, temperature, humidity, air velocity, air changes and the air flow direction is also a
described in this protocol.
4.0 Change Control:

Any modification, fabrication or relocation of HVAC system which directly or indirectly effect the
performance of the HVAC system or the quality of the product should be controlled and in accordance
with the Change Control Procedure, and it will be required to re-qualify the HVAC system.
Minor changes or the changes have no direct impact of the in process or final product quality should be
handled through the system of the preventive maintenance programme.
5.0 Responsibility:
To manage all the activities of validation of HVAC system and to coordinate
with the Validation Team throughout validation process by scheduling,
Validation manager reviewing the validation protocols by supervising the validation process and
analyzing the validation data and test results and finally reviewing the Final
Validation Report.
He is responsible to prepare the validation protocol for HVAC system,
monitoring the validation process, compiling and analyzing the validation
Officer validation) data and results and to report the final results to validation team leader.
He is responsible for the Calibration of Machine & Equipments and HEPA
filters and to help validation manager in performing IQ, OQ & PQ.
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The Representative of Engineering Department is responsible for defining

the necessary HVAC system specifications, limitations, capacity, and
maintenance requirements, and providing the necessary training on the
proper operation and maintenance of the HVAC system. He is also
Representative of responsible for providing the necessary utilities, equipment accessories and
Engineering Department the facilities. The representative of Engineering Department is also required
to participate in HVAC system Installation and Operational Qualification and
to provide technical support to ensure proper and efficient function during
validation process.
The representative from Quality Control (Microbiological Lab.) is
Representative Quality
responsible to perform the microbiological testing and environmental
Control (Microbiological
monitoring during the HVAC system validation process and. Analysis of the
Lab)
samples
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6.2 HEPA Filter Integrity Test :

Purpose :
The purpose of the test is to challenge the integrity and installation of filters and its housing with a
known concentration of smoke particles.
Equipment :
Aerosol photometer ATI (External Source Biotroll)
Division of Hamilton Association INC.
Air Technique
Model 2H USA.
Operating Method :
 Start the cabinets circulating fan for Laminar Flow Hood or blower for ceiling HEPA filters.
 Using the Velometer take 12 velocity measurements per filter of the downward supply air on
the horizontal plane defined by the bottom edge of the window frame. Adjust the blower speed
so that the velocities are in the range of 90 FPM + 20 %.
 Place the aerosol generator so that the aerosol is introduced into the cabinet upstream of the
HEPA filter of Laminar Flow Hood and blower duct for ceiling HEPA filter. Introduce the aerosol
in a manner that will produce uniform concentration at each of the HEPA filter being tested.
 Scan the downstream side of the HEPA filters and parameters of each filter pack by passing
the photometer probe in slightly over lapping strokes over the entire surface of the HEPA filter
at a traverse rate of not more than two inches per second. Hold the nozzle of the probe no
more that one-inch from the surface.
 Make separate passes around the entire periphery of the filter, along the bond between filter
media and frame, along the gasket seal between filter frame and installation clamping frame,
and along all other joints in the installation through which leakage might bypass the filter
media. Periodic surges are not indicative of leaks but are merely burst releases form crevices
or from flaking the suspected areas to verify the absence of a leak.
 After pin pointing a leak in the filter media, mark a 1-2 inch area around the leak parallel to the
media and between the separators with silicon adhesive sealant.
 Minor leak at the gasket frame interface can be repaired with silicon grease. If major gasket
leaks are found, remove the filter access panel and check the filter clamps. Tighten the clamps
as necessary. If the gasket still leaks, remove the HEPA filter. Smooth or clean the matting
surfaces, or replace the gasket. Re-install the filter.
 Approximately 5 % of the filter media may be sealed. If more sealant is required, the filter must
be rejected and a new one to be installed by engineering Department.
 Diagram all leaks and record them on the certification report form.
Routine Frequency :
Once a year
Acceptance Criteria :
The leakage should be less than 0.01 %
Minor leaks approx. 5%- 10% is avoidable, to be closed by silicone grease.
Major leakage i.e. above 10 % leakage of the upstream PAO concentration, is critical and needs
to change the filter.
Results :
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6.3 Room Particulate Classification (RPC)
Purpose
To establish that the quality of air meets class A specification with system returning to class A within 30 minutes of
staff leaving the area.
The quality of air at specified locations of the aseptic filling room will be monitored. This in turn would
 Provide assessment of the ‘cleaning action’ of air
 Verify the quality of air supplied to the aseptic area
Equipment
Operating Method
 Connect the particle counter with UPS.

 Connect the UPS with power supply if necessary.
 Connect the particle detecting probe with particle counter.
 Power ON the particle counter & check the data on screen.
 Adjust the location and number of replicates required.
 Run the instrument.
 Check the number of particles at different locations.
 Prepare reports according to the locations separately and submit to Validation department
Developing a “Location Matrix”
Min. No. of sample locations = X = Floor area of clean Zone (A)
Airborne Particulate Class Designated
= X = A (m2)
100
= X = A (m2)
10
The no. of sample locations will be uniformly spaced horizontally and vertically, throughout the clean zone.
Routine Frequency
Monthly
Acceptance Criteria
S.
Class Particle Size μm Limits Rest Limits Operation
No.
0.3 3500 3500
0.5 μm 3500 3500
A 1.0 μm 01 01
5.0 μm 01 01
0.3 3500 350,000
0.5 μm 3500 350,000
2 B
1.0 μm 01 2,000
5.0 μm 01 2,000
0.3 350,000 3500,000
0.5 μm 350,000 3500,
3 C
1.0 μm 2,000 20,000
5.0 μm 2,000 20,000
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6.4 Microbiological Assessment of Air.
Purpose :
The purpose of the test is to challenge the air provided by the HVAC system is clean and meets the
requirements of class 100.
Equipment :
Operating Method :
 Disinfect the items with an approved disinfectant before taking in sterile area. Allow to dry for 10 – 15 minutes.
 Transfer the EQp air sampler to the production area.
 Transfer the media filled plates 90x20 mm (disposable) in Production area.
 Transfer the air sampler to the required location.
 Place a sterile 90 x 20mm media filled plate in the air sampler as per SOP.
 Run the air sampler to sample 1000 liter of air in filling room and under laminar flow.
 Run the air sampler to sample 250 liters of air in entrance and change room
 After completion of the sampling time remove the plate from the slot and cover it.
 Label the plate with date, site and area.
 Transfer the plates and the air sampler to the Q.C. Micro Lab.
 Incubate the air sampled plates at 32.5 + 2.5°C in an incubator as per SOP for 48 hours.
 Count the No. of CFU and report as CFU per cubic meter of air.
Routine Frequency :
Weekly for Injectable sterile area
Twice a month for micro lab.
Under Laminar Flow <1cfu
Filling Room <5cfu
Entrance Room <7cfu
Change Room <10cfu
Sterility Test Room Micro lab. <15cfu
Results :
Will be given in report
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6.5 Air Velocities and Air Changes
Purpose
The purpose of this exercise is to determine the operating range for number of air changes and air velocities in
each room. Data generated will be analyzed to
a) Confirm that the air changes in each room are more than 20/hr.
b) Determine the air velocity in each room
Equipment
Anemometer
Operating Method
 Measure the air velocity with the Velometer at multiple points (12 points) across the Workspace below the HEPA
filter.
 Take measurement for a minimum of 15 seconds & record the reading.
 When air velocities fall below the lower limits i.e. 70 FPM, the pre-filters should be Checked / cleaned (as required)
and replaced if exceptionally dirty. If the average air flow velocity is still below the lower limit, increase the blower
speed until the acceptance Criterion is met. When the blower speed control is turned to the maximum setting
and the average air flow velocity is below the recommended value, assess the need for the blower repair and
HEPA filter replacement.
 If blower motor repair or HEPA filter replacement is necessary, repeat the above steps Report all the results on the
certification report form.
 After measuring the air velocity, the No, of Air changes can be measured by the following calculations.
No, of Terminal Filters = Average Air velocity of Terminal Filter A =

Average Air velocity of Terminal Filter B= Net Average Air Velocity (AV) A + B =
2
Air Changes Formula : AV Χ FA Χ 60
VA
Where;
AV = Average Air Velocity

FA = Filtration Area of HEPA Filter
VA = Volume of Area
Routine Frequency :
The frequency of air velocity is after three months.
The frequency of air changes is after 06 months
Air change/hr. : Must not be less than 20 per hour
Air velocity : 90± 20 FPM
Results :
Will be given in report.
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6.6 Differential Pressure Balancing :

Purpose :
To measure the pressure differential between
 Sterile filling and sterile associated rooms
 Sterile filling and non-sterile areas
Equipment :
Mounted Magnehelic Gauge
Routine Frequency :
Daily
Pressure differential between
 Sterile filling and non-sterile areas is 0.06 in. H 2O.
Results
Will be given in report
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Attachment I
A conventional Air Handling System has 4 sub-systems:
Exhaust air treatment
Fresh air treatment

(make-up air) Terminal air treatment
+ at production room level
Production Room
Central air handling unit
Attachment II
A conventional Air Handling System:
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Silencer Flow rate controller Fan Filte

r
Weather Control damper

louvre
+
Humidifier
Prefilter Terminal
filter
Cooling Production Room

coil
Heating With
Coil droplet
separator
8.0 Facilities/Responsible Personnel/Equipment/Material & Documentation:
8.1
Facilities:
The Performance Qualification of HVAC system will be performed in Hygeia Pharmaceuticals. In

ISLAMABAD, PAKISTAN.
8.2 Identification of Responsible

Personnel:
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Name Job Title Signature
Officer
Validation/Calibration
Validation Manager
Production Manager
Supervisor Engineering
QA Officer
QC Microbiologist
8.3 Identification of Documentation:

Description of Document SOP/ QF No.
System/ Equipment Qualification (SOP)
Calibration (SOP)
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Undertaking
Subject: CHANGE OF BRAND NAME IN CASE OF RESEMBLENCE.
It is stated, on behalf of ms Hygeia Pharmaceuticals, Plot no 295 – Industrial Triangle Kahuta Road
Islamabad. That, the brand name of drug Hytrex injection 250mg IM (Ceftriaxone sodium), applied for
registration, and has been assigned the given name keeping in mind that it does not resemble with any
other brand name. In case of any resemblance with any existing brand name, we shall change the
name of our drug.
Thanks and regards.
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ANNEXURE II
RAW MATERIAL:
Ceftriaxone Sodium
C18H16N8Na2O7S3·3½H2O 661.60
5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[[(2-amino-4-thiazolyl)

(methoxyimino)acetyl]amino]-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5-, 6-dioxo-1,2,4-triazin-3-
yl)thio]methyl]-, disodium slat, [6R-[6 ,7 (Z)]]-, hydrate, (2:7).
(6R,7R)-7-[2-(2-Amino-4-thiazolyl)glyoxylamido]-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-
dioxo-as-triazin-3-yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7 2-(Z)-
(O-methyloxime), disodium salt, sesquaterhydrate [104376-79-6].
Anhydrous 598.56
» Ceftriaxone Sodium contains the equivalent of not less than 795 µg of ceftriaxone
(C18H18N8O7S3) per mg, calculated on the anhydrous basis.
Packaging and storage— preserve in tight containers.
Labeling— where it is intended for use in preparing injectable dosage forms, the label states
that it is sterile or must be subjected to further processing during the preparation of injectable
dosage forms.
Identification—
A: Infrared Absorption 197K .
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B: The chromatogram of the Assay preparation obtained as directed in the Assay exhibits a

major peak for ceftriaxone, the retention time of which corresponds to that exhibited in the
chromatogram of the Standard preparation obtained as directed in the Assay.
C: It responds to the tests for Sodium 191 .
Crystallinity 695 : meets the requirements.
pH 791 : between 6.0 and 8.0 in a solution (1 in 10).
Water, Method I 921 : between 8.0% and 11.0%.
Other requirements— where the label states that Ceftriaxone Sodium is sterile, it meets the
requirements for Sterility and Bacterial endotoxins under Ceftriaxone for Injection. Where the
label states that Ceftriaxone Sodium must be subjected to further processing during the
preparation of injectable dosage forms, it meets the requirements for Bacterial
endotoxins under Ceftriaxone for Injection.
Assay—
pH 7.0 Buffer— Dissolve 13.6 g of dibasic potassium phosphate and 4.0 g of monobasic
potassium phosphate in water to obtain 1000 mL of solution. Adjust this solution with
phosphoric acid or 10 N potassium hydroxide to a pH of 7.0 ± 0.1.
pH 5.0 Buffer— Dissolve 25.8 g of sodium citrate in 500 mL of water, adjust with citric acid
solution (1 in 5) to a pH of 5.0 ± 0.1, and dilute with water to a volume of 1000 mL.
Mobile phase— Dissolve 3.2 g of tetraheptylammonium bromide in 400 mL of acetonitrile, add
44 mL of pH 7.0 Buffer and 4 mL of pH 5.0 Buffer, and add water to make 1000 mL. Filter
through a membrane filter of 0.5 µm or finer porosity, and degas. Make adjustments if
necessary (see System Suitability under Chromatography 621 ).
Standard preparation— Dissolve an accurately weighed quantity of USP Ceftriaxone Sodium
RS in Mobile phase, to obtain a solution having a known concentration of about 0.2 mg per
mL. Use this solution promptly after preparation.
Resolution solution— Dissolve a suitable quantity of USP Ceftriaxone Sodium E-Isomer
RS in Standard preparation, and dilute with Mobile phase to obtain a solution containing about
160 µg of USP Ceftriaxone Sodium E-Isomer RS per mL and 160 µg of USP Ceftriaxone
Sodium RS per mL. Use this solution promptly after preparation.
Assay preparation— Transfer about 40 mg of Ceftriaxone Sodium, accurately weighed, to a
200-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix. Use this
solution promptly after preparation.
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Chromatographic system (see Chromatography 621 )—the liquid chromatograph is

equipped with a 270-nm detector and a 4.0-mm × 15-cm column that contains 5-µm packing
L1. The flow rate is about 2 mL per minute. Chromatograph the Resolution solution, and
record the peak responses as directed under Procedure: the resolution,R, between the
ceftriaxone E-isomer and ceftriaxone peaks is not less than 3. Chromatograph the Standard
preparation, and record the peak responses as directed underProcedure: the column
efficiency determined from the analyte peak is not less than 1500 theoretical plates; the tailing
factor for the analyte peak is not more than 2; and the relative standard deviation for replicate
injections is not more than 2%.
Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and
the Assay preparation into the chromatograph, record the chromatograms, and measure the
responses for the major peaks. Calculate the quantity, in µg, of ceftriaxone (C 18H18N8O7S3) per
mg of the Ceftriaxone Sodium taken by the formula:
200(CP / W)(rU / rS)
in which C is the concentration, in mg per mL, of USP Ceftriaxone Sodium RS in the Standard
preparation; P is the designated potency, in µg of ceftriaxone per mg, ofUSP Ceftriaxone
Sodium RS; W is the quantity, in mg, of the Ceftriaxone Sodium taken to prepare the Assay
preparation; and rU and rS are the ceftriaxone peak responses obtained from the Assay
preparation and the Standard preparation, respectively.
REFERENCES: AS PER USP SPECIFICATIONS
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SOP FOR CLEANING VALIDATION
1. OBJECTIVE
The objective of the cleaning validation is to provide evidence that the cleaning materials used, the
procedure employed, the reliability of the equipment used, and the training of the operators executing
the procedure are effective, reproducible, and adequate to achieve consistent predetermined product,
cleaning agent and microbial residue levels.
2. SCOPE
This procedure will be applicable to all critical equipment comes in direct contact with the product.
Non-contact parts should also be considered wherever a risk of contamination is possible. Following
the production of the three batches and the cleaning of the relevant equipment, cleaning validation will
be performed by swab sampling of pre-selected sites and testing for Active Drug Substance
Contamination.
The selected sample sites will represent the areas that have the highest risk of contamination
(identified as areas that are difficult to clean) but also areas with a low risk of contamination. These
sites are also selected with regards to accessibility of the sampling sites.
3. Validation Teams
Plant Manager of the Site will nominate the Validation Coordinator (preferably Head
of Quality & Compliance) and Team Members (cross functional).
For Cleaning Validation

- Quality Assurance Manager or his Deputy
- Quality Assurance Officer
- Quality Control Manager or his Deputy
- Quality Control Officers (2)
- Microbiologist
- Production Manager or his Deputy
- Engineering Manager or Officer
4. RESPONSIBILITIES
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Plant Manager
- To provide resources for execution of cleaning validation program.
- Nomination of cleaning validation team.
- Ensure that cleaning validation procedures are validated.
Production Manager
- To assist with the completion of this Cleaning Validation where necessary.
- Ensuring that production is performed according to the written procedures and
That the cleaning process is done as per procedure
- Responsible for assuring that all the involved equipment is available, well kept
And cleaned before their use.
- Is also responsible for assuring that the relevant SOP’s are available for
Producing batches according to the production records.
Quality Control Manager / Quality Assurance Manager
- Preparation, compilation and completion of cleaning validation protocols.
- Ensure that all pre-requisites are met and protocol information is completed.
- Writing of the Cleaning Validation Report after completion of this protocol.
- Responsible for performing all physical and chemical tests necessary for the
quantification of the chemical residues.
- Ensuring that all Analytical Methods used are validated.
- Ensure that all procedures required to execute cleaning validation protocol are
available and updated.
5. PREREQUISITES
To ensure that the cleaning procedure and method used leads to thorough andreliable cleaning of the
equipment as per existing core documents.
5.1 Installation / Operation Qualification Completion

The various equipment involved in the production has to qualified (Installation and Operation
Qualification) to ensure that it meets its predetermined specifications and fulfills the
requirements to produce the batches according to GMP.
The IQ and OQ activities are compiled in respective protocols, which have to be available
completed and approved before cleaning validation commences.
5.2 Standard Operating Procedures (SOP’s)

In order to ensure proper operation of equipment during the manufacturing process of drug
product by the operators, as well as to ensure reliable and reproducible cleaning of the equipment
used it is required to follow relevant SOP’s.
5.3 Personnel Training Records
To ensure reproducibility and minimize inter-operator variability within the execution of the
cleaning procedure each operator has to be trained. This training has to be documented by the QA
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and refresher training has to be done to ensure that there are no deviations from the cleaning
procedure. Only trained personnel should be allowed to carry out this procedure.
5.4 Purified Water
For successful Cleaning Validation, the cleaning agent used has to be validated (ability of the
chemical agent used to reduce microbial contamination).
5.5 Validation of Sampling Techniques
To ensure that the samples that we take are indeed representative of the actual level of
chemical residue we also need to validate the sampling methods. We need to be sure that the type of
swab we use will lift the contaminants from the surfaces and that the swabbing solvent in combination
with the type of swab will give a good recovery
The sampling technique is also very important, thus the sampler must be trained to ensure
reproducibility.
5.6 Analytical Method Validation

This is one of the most import prerequisites of Cleaning Validation. If the methods by which we
measure the samples taken are not validated there is no point in performing validation as we will either
never achieve the acceptance criteria or even worse, we will have a false sense of security.
It is very important that the level of sensitivity produced is at least equal to acceptable residual
level for each component. Limit of detection, Limit of quantification, specificity, ruggedness, sample
handling and storage, etc. will be included in each protocol.
5.7 Visual Inspection
In order to complete the sampling of the equipment by means of surface swabs (where
applicable) the equipment cleaned should be clean on visual inspection. If the surface fails visual
inspection the subsequent surface swabs may also fail the acceptance criteria. Thus as a prerequisite
to surface swabs the equipment has to pass visual inspection.
5.8 Calibration Status Check
At the time of performing the Cleaning Validation it is also necessary to check that all the critical
instruments are still within their calibration period.
6. PROCEDURE
To perform the cleaning validation, a campaign of Three Consecutive, Identical Batches will be used to
demonstrate that the limits as indicated in this protocol are met.
All the equipment, procedures and personnel involved in the Cleaning Validation will be the same as
those used for normal production batches. Procedures and Documentation will comply with the
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current GMP and Global Quality Directives. The production batches will be manufactured utilizing the
normal equipment as per BMR; afterwards the equipment will be cleaned as per the Cleaning SOP.
Swab Sampling
- Identify the sampling points of major equipments for direct surface sampling for Residues.
- Carry out Swab sampling on at least three direct surface sites of the equipment, which are critical
due to potential contamination by active substance.
- The size of the direct surface samples will be 100 cm2 swabs and will be taken with
the help of template that is cut to represent a 10 x 10 cm area.
- Take the sample from the equipment by gentle sweep 3 times to cover the area once
horizontally & once vertically with the swab and avoid repeated rubbing.
- Analyze the swab samples for the target substance as per individual product test
method.
- Wear powder free latex glove, dip the swab into distilled water. Press and twist the swab
against the sides of a 100ml beaker to eliminate the air and fully wet the swab
tip.
Final Rinse Sampling

- Collect the sample of the final rinse according to the method given in the individual
product monograph.
- Ensure that appropriate information of the last product manufactured is written /
labeled on the sample container.
Storage of Samples
- Samples will be stored in Glass Culture Tubes 20mm x 150mm
- Samples will be stored for maximum up to 24 hrs.
Campaigns to be studied
- Minimum 03 consecutive applications of the cleaning procedure will be performed
in order to declare the method as validated.
- Manufacture of a new product
- Installation of new equipment
Studies to be carries out
Following studies will be carried out during validation exercise.
- Effectiveness of the cleaning processes
- Effectiveness of cleaning after long campaigns.
- Effectiveness of process for removal of Water insoluble materials
- Maximum / Existing holding period between cleaning and use
- In case if holding time is not possible to validate, cleanliness of the cleaned equipment will be
ensure through area monitoring will be carried out for 5 working days & ensure its compliance.
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7. SAMPLING TECHNIQUES
For recovery of residual active(s) following sampling techniques will be employed :
Direct Sampling
Swabbing of the selected surface of equipment will be conducted using appropriate swab. Swab
material may be polyester, cotton, glass wool or filter paper. The swabs will be treated with a liquid
medium (good solvent) to facilitate the absorption of the residue. Squeegee method will be
employed as described below
OR
First Second
Indirect Sampling
It is commonly known as rinse method. This method will be used for difficult to access area. A known
volume of solvent will be passed through a large area and then recovered solution will be analysed.
Microbiological Sampling
Direct contact, swabbing and rinse sampling will be performed as appropriate for the equipment
wherever required. A thorough cleaning with water followed by wiping with ethanol is necessary to
remove residual sampling material from the surface area.
7. ACCEPTANCE CRITERIA
The residue limits determination will be based on “Visually Clean Analytically Clean” acceptance
criteria and a logical approach to which equipment are critical with regards to potential contamination
by
Active Ingredient. Where it is not possible to perform direct surface sampling or final rinse sampling
the equipment have to pass a “visually clean” inspection.
HOLDING TIME
Swab sampling must be performed immediately after cleaning of the equipment’s.
VISIBLY CLEAN CRITERIA
Visual examination should not reveal any evidence of contamination.
ODOUR
No odor of the previous product must be detectable.
ANALYTICALLY CLEAN CRITERIA
The requirement for this component applies to all product contact surfaces of any equipment that has
been cleaned and dried. The requirement is composed of the following two elements:
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1) The average active residue test results of all samples per each major piece of equipment must be:
- Standard therapeutic dosage actives
Not more than 100 micrograms per 100 cm2
a dose less- Low therapeutic dosage actives. Not more than 10 micrograms per 100 cm2
2)The individual active residue test results must be:

- Standard therapeutic dosage actives
- Low therapeutic dosage actives
Typically, low therapeutic dosage actives are compounds that have high to very high pharmacological
activity or toxicity. As a general rule, these actives have an oral LD50 in rats of less than 100 mg/kg
and have than 1 mg.
Standard therapeutic dosage drug actives are all other compounds that do not meet the definition for
low therapeutic dosage actives.
8. Required Testing and Rationale

Major equipment will be identified in the protocol for direct surface sampling of residues . If it is not
possible to sample at least three direct surface sites then samples may also be taken by final rinse
sampling. The size of the direct surface samples will be 100cm 2 where possible for chemical swabs
and will be taken with the aid of a template that is cut to represent a 10 x 10 cm area. Final rinse
samples will also be taken if the equipment isn’t all that accessible to surface sampling.
9. Supporting Data and Documentation
Any test reports or additional information may be filed under the section for Supporting Data and
Documentation.
10. Cleaning Validation Summary
Data generated during the CV will be summarized in Document any discrepancies or variations noted
during the performance of the Cleaning Validation include the resolution of these items and/or any
item outstanding that will require further attention to resolve.
11. Change Control and Revalidation
Any modifications or changes to the Cleaning SOP or its equipment will be documented on a
modification/change control form, in accordance with established SOP. An assessment will be made
after any modification or change to the Cleaning or equipment to determine if revalidation is required.
All modification/change control forms for the Cleaning and equipment will be filed.
Cleaning Validation – Visual Cleanliness Check
Object of test. To ensure that the required equipment meet the specification of visual
cleanliness.
Description of Test Inspect each of the equipment listed below and ensure that no visible traces
of the drug product exist.
Acceptance Criteria Visual cleanliness
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Test Result Equipment Complies with visual cleanliness

Product:  Yes  No
Batch No.:
Comments:
Test Requirements met  Yes  No
Done by:_____________ Checked by:____________________
Cleaning Validation – Chemical Cleanliness

Active Drug Substance Residual quantity: Large
Object of test and To determine the amount of residual active drug substance on the equipment.
Samples to be taken.
Description of Test Direct surface samples will be taken as indicated at the selected sites and will
be analyzed for compliance to the Analytically Clean criteria.
Acceptance Criteria Active Drug substance:

EQUIPMENT X :  x g / 100 cm2
EQUIPMENT Y :  x g / 100 cm2
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EQUIPMENT Z :  x g / 100 cm2
Test Result Equipment’s g / 100 cm2

Product: XXX Location 1 Location 2 Location 3
Batch No.: XXX
Comments: Complies
Test Requirements met  Yes  No
Done by:____________________ Checked by:_______________________
Cleaning Validation Summary

The following table summarizes the data collected in the study.
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Equipment Description Chemical Level of API

Active Drug substance:
Description of Deviation (if any)
Evaluation: Validated Maximum Time for equipment cleaning = Minutes

Validated Cleaning Agent to be used for equipment cleaning =
Conforms to CV specification: Yes No

Comments:
Done by: Date:
QC Manager: Date:
QA Manager: Date:
Production Manager Date:
Head IQC Date:
Dy. Director Industrial Site Date:
Identification and description of Critical steps

Controls of critical steps
The tests performed during the manufacturing process are the following:
Stage 1 –Weighing of sterile powder.
- To be used raw material quantity (admissible criteria: conform with manufacturing
formula)
Stage 2 – Preparation and maintenance of sterile area.
Critical parameters are
 Extent of temperature and humidity control.
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 Maintenance of area in all aspect preferably microbiological tests like microbiological

contaminant, particle count, Settle Plates etc.
Stage 3 – washing Sterilization and depyrogenation of vials.
Critical parameters are;
Temperature Control of sterilizer .Water quality (ro2) used for washing.Time control of
sterilization process and control.
Stage 4 – entrance to sterile area

Critical parameters are;
 Sterilization of uniform, gloves and masks used during filling process.
 Prevention of irregular and unnecessary movement of operators in and out.
Stage 5 –Filling
Critical Parameters are:
 Temperature (NMT 25ºC) and humidity (NMT 45% control of area.
 Physical description of powder.
 Filling weight of powder as recommended by Quality control.
 Weight Variation of the filled vials.
 Appearance of the powder.
 Assay
 Endotoxin test
 sterility testing
 optical testing
Stage 7 – Packaging.
Finished drug product control is performed according to the Release Quality
Specification for the drug product.
During this stage are verified the following parameters:
 Vials mark;
 Vials sealing, rubber stopper and flip of seal of vial
 vials tightness;
 No of vials in the carton box;
 the presence of the package insert leaflet;
 carton box mark;
 collective packaging;
Collective packaging labelled
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Microbiological program used during sterile production
PROCEDURE
Monitoring Schedule
Particles count…….. Once on working day
Settle Plate…….. Once on working day
Finger Dabs........ Once on working day
Swab Test……….. Once on working day/week
PARTICLES COUNT
Particles count is necessary to know about the condition of the HEPA filters of sterile area. And
performed daily on each filling day before working
Class Limits Area to be tested Results Remarks
A 0.5µm
Room 100/ft³ Under Laminar /ft³
5.0 µm
00/ft³ Under Laminar /ft³
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Filling Room Inlet I /ft³
B Filling Room Inlet II /ft³

Room
0.5µm Cool Down Room Inlet /ft³
1000/ft³
Transfer Window Inlet /ft³
Filling Room Inlet ft³

5.0 µm
07/ft³ Filling Room Inlet II /ft³
Cool Down Room Inlet /ft³
Transfer Window Inlet /ft³

C 0.5µm Buffer Inlet
Buffer 10,000/ft³ Solution Room Inlet /ft³
Buffer Inlet /ft³

5.0 µm Solution Room Inlet /ft³
70/ft³
SETTLE PLATE
Settle plate method performed daily on each filling day for the conformation of area status.
PROCEDURE
For this purpose nutrient agar and dextrose agar is used for bacterial and fungal growth. Weigh the
desired quantity of the medium according to the manufacturer’s direction on digital balance and
dissolve in the required quantity of Distilled water Sterilize at 121ºC for 20 minutes in an autoclave.
Then allow cooling up to 40-45 ºC, then media pour into the plates and allow to solidifying. Plates are
prepared under laminar flow hood in Microbiology Lab (in sterile area). Now these prepared plates are
exposed to sterile filling area at different location for 3-4hours and then incubate this nutrient agar
plates in hot incubator at 30-35 ºC and dextrose agar plates in cold incubator at 20-25 ºC. And
observed the result after 48hrs of incubation
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Results
Sr.# Area Grading Limits
Bacteria Molds
1 Laminar Flow Hood Class A <1 Cfu
2 Sealing Table Class B <5 Cfu
3 Air duct Inlet Class B <5 Cfu
4 Cooling Area Class B <5 Cfu
5 Transfer Window Class B <5 Cfu
6 Buffer 01 Class C <25 Cfu
7 Buffer 02 Class C <25 Cfu
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SWAB TEST (Surface Culture)
PROCEDURE
To check the sterile conditions of production vessels (equipments), and personnel hygiene
and also clean rooms
The swabs are used to monitor the microbial load in equipments involved
in production activities, clean rooms’ walls n floors and uniform worn by
the operators and filling machines (under L.F.C.) as well.
For this purpose wet the swab in sterile NaCl / Phosphate Buffer pH 7.2.
Results
Sr.# Area Grading Limits Bacteria Molds
1 Under Laminar Table Class A <1 Cfu
2 Sealing Table Surface Class B <5 Cfu
3 Filling Room wall Class B <5 Cfu
4 Filling Room Floor Class B <5 Cfu
5 Cooling Area Wall Class B <5 Cfu
6 Cooling Area Floor Class B <5 Cfu
7 Transfer Window Class B <5 Cfu
8 Buffer Wall Class C <25 Cfu

Swab an area of 2.5x2.5 inch² on the desired place (filling nozzle / hoper, table, walls, floors,
LFC etc.)
Put the swab back in the same test tube containing phosphate buffer pH 7.2 and label with
site tested and date.
Test all the required and desired sites in similar manner.
After completion of the testing, incubate all the swabs containing tubes at 30-35ºC
for 48 hrs.
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After the incubation time identify microbes if needed with the help of microbial identification
protocol file. And record the data.
FINGER DABS TESTS

For this purpose nutrient agar and dextrose agar is used for bacterial and fungal
growth. Weigh the desired quantity of the medium according to the manufacturers
direction on digital balance and dissolve in the required quantity of Distilled water
Sterilize at 121ºC for 20 minutes in an autoclave. Then allow cooling up to 40-45 ºC,
Then media pour into the plates and allow to solidifying. Plates are prepared under
Laminar flow hood in Microbiology Lab (in sterile area).
The finger prints with sterile gloves inside the filling sterile area will be taken on Prepared
nutrient and dextrose agar plates as per procedure from flowed right and left hand of
the filling operator/s and helpers as well. Then incubate these plates in hot incubator at
30-35ºC dextrose agar plates in cold incubator at 20-25 ºC. And observed the result
after 48hrs of incubation.
Results
Sr.# Area Grading Limits
Bacteria Molds
Filling Operator Right
1 Class A <1 Cfu
Hand
Filling Operator Left
2 Class A <1 Cfu
Hand
Sealing Operator
3 Class B <5 Cfu
Right Hand
Sealing Operator Left
4 Class B <5 Cfu
Hand
5 Helper Right Hand Class B <5 Cfu
6 Helper Left Hand Class B <5 Cfu

CRITERIA AND ACTION
Perform the test carefully, before start working. Operators must be trained.To perform the
test. If growth observed, inform the sterile area incharge about the test Failure. Sterile area
incharge will investigate the source of contamination and takes
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The corrective action immediately.
“FORM 5”
[See rule 26(1)]
APPLICATION FOR REGISTRATION OF A DRUG FOR LOCAL MANUFACTURE
HAVING THE SAME ACTIME INGREDIENT OR SALT THEREOF, THERAPEUTIC USE,

DOSAGE FORM AND ROUTE OF ADMINISTRATION THAT HAS ALREADY BEEN
APPROVED BY THE DRUG REGULATORY AUTHORITY OF PAKISTAN, ALREADY ON
SALE IN LOCAL AND/OR INTERNATIONAL MARKET.
I M/s Hygeia Pharmaceuticals of Plot No. 295 Industrial Triangle, Kahuta Road,
Islamabad hereby apply for registration of the drug, namely
HYTREX INJECTION 250MG (INTRAMUSCOLAR) IM details of which are enclosed.
Date ……………… Signed:

Raja Farukh Javed
Place: ___________ Chief Executive
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ENCLOSURES OF THE APPLICATION FOR REGISTRATION OF A DRUG FOR LOCAL

MANUFACTURE
Dosage Form: Injection
1. Name and address of the manufacturer (applicant):

Hygeia Pharmaceuticals, Plot # 295, Industrial Triangle, Kahuta Road Islamabad.
2. Brand (Proprietary) name of Drug
Hytrex 250mg Injection IM
3. The chemical name(s) and, as appropriate and available the established (generic)
names and synonyms of the drug.
Ceftriaxone sodium
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a) Chemical name (IUPAC):

5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[[(2-amino-4-thiazolyl)
(methoxyIMino)acetyl]amino]-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5-, 6-dioxo-1,2,4-triazin-3-
yl)thio]methyl]-, disodium slat, [6R-[6 ,7 (Z)]]-, hydrate, (2:7).
b) Structure:
C18H16N8Na2O7S3·3½H2O 661.60
Strength of active ingredient(s) per unit, e.g. each tablet or 5 ml, etc. contains.
Each Injection Contains:
Ceftriaxone sodium sterile equivalent to Ceftriaxone……….250mg
4. Pharmacology:
Attached below (taken from BNF 61)
5. Proposed route of administration.
Parenteral
6. Proposed shelf life of the drug.
02 Years
7. Proposed storage conditions of finished product.
Keep in Cool and dry place.
8. Unit price of the drug, e.g. per tablet, per capsule, per 5ml, etc.
As recommended by the PRC (MOH)
9. In case of international availability, provide the following information, namely:
i. Name of the drug;

Rocephin Injection IM
ii. Country where sold / registered; and
UK/USA and Switzerland
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iii. Name of company selling the drug or having registration to manufacture (include
supporting documents/proof of International registration).
Hoffman-La Roche ltd Switzerland
10. Brand name(s) of drug available in Pakistan
Oxidil injection
Getofin injection
11. Name(s) of company(s) manufacturing in Pakistan.
Sami Pharmaceuticals.
Getz Pharmaceuticals
12. Composition (active & expedients) including statement of the quantitative
composition, giving the weight or measure for each active substance used in the
manufacture of the dosage form.
The qualitative and quantitative composition of Hytrex IM injection 250mg is presented in the
following table:
Batch size: 10,000 Vials Pack size: 250mg
Quantity / Vial Quantity / Batch Quality

Raw Material * Function
( gm) (Kg) specification
ActIMe substance
Ceftriaxone sodium
equivalent to ceftriaxone 0.300 3.00 Active substance USP 36
base.
Excipients
-
Solvent for
Water for injection 5ml - 10000 Phr. Eur 2006
constitution
-
Total Weight (Vial) 0.300 - -
*** Ceftriaxone sodium Raw material received as a sterile, pyrogen free ready to use powder for
injection, so manufacturing and processing is not required, only the powder is to be filled in sterile
condition.
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EQUIPMENT USED:
 Analytical and semi-analytical scales/Tables;
 Filtration assembly
 Vial washing machine;
 Water storage tank;
 Laminar flow hood;
 Autoclave
 Dry heat Sterilizer
 Automatic filling and sealing machine
 Optical check boxes
 Automatic labeling machine
 stainless steel; Utensils ,scoop etc (16 gauge Grade 316)
13. Facility of water processing with specifications.

Annex E
14. Environment control processing with details.
Annex F
15. Type of container/packaging.
1 vial of 250mg ceftriaxone sodium powder/1 vial of 5ml water for injection
A copy of last inspection Report conducted by the Ministry of Health.
Annex G
16. Undertaking regarding resemblance in brand name, label and
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Attached
UNDER TAKING
I / We hereby undertake that the above given information is true and
correct to the best of my / our knowledge and belief.
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______________________ ______________________
Production Manager Quality Control Manager
TAGE
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ISO 9001-2008 CERTIFIED
Brand name: Hytrex Injection IM

Active ingredient: Ceftriaxone sodium
Strength: 250mg/Vial
Dosage form: Parental
Pack size: 250mg
Hygeia Pharmaceuticals
Plot # 295, industrial Triangle Kahuta Road, Islamabad-PAKISTAN
ISO 9001-2008 CERTIFIED
Brand name: Hytrex Injection IM

Active ingredient: Ceftriaxone sodium
Strength: 250mg/Vial
Dosage form: Parental
Pack size: 250mg
Hygeia Pharmaceuticals
Plot # 295, industrial Triangle Kahuta Road, Islamabad-PAKISTAN
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IN PROCESS CHECKS STERILE

Sr.
NO. MANUFACTURING TESTS SPECIFICATIONS TEST METHOD TESTING
STAGE FREQUENCY
Temperature :NMT
01 (Before starting): Environment 30°C By digital thermo Once
control RH : NMT 40% hygrometer
Cleanliness of Should be Visual Inspection Once
equipment & Cleaned
utensil
Environment Temperature :NMT By digital thermo Once
control 30°C hygrometer
RH : NMT 40%
Cleanliness of Should be Visual Inspection Once
equipment & Cleaned
Machine parts
Manufacturing:
02 Temperature of NMT 30° C By digital Throughout the
the filling room thermometer process at regular
intervals
Microbiological N/A N/A N/A
test
1 Bacterial
Endotoxin
2. Sterility
Assay It contains not less USP-36 Once
than 90.0% and
NMT 115.0% of the
labeled amount of
Ceftriaxone sodium
Identification Ceftriaxone sodium USP-36 Once
is positive
Filling and sealing: Filled weight 250mg±10% Measuring Throughout the
04 balance process at regular
intervals
Sterilization 121°C/15psi By digital device Before the start of

temperature/ sterilization process
Pressure and throughout the
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(only uniform & process at regular

utensils) intervals
Sterilization time 45 minutes Stop watch Throughout the
process at regular
intervals
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ii. Sterility It meets the USP-36 Once for 14 days
requirements when
tested as directed for
Membrane Filtration
under test for sterility
of product to be
Examined.
It contains not less
than 90.0% and NMT USP-36 Once
Assay 115.0% of the labeled
amount of ceftriaxone
sodium
ceftriaxone sodium Is
Identification positive BP -2013 Once
05 Packing 01 Visual Through the Batch

( labeling and No of Vials/Packs Inspection
packaging of
bottles in printed If for 10 saeled vials Appearance Through the Batch
unit cartons) Seal tightness is detected a single type vaccume at regular intervals
(packaging in vial with tightness Leakage tester
shipping box) defect the test is F18
repeated on other 10
vials
Correctness & As per running batch. Visual Through the Batch
legibility of batch Inspection at regular intervals
number & expiry
date on labels
before packaging
Correctness & As per running batch.
legibility of batch
number & expiry
date on labels
before packaging
Correctness of Should be correct
leaflets during
cartooning
Correctness & All information’s
legibility of batch should be correct &
number & expiry legible
date on labels
before packaging
Quantity 100 Packs of 1 x 1’s
Properly sealed with

Proper Sealing. tape
The tests are performed according to the released quality specifications for the drug product.
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LIST OF TECHNICAL STAFF IN PRODUCTION AREA
University/Colleg
S# Name S/O, D/O Year Designation
e
1. Mohammad Baluchistan
Mr. Iftikhar Khan 1998 Production Manager
Anwar khan University
2. Ghulam Urdu university
Mr.Irfan Murtaza 2011 Sterile area Pharmacist
Murtaza Karachi
3. Ms. Najma Naz Gul nawaz Riphah University 2012 Production Pharmacist
4. Mr.Hasan Bilal Mohammad Riphah University 2013 Production Pharmacist

Ashraf
5. Mr. Qasim Bahadur Hazara University 2013 Production Pharmacist
Zaman Zaman
6. Mr.Hanif Mohammad Sarhad University 2012 Production Pharmacist
shoaib
7. Mr.Mursalen Sarwar Hazara University 2013 Store Pharmacist
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MANUFACTURING OPERATIONS:
General Compliance Conditions.
The manufacture method shall only start when the working area, equipment, personnel and raw
materials involved are in compliance with the Good Manufacturing Practices (GMP’s), namely:
a) The working area must be perfectly clean and free from any document, material or component non-
required for the operation being performed and/or of previous batches;
b) The equipment must be perfectly clean and duly identified;
c) The personnel must wear sterile personal protection, including cap, gloves, boots, mask and
glasses;
d) The starting materials must be previously approved by the Quality Control;
e) All documents and equipments necessary for the batch manufacturing must be in the working area.
f) All operations should be carried out under strict aseptic condition.
g) All machine parts, uniforms (Jump suits), Mask and surgical gloves should be sterilized before use.
h) Check and ensure the L.F.H. is working.
i) Check and ensure the required +ve pressure inside the sterile area.
j) Check and record the temperature & +ve pressure of the filling room.
i. Temperature should be less than 25°C.
ii. +ve Pressure more than 0.06 water inches.
iii. Humidity should be less than 45%.
Description of the manufacturing process

Stage 1– Vial Washing and Sterilization (Processing of Packing materilas)
A. Dry Heat Sterilization.
In dry-heat processes, the primary lethal process is considered to be oxidation of cell constituents. Dry-
heat sterilization requires a higher temperature than moist heat and a longer exposure time. The method
is, therefore, more convenient for heat-stable, non-aqueous materials that cannot be sterilized by steam
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because of its deleterious effects or failure to penetrate. Such materials include glassware, powders,
oils, and some oil-based injectables.
Preparations to be sterilized by dry heat are filled in units that are either sealed or temporarily closed for
sterilization. The entire content of each container is maintained in the oven for the time and at the
temperature given in the table below. Other conditions may be necessary for different preparations to
ensure the effective elimination of all undesirable microorganisms. The ovens should normally be
equipped with a forced air system to ensure even distribution of heat throughout all the materials
processed. This should be controlled by monitoring the temperature. Containers that have been
temporarily closed during the sterilization procedure are sealed after sterilization using aseptic
techniques to prevent microbial recontamination.
The bioindicator strain proposed for validation of the sterilization process is: spores of Bacillus
subtilis (e.g. var. Niger ATCC 9372 or CIP 77.18) for which the D-value is 5-10 minutes at 160
°C using about 106 spores per indicator.
Place the Vials in washing trays of the vials washing machine and ensure appropriate washing
to remove any particulates.
Place the trays of washed, ampoules in Hot Air Oven for sterilization and operate the Hot Air
Oven.
Standard Parameters include:

 Temperature 240°C
 Time 02 Hrs
B. Autoclave.
Exposure of microorganisms to saturated steam under pressure in an autoclave achieves their
destruction by the irreversible denaturation of enzymes and structural proteins. The temperature at
which denaturation occurs varies inversely with the amount of water present. Sterilization in saturated
steam thus requires precise control of time, temperature, and pressure. As displacement of the air by
steam is unlikely to be readily achieved, the air should be evacuated from the autoclave before
admission of steam. This method should be used whenever possible for aqueous preparations and for
surgical dressings and medical devices.
The recommendations for sterilization in an autoclave are 15 minutes at 121-124 °C (200 kPa). 1 the
temperature should be used to control and monitor the process; the pressure is mainly used to obtain
the required steam temperature.
Minimum sterilization time should be measured from the moment when all the materials to be sterilized
have reached the required temperature throughout. Monitoring the physical conditions within the
autoclave during sterilization is essential. To provide the required information, temperature-monitoring
probes should be inserted into representative containers, with additional probes placed in the load at the
potentially coolest parts of the loaded chamber (as established in the course of the validation
programme). The conditions should be within ±2 °C and ±10 kPa (±0.1 atm) of the required values.
Each cycle should be recorded on a time-temperature chart or by other suitable means.
Porous loads, such as surgical dressings and related products, should be processed in an apparatus
that ensures steam penetration. Most dressings are adequately sterilized by maintaining them at a
temperature of 134 - 138 °C for 5 minutes.
In certain cases, glass, porcelain, or metal articles are sterilized at 121 - 124 °C for 20 minutes. Place
Machine Parts, Rinsing Water, Uniforms, Mask and Surgical Gloves in Autoclave for sterilization and
Operate the Autoclave.
 Steam Pressure 15 PSI
 Temperature 121°C
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 Time 45 min
Stage 2 – Sterile Filling and Sealing.
After the analysis by Quality Control on the sample vial provided along with the bulk material of
the same lot and the results obtained thereof, take area clearance report from QA on Technical
Information sheet. Switch ON the Laminar Flow Hood of the filling machine. Check the
parameters of temperature, +ve pressure and humidity inside the sterile filling area and record
it (during filling record the temperature and humidity after every 30 minutes. Open the valves of
Oxygen and natural gas and fit the sterile parts in aseptic environment. Adjust volume and
sealing by first running 0.4% formalin solution on the machine. Remove the formalin flask and
flush the line with sterile water and separate the Vials. After flushing, attach the lining to the
bulk container under aseptic conditions. Adjust the weight (Amount) of the powder to be filled
on the machine and start the filling and sealing operation. First approx. 10 Vials are discarded
and identified as rejected. The vials are filled and sealed using butyl rubber stoppers and flips
off seals that have been sterilized already. In process checking of volume is done after each 30
minutes and is recorded in.
QA takes sample every half an Hour. Send the filled vials for visual testing in S.S container.
After visual testing these vials closed in card board boxes and stored in In-process cold room.
Intimate QA for Release for Packing. QA takes the filled sample to QC for Chemical and
Microbiological analysis. Quality Control analyses the product according to its protocols and
testing parameters and releases or rejects the product depending upon the results of analytical
methods of both chemical and microbiological attributes.
Stage 3– Packaging.
The primary packaging is executed on vial filling and sealing machine (productivity 70 to
80Vials / minute) and the secondary packaging is executed on Packaging Machine HD 80 type
(productivity 50-80 carton box / minute).
a) Primary packaging
Each vial contains powder equivalent to 250mg of CEFTRIAXONE.
The vial is labeled and the label contains all data for the identification of the medicinal
product.
b) Secondary packaging
One labeled vial containing the powder, accompanied by a patient information leaflet, and the
reconstitution solvent are introduced in a carton pack.
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c) Collective packaging
50 or 100 commercial units are packed in labeled collective carton Packs.
After packaging the entire batch, this is stored in quarantine of finished products, until the
Quality Control Laboratory releases the Certificate of Analysis.
Stage 4– Testing and Release the finished product
Batch release for the market, of the finished drug product is executed after all documentation
for every batch (batch recording data, manufacturing batch file), is verified, approved and
signed by the responsible person with batch release.
Release of the finished parenetral product is subject to chemical and microbiological testing
prior to release for the market.
A. Chemical tests:
Involves assay, identification and other tests; details of which are provided in the concerned
section.
B. Microbiological tests:
These involve Bacterial Endotoxin and Sterility testing for an incubation period of 14 days;
D IN PROCESS CONTROL
PROCESS VALIDATION REPORT
Post-approval validation protocol on manufacturing batches

Validation study objective:
Technological process for: Ceftriaxone sodium 250mg/vial
Process validation is done to ensure process (within their specified design parameter) consistently,
repeatedly and reliably produce the product of required quality.
Validation Type:
Prospective validation for three pilot batches.
Validation has been performed throughout manufacturing of Pilot batches of Ceftriaxone sodium
250mg/vial
The validation based on the evaluation of the Process Control and Physical-Chemical Results
pertains to the Critical steps of the Manufacturing Process highlighted and pointed out in the
Validation Protocol.
Documents associated with this validation study include:
- Standard Operating Procedures (SOPs);
- Quality Specification for Ceftriaxone sodium 250mg/vial.
- Validation Master Plan (VMP);
- Batch Manufacturing Documentation for 3 (three) pilot batches of Ceftriaxone sodium 250mg/vial.
Made by Hygeia pharmaceuticals
- Qualification protocols and reports;
- Validation Guidelines for Pharmaceutical Dosage Forms;
- Supplementary Guidelines on Good Manufacturing Practices (GMP): Validation;
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Marketing authorization dossier for Ceftriaxone sodium 250mg/vial.
The scope of the validation study.

To provide documentary evidence that the manufacturing process of Ceftriaxone sodium 250mg/vial.
(within its specified design parameters) is capable of repeatedly and reliably producing a finished
product of the required quality.
Methods for recording and evaluating results, including statistical analysis and formulas
The following appendixes will be included in the validation report:
1. Appendix no. 1 :
 the batch number of the raw materials used and their number of certificates of analysis
2. Appendix no. 2:
 the batch numbers of the final product
 the size of the batches
 the number of the certificates of analysis for:
 Solution preparation and Filling;
 Finished product.
3. Appendix no. 3:
 The registering of the calculated parameters / productivity of the batches carried out.
Table no. 2 – The meaning of the terms used in formulas
S. Parameter [MU]
No.
1 CSAIP - the amount of the active substance entered in the process Kg
2. CTSP - the total amount of the substance entered in the process Kg
3. CTSO - the total amount of the substance entered in the filling Kg

process
CTP - the total amount of filled vials obtained after filling Kg

4 process
5. PSO - the loss of substance in the filling process Kg
SAP - the amount of active substance assayed in the filling Kg

6 process
7 MCSG - the amount of Vials introduced in the filling process Kg
8 CS - the amount of filled vials obtained Kg
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9 SACS - the active substance assayed for one vial mg/cps
10 MMC - the average fill weight of the vials ml
11 MCS - the average weight of the filled vials kg
12 PSC - the loss of substance during filling process kg
13 NUC - the number of commercial units obtained -
14 NCS - the number of vials obtained -
15 CPF - the amount of finished product obtained Kg
16 PSA - the loss of substance during packaging kg
17 NBF - the number of vials/ commercial unit -
18 NCSBF - the number of vials/ master pack -
19 RSAO - the active substance productivity for the filling process %
20 RSO - the substance productivity for the filling process %
21 RSC - the substance productivity for the filling process %
22 RSA - the substance productivity for the packaging process %
23 RCSA - reconciliation regarding the active substance for the entire %

technological process
24 RGSA - total productivity for the active substance %
25 RCS - reconciliation of the substance for the entire technological %

process
26 RGS - total productivity of the substance %
4. Appendix no. 4: the tables will contain all parameters specified in quality specifications with their
acceptance criteria regarding: filling process, bulk vials and the values obtained practically.
5. The following formulas will be used:
a. Loss of substance in the filling process (PSO):
PSO = CTSO  CTP
b. Loss of substance in the filling process (PSC):
PSC = (CTP + MCSG) - CS
c. Number of obtained vials (NCS):
NCS = NUC  NBF  NCSBF
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d. The amount of the finished product obtained (CPF):

CPF = NCS  MCS  10 6
e. The loss of substance during packaging (primary and secondary) (PSA):
PSA = CS  CPF
f. The active substance productivity for the filling process (RSAO):
SAP  CTP
RSAO() =
CSAIP
g. the substance productivity for the filling process (RSO)
CTP
RGO  =  100
CTSO
h. The substance productivity for the filling process (RSC):
CS
RSC() =  100
CTP + MCSG
i. The substance productivity for the packaging process (RSA):
NUC  NBF  NCSBF  MCS
RSA() =  10  4
CS
j. Reconciliation regarding the active substance for the entire technological process (RCSA):
 =  NUC  NBF  NCSBF  SACS  10  +  PSO  SAP  10   +   PSC / MMCSXSACS  +  PSA / MCSXSA
6 2
RCSA
CSAIP
k. The total productivity for the active substance (RGSA):
NUC  NBF  NCSBF  SACS  10 6

RGSA() =  100
CSAIP
l. Reconciliation of the substance for the entire technological (RCS):
CPF + PSO + PSC + PSA

RCS() =  100
CTSP + MCSG
m. Total productivity of the substance (RGS):
CPF
RGS() =  100
CTSP + MCSG
n. The mean value ( x ) for a set of values x1, x2, x3, …, xi, …, xn, is calculated using:
x
1
i
x=
n
o. The standard deviation (s) for a set of values x1, x2, x3, xi, xn, is calculated using:
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2
 n 
n
 x
1 x   1 n 
2
s=
n 1
p. Relative standard deviation (RSD %) for a set of values x1, x2, x3, …, xi, …, xn,:
s  100
RSD() =
x
C.P.3.4.1.4 sampling plan
Sampling plan is performed in accordance with the Quality Assurance procedure QA_019 “Sampling”.
C.P.3.4.1.5 Admissible criteria:

Acceptance limits for the productivity/reconciliation:
Table no. 3
Productivity / Reconciliation Optimum interval Not accepted
(%)
The substance productivity for the filling process 92.5 – 107.5 < 92.5 >107.5
The active substance productivity for the filling process 92.5 – 107.5 < 92.5 >107.5
The substance productivity for the filling process 92.5 – 107.5 < 92.5 >107.5
The substance productivity for the packaging process 92.5 – 107.5 < 92.5 >107.5
Reconciliation regarding the active substance for the

92.5 – 107.5 < 92.5 >107.5
entire technological process
Total productivity for the active substance 92.5 – 107.5 < 92.5 >107.5
Reconciliation regarding the substance for the entire

92.5 – 107.5 < 92.5 >107.5
technological process
Total productivity of the substance

92.5 – 107.5 < 92.5 >107,5
C.P.3.4.1.6 The presentation of the results

The results of the study of the concurrent validation for the drug Ceftriaxone sodium 250mg/vial. will be
recorded in the validation report, Appendixes no. 1-4. RSD values must be less than 5%.
VALIDATION REPORT
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Validation Summary
For the prospective validation of the process used for the manufacturing of the drug CEFTRIAXONE
SODIUM 250mg/VIAL were processed 3 pilot manufacturing batches. The pilot size of the batch is
1,000 vials.
The productivity values calculated for each stage in the manufacturing process fit in the interval (92.5-
107.5 %).
The critical parameters values measured for the products obtained in the stages:
Sterile filling process for the three pilot batches fit in the interval stipulate in the product quality
specification, for each stage.
● In the phase of sterile filling the following parameters were controlled: appearance of the filled vial
and of the vial's content, average fill weight of the vial, assay of CEFTRIAXONE.
for the finished product were controlled the following parameters: appearance of the vial and of the
vial's content, average fill weight of the vial, , identification of CEFTRIAXONE, assay of
CEFTRIAXONE.
Conclusion: It has been proved that the technological manufacturing process of CEFTRIAXONE
SODIUM 250mg/VIAL is characterized by homogeneous and uniform batches that follow the imposed
quality specifications and can be considered validated.
The validation study method

The validation of the technological process of Ceftriaxone sodium 250mg/vial. has been performed on
three pilot batches according to The Batch Manufacturing File no.: TSP001, TSP 002, TSP 003.
Raw material batches (active substances and excipients) used in the manufacturing process is
registered in appendix no. 1.
The appendix no. 2 contains the numbers of manufacturing batches used in the validation study, the
numbers of certificates of analysis released by the Control Laboratory for: sterile filling, bulk vials,
finished product.
The productivity on each phase of the manufacturing process, as well as the reconciliations is
presented in appendix no. 3.
In appendix 4 there are recorded all the critical parameters as specified in the quality specifications
and their practical values for all phases.
In the technological manufacturing process of Ceftriaxone sodium 250mg/vial.
all equipments, the production environment and analytical testing methods used have been fully
validated.
The quality specifications for all phases of the manufacturing process of CEFTRIAXONE SODIUM
250mg/VIAL can be found in Marketing Authorization Dossier.
Analytical methods used in the validation process are specified in the Validation Protocol.
The productivity for every stage, as well as the global productivity was calculated. The mathematical
formulas used to calculate the productivity are also presented in the Validation Protocol.
The statistical calculation of the results was done by calculating the descriptive statistical parameters
(mean, spread, standard deviation) as well as by applying the t Student (bi-dimensional test) to
compute the data; the differences were considered significant for p < 0.05.
The mathematical formulas used for the calculation of the mean, standard deviation, spread and
relative standard deviation are the following:
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2
 n 
n n n
 x
1 xi , 2 1 (xi  x) ,
2
1 x   1 n  , RSD = s  100
2
x= s = s= x
n n 1 n 1
Where:
n- Represents the number of determinations
Xi- represents the individual values
x - represents the average value
s- Represents the standard deviation
s2 - represents the variance
RSD – represents the relative standard deviation.
The t statistic has a Student distribution with n-1 degrees of freedom, t ~ t (n-1).
The spread (∆x) of the individual xi values, for a random independent variable is:
Δx = ±t(P, v)  s
Where:
∆x –represents the spread;
t - Represents the critical value of the Student distribution;
P= 1-α represents the probability with which it is guaranteed that the interval covers the xi
value;
α = n – 1- represents the degrees of freedom;
n – Represents the number of determinations;
s – Represents the standard deviation;
The ∆x interval represents the spread of xi and it depends on the probability with which it is
guaranteed that the confidence interval contains the xi.
The confidence interval is defined using the confidence limits:
Li = x - ∆x and Ls = x + ∆x:
x - ∆x < x i < x + ∆x
Li, Ls – the confidence limits: upper and lower;
∆x – represents the spread;
P = 100-α – represents the probability with which it is guaranteed that the interval covers the xi
value;
p – Represents the risk;
Xi – represents the individual values;
x – represents the average value
We consider the 95% probability P that a random variable xi can be found in the confidence
interval. The value xi will be found in the interval x ± ∆x with a given statistical confidence
(probability P), thus at a significance level of
p = 2 x α = 100 – P%
The critical value of the t Student distribution for a 95% probability with 2 degrees of freedom is
4.3.
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P = 95% and α = 2 value t (95%,2) = 4,3

We can conclude with a 95% probability that a random variable xi will not distance itself from
the average value by more than 4.3 standard deviations.
C.P.3.5.1.3 Batch production records and statistical analysis
During the manufacturing process the productivity values for the active substance the final
reconciliations were calculated. The results obtained for each stage of the technological
process, Sterile filling and packaging can be found in the following tables (1 – 4):
Table no.1 – The values of productivity obtained in the filling stage
The substance The active substance

Complies/d
productivity for the productivity for the Admissible criteria
Batch no. o not
filling stage filling stage (%)
complies
(RSO) (RSAO)
TSP001 97.20 97.99 Complies
Complies
TSP002 97.60 98.60
92.5 – 107.5
Complies
TSP003 97.85 98.22
RSD% 0.392 0.320 <5 Complies
Table no.2 – The values of productivity obtained in the packaging stage
Batch no. The substance Admissible

productivity for packaging criteria Complies/do not complies
process (RSA) (%)
TSP001 99.75 Complies
TSP002 99.30 Complies
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98.79 Complies
TSP003
92.5 – 107.5
RSD% 0.4837 <5 Complies
Table no.3 – Reconciliations and total productivity for CEFTRIAXONE SODIUM 250mg/VIAL for the
entire technological process
Table no.4 – Reconciliations and total productivity for the raw materials for the entire technological
process
The results obtained by calculating the productivity for each phase of the manufacturing process
were within the imposed acceptance limits and the value of the RSD calculated was less than 5
%.
During the mixture of powders stage the following critical parameters have been controlled:
appearance and assay of the active substance. All the values obtained comply with the specified
limits within the quality specifications.
The results documented by the certificates of analysis for the mixture of powders stage are
presented in appendix no.4.
In table no. 5 are presented the results obtained experimentally at the assay of the active
substance and the statistical parameters (average, standard deviation, the variance, relative
standard deviation) for the three batches that were considered.
Table no. 5 – The results obtained for the assay of the active substance during the filling stage
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The results obtained for the assay of the active substance during the
Batch no.
filling stage (%)
TSP001 98.65
TSP002 97.98
TSP003 98.24
x 98.29
s 0.337787
s2 0.1141
RSD 0.337787
In table no. 6 are presented the results obtained experimentally at the calculation of the assay of
the active substance in the bulk vials and the statistical parameters (average, standard deviation,
the variance, relative standard deviation).
Table no.6 – The results obtained for the active substance assay in the bulk vials
Batch no. The results for the active substance assay in the bulk vials (mg/vial)
TSP001 555.15
TSP002 555.95
TSP003 556.56
x 555.553
s 0.400042
s2 0.160033
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RSD 0.400042
For the finished product the following parameters been controlled: appearance of the vial,
appearance of the vial's content, average fill weight of the vials, identification and assay of the
active substance (CEFTRIAXONE). All the values obtained complied with the specified limits
within the quality specifications.
The results documented by the certificates of analysis for the finished product CEFTRIAXONE
SODIUM 250mg/VIAL are presented in appendix no. 4.
In table no. 7 are presented the results obtained experimentally at the calculation of the average
weight of the Vial – finished product and the statistical parameters (average, standard deviation,
the variance, relative standard deviation).
Table no.7 – The results obtained for the average weight of the Active in the finished product.
Batch no. The results for the average weight of the finished product (mg)
TSP001 555.65
TSP002 555.75
TSP003 555.29
x 555.553
s 0.400042
s2 0.160033
RSD 0.400042
In table no. 8 are presented the results obtained experimentally for the Microbiological Analysis
of the active substance from the finished product and the statistical parameters (average,
standard deviation, the variance, relative standard deviation). The RSD value for the
Microbiological Analysis must be less than 5%.
Table no.8 – The results obtained at the Microbiological Analysis of the active substance for
the finished product
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Endotoxin
Batch no. Endotoxin results
Limits
TC001 0.2Eu/mg 0.08Eu/mg
TC002 0.2 Eu/mg 0.08Eu/mg
TC003 0.2 Eu/mg 0.08Eu/mg
x ---- 0.47
s ---- 0.070238
s2 ---- 0.004933
RSD ---- 0.070238
For Sterility testing a 14 days sterility test was placed and a batch that confirmed the test was
considered validated.
Calculating the value of the relative standard deviation for the CEFTRIAXONE Microbiological
Analysis values, it was obtained a minimum RSD value of 0.070238. For the all three
manufacturing batches analysed, the RSD values were less than 5% so they fit in the
admissible limits.
Conclusions
The manufacturing process of the drug CEFTRIAXONE SODIUM 250mg/VIAL is characterized
by the uniformity and the reproducibility of the manufactured batches, obtaining a finished
product of corresponding quality, according to the Quality Specification.
 The quality parameters controlled during the solution preparation and filling stage (appearance,
assay of CEFTRIAXONE), fit in the admissible limits according to the Quality Specification; the
results obtained at the assay of the active substance in filling stage for the three manufacturing
batches presented a reduced variability, indicating that the results will be reproducible in the
same working conditions;
 The quality parameters controlled for the bulk vials and for the finished product (vial's
appearance, vial's content appearance, average fill weight, identification of CEFTRIAXONE,
related substances, microbiological analysis and assay of the active substance), fit in the
admissible limits according to the Quality Specification; the quantified results were
characterized by homogeneity (average fill weight, microbiological analysis and assay of
CEFTRIAXONE parameters have RSD values less than 5%); fits in the admissible limits,
indicating the reproducibility of the results in the same working conditions;
 The productivity values calculated for each stage are homogeneous, with a reduced variability
(reduced values of RSD: RSD<5%).
The manufacturing process of CEFTRIAXONE SODIUM 250mg/VIAL can be considered
validated.
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MASTER FORMULATION SHEET OF CEFTRIAXONE SODIUM

INJECTION 250MG IM.
Composition (active & excipients) including statement of the quantitative composition, giving
he weight or measure for each active substance used in the manufacture of the dosage form
The qualitative and quantitative composition for each Vial and a batch size of 10,000 Vials is presented
in the following table:
Quantity Quantity (Kg)/ Quality
Raw Material * % W/W Batch Function
(mg)/Vial specification
Active substance
Ceftriaxone sodium Active
300.00** 100 3.00 USP 32
equ. To Ceftriaxone substance
Total 300.0 100.00 3.00 - -
* None of the raw materials is overaged.

**The quantity of the active material is subjected to the % age assay and moisture content.
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SPECIFICATION OF PACKAGING MATERIAL
TECHNICAL SPECIFICATION FOR PRIMARY PACKAGING MATERIAL

GLASS VIALS TYPE II
General data
1.1 The present specification reffers to the analysis of the glass vial used for filling
in the production site.
Reference method
2.1. Reference method: AS PER USP: (method code: HP/PMS/H-037)

3. Physical properties
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Glass Vials
Tests Admissibility conditions

Description Empty Clear Glass Vials 100 ml
Glass Quality
Colorless
i) Glass Color Should be free from scratches, particles,
bubbles and extra color dots
ii) Glass Surface
Glass Type EP Glass type II
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Bottles must be free from dirt, dust &

Dirt & dust
internal contamination.
Volume (Capacity) 5.0ml±0.2ml and 10.0ml±0.2ml
Alkalinity of glass Not more than 4.8ml of 0.01N HCl
Mass (g) Approximately equal to 100
Dimensions
 Total Height
 Finish Height (60±1.0)mm
(7.7±0.3)mm
 Outer diameter of Body
(29.0±0.7 )mm
 Outer diameter of Lip
(22.6±0.6)mm
 Outer diameter of Neck
(≤20.0)mm
 Thickness of Bottom (≥2.5)mm
 Vertical Axle deviation (≤2.0)mm
 Thickness of Body (≥1.0)mm
Rubber Stopper
Tests Technical Standards
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Apearance Almost gray colored Complies

1.Falling leftover puncture ≤20pcs of granularity
2. Power of puncture ≤75N
Physical
3. Fit function of butyl rubber Should egree with the
function
stoppers and container requirements
4. Ash Residue left ≤45%
1. Clarity and color Clarity ≤No.2
Color ≤ kelly No.5
Chemical 2. pH Disparity between with blank
function liquid ≤ 1.0
3. UV Absorbancy (220-360mm) Amax ≤0.10
4. Heavy metals ≤1ppm
5. Ammonium ≤0.0002%
6. Zinc ≤0.0003%
7. Conductivity ≤40.0µs/cm-1
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Flip off Seals

Tests Technical parameters
Appearance Almost red colored flipoff seal engraved with
hygeia monogram.
Total Height（H） 12.5±0.4mm
Inner Height(H) 12.0±0.1mm
Inner Diameter(D) 20.0±0.1mm
Opening Force Complies
i. Patient Information Leaflets:

Text matter As per Approved Art Work.
Dimensions. Length NLT 160mm
Breadth: NLT 103mm
ii. Unit Carton:

Text matter. As per Approved Art Work.
Mfg.License No. As per drug manufacturing License
Registration No. As per registration letter.
Dimensions. LxBxH
Grammage. NLT 250 g/m2
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PHARMACOLOGICAL GROUP OF APPLIED DRUG
Ceftriaxone is a third generation, semisynthetic cephalosporin antibiotic. Cephalosporins are

derivatives of 7-aminocephalosporic acid and are closely related to penicillins in structure.
Cephalosporins have a six membered sulfur containing ring adjoining a ß lactam ring.
Ceftriaxone is a broad spectrum, ß lactmase resistant antibiotic effective against a wide range
of gram positive and gram negative bacterias. Antibiotics require constant drug level in body
for therapeutic effect.
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1.0 DRY POWDER INJECTION CEPHALOSPORIN

EQUIPMENTNAME: Model NUMBER CAPACITIES cGMP Compliance
Washing machine HP/Pro/DV/001 Manual 18 Yes
Headed machine
Autoclave HP/Pro/DV/002 50 Liter Yes
Dry Heat Sterilizer HP/Pro/DV/003 15000Vials Yes
Filtration assembly HP/Pro/DV/004 142mm Yes
Laminar flow hood HP/Pro/DV/005 24/48 inches Yes
with HEPA filters filter
Water storage tank HP/Pro/DV/006 SS 300 Liter Yes
automatic Vials filling HP/Pro/DV/007 70-80 Vials/min Yes
& sealing machine 250mg-2gm
Automatic Labeler HP/Pro/DV/008 --- Yes
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RO Distillation unit HP/Pro/DV/009 300Liter/hour

Optical checking box HP/Pro/DV/010 Manual Yes
Electric Balance HP/Pro/DV/0011 0.01gm-600gm Yes
2.0 CREAM SECTION

EQUIPMENTNAME: COAD NUMBER CAPACITIES cGMP Compliance
Mixer/Homogenizer HP/Pro/CM/012 100 kg Yes
Steam Jacket Kettle HP/Pro/CM/013 20 kg Yes
Plastic/Aluminium filling HP/Pro/CM/014 5gm-40gm Yes
tube & sealing machine
Milling machine HP/Pro/CM/015 2800 rpm Yes
S.S Container HP/Pro/CM/016 Yes
Electric Balance HP/Pro/CM/017 0.01gm- Yes
600gm
RECOMMENDED CLINICAL USE AND DOSAGE:
CEFTRIAXONE SODIUM INJECTION:
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INDICATIONS: infections due to sensitive Gram-positive and Gram-negative bacteria, but

the cephalosporin’s are broad-spectrum antibiotics which are used for the treatment of
septicemia, pneumonia, meningitis, biliary-tract infections, peritonitis, and urinary-tract
infections. The pharmacology of the Cephalosporin’s is similar to that of the penicillins,
excretion being principally renal. Cephalosporins penetrate the cerebrospinal fluid poorly
unless the meninges are inflamed; surgical prophylaxis; prophylaxis of meningococcal
meningitis [unlicensed indication]
CAUTIONS: sensitivity to beta-lactam antibacterials (avoid if history of immediate
hypersensitivity reaction, urinary glucose (if tested for reducing substances) and false
positive Coombs’ test; interactions: may displace bilirubin from serum albumin, administer
over 60 minutes in
Neonates (see also Contra-indications); treatment longer than 14 days, renal failure,
dehydration—risk of ceftriaxone precipitation in gall bladder; interactions:
CONTRA-INDICATIONS: cephalosporin hypersensitivity, neonates less than 41 weeks
postmenstrual age; neonates over 41week’s postmenstrual age with jaundice,
hypoalbuminaemia, or acidosis; concomitant treatment with intravenous calcium (including
total Parenteral nutrition containing calcium) in neonates over 41 weeks postmenstrual age
—risk of precipitation in urine and lungs
HEPATIC IMPAIRMENT: reduce dose and monitor plasma concentration if both hepatic and
severe renal impairment
RENAL IMPAIRMENT: reduce dose if eGFR less than 10 mL/minute/1.73m2 (max. 2 g
daily); monitor plasma concentration if both hepatic and severe renal impairment
Pregnancy: not known to be harmful
Breast-feeding: present in milk in low concentration, but appropriate to use
SIDE-EFFECTS: diarrhea (rarely antibiotic-associated colitis), nausea and vomiting,
abdominal discomfort, headache; allergic reactions including rashes, pruritus, urticaria,
serum sickness-like reactions with rashes,
fever and arthralgia, and anaphylaxis; Stevens-Johnson syndrome, toxic epidermal
necrolysis reported; disturbances in liver enzymes, transient hepatitis and cholestatic
jaundice; other side-effects reported include eosinophilia and blood disorders (including
thrombocytopenia, leucopenia, agranulocytosis,aplastic anemia and haemolytic anemia);
reversible interstitial nephritis, hyperactivity, nervousness, sleep
Disturbances, hallucinations, confusion, hypertonia, and dizziness
DOSE:
250 mg every 8 hours, doubled for severe infections; max. 4 g daily; CHILD over 1 month, 20
mg/kg daily in 3 divided doses, doubled for severe infections, max.1 g daily; or 1 month–1
year, 62.5 mg every 8 hours;1–5 years, 125 mg; over 5 years, 250 mg; doses doubled for
severe infections.
.By deep intramuscular injection, or by intravenous injection over at least 2–4 minutes, or
by intravenous infusion, 1 g daily; 2–4 g daily in severe infections; intramuscular doses over
1 g divided between more
than one site; single intravenous doses above 1 g by intravenous infusion only/.
NEONATE, by intravenous infusion over 60 minutes, 20–50 mg/kg daily (max. 50 mg/kg
daily); INFANT and CHILD under 50 kg, by deep intramuscular injection, or by intravenous
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injection over 2–4 minutes, or by intravenous infusion, 20–50 mg/kg daily; up to 80 mg/kg
daily in severe infections; doses of 50 mg/kg and over by intravenous infusion only; 50 kg
and over,
Adult dose. Endocarditic caused by haemophilus, actinobacillus,
Cardio bacterium, eikenella, and kingella species (‘HACEK organisms’) (in combination with
another antibacterial, see Table 1, section 5.1; [unlicensed indication]), by intravenous
infusion,
2–4 g daily. Early syphilis [unlicensed indication], by deep intramuscular injection, 500 mg
daily for 10 days. Uncomplicated gonorrhea, pelvic inflammatory Disease (see also Table 1,
section 5.1) by deep intramuscular injection, 250 mg as a single dose. Surgical prophylaxis,
by deep intramuscular injection or by intravenous injection over at least 2–4 minutes,1 g up
to 30 minutes before the procedure; colorectal surgery, by deep intramuscular injection or
by intravenous infusion, 2 g up to 30 minutes before the procedure; intramuscular doses
over 1 g divided between more than one site.
REFRENCE: AS PER BNF-61
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Sterility Test Final

PROCEDURE
Equipment and Material Required
1. Incubator
2. Laminar Flow Cabinet
3. Filtration Assembly
4. Autoclave
5. Vacuum Pump
6. Forceps, sterile wrapped in Aluminum foil / Butter paper
7. Scissors, sterile wrapped in Aluminum foil / Butter paper
8. Bunsen burner
9. Ampoule cutter / rail opener sterile wrapped in Aluminum foil /Butter paper
10. Butter paper sheets
11. Facemask sterile wrapped in butter paper
12. Aluminum foil
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13. Surgical gloves sterile
14. Peptone water 0.1% 200 ml sterile in flask or bottle
15. Tryptic Soy broth sterile, 100 ml in screw capped bottles or culture tube.
16. Fluid Thioglycolate Medium USP sterile, 100 ml in screw capped bottle or culture tube.
17. Sterile membrane filters 0.45µm pore size and 47 mm diameter (individually packed).
SAMPLE REQUIREMENT
 FILLED PACKS
1. For Injectables having a filled volume of 1.0 ml or less than 1.0 ml 40 ampoules from each
filling operation.
2. For Injectables having a filled volume of more than 1.0 ml, 20 vials/ Ampoules from each
filling operation.
 BULK
1. For liquid bulks not less than 30 ml of sample from each bulk container.
2. For powder bulk not less than 06gms from each bulk container.
3. Number of containers to be sampled is √n+2
 STABILITY STUDIES
1. For liquid finished not less than 50 ml except of ampoules.
2. For powder finished not less than 03 grams.
1. PREPARATION OF MEDIA
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 PEPTONE WATER 0.1 %
Weight 0.2 gm peptone on digital balance and dissolve in 200 ml distilled water in a
conical flask. Sterilize at 121ºC for 20 minutes in an autoclave.
 TRYPTIC SOY BROTH
Weigh the desired quantity according to the manufacture direction of the medium on digital
balance and dissolve in the required quantity of distilled water. Adjust pH on pH meter 7.3 ±
0.2 with 0.1N NaOH or 0.1N HCl.
Dispense 100 ml each in screw-capped bottles or culture tubes. Sterilize at 121ºC for 20 minutes
in an autoclave. Pre incubate at 32ºC ± 2.5 ºC for at least three days. After pre incubation, store
medium at room temperature under dark. Properly sterilize media stored at room temperature
can be used for a period of three months. Discard any tube or bottle showing any turbidity.
 FLUID THIOGLYCOLLATE MEDIUM
Weigh the desired quantity of the medium according to the manufacturer’s direction
on digital balance and dissolve in the required quantity of Distilled water. Adjust pH
on pH meter (as per SOP) to 7.1 ± 0.2 with 0.1N NaOH or 0.1N HCl.
Dispense 100 ml each in screw-capped bottles or culture tubes. Sterilize at 121ºC for 20 minutes
in an autoclave. Pre-incubate at 32.5ºC ± 2.5ºC for 48 hours. After pre incubation, store medium
at room temperature under dark. Properly sterilize media stored at room temperature can be
used for a period of three months. Discard any tube or bottle showing any turbidity.
 SAMPLE SIZE
For sterility test of bulk material, at least 10ml of sample must be used for each culture medium
kept at different temperatures of incubation.
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TABLE 1 Quantity for Liquid Articles.
Minimum volume of each medium

Minimum
Used for direct Used for membrane or half
Container volume
transfer of membrane representing total
content(ml) taken from each
volume taken volume from the appropriate
container
from each no. of containers (ml)
each medium
Container2(ml)
1 ml or entire if
Less than 10 15 100
<1 ml
10 - <50 5 ml 40 100
50 - <100 10 ml 80 100
50 - <100 intended
for intravenous ½ content 200 100
administration
100 – 500 ½ contents N/A 100
Over 500 500 ml N/A 100
Antibiotics
1ml N/A 100
(liquid)
1
Constitute powder products according to the Manufacturer’s instructions and then treat as liquid
products.
2
For products that cannot be tested by the membrane filtration procedure
Table 2… Minimum No. of Articles to be tested in relation to No. of Articles in Batch
No. Of Articles In The Batch No. Of Articles Be Tested

N.M.T 100 Articles 10% or 4 Articles whichever is greater
M.T 100 But N.M.T 500 Articles 10 Articles
M.T 500 Articles 2% or 20 Articles whichever is less
For large volume Parenteral 2% or 10 Articles whichever is less
 QUANTITIES FOR SOLIDS
Take at least 06 gm of the dry product and dissolve in sterile 0.1% peptone water when
using membrane filtration. For direct method use not less than 300 mg from each container
being tested, or the entire contents of each container if it contains less than 300 mg of
solids to each bottle of the medium. Test 20 containers of Thioglycolate medium and 20
containers of TSB medium.
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 STERILITY TESTING PROCESS
Membrane Filtration Technique is used for sterility test.
o Disinfection of Sample Containers
Disinfect the exterior of all sample containers with an approved disinfectant i.e., 70 % Isopropyl
alcohol and place them under L.F.H.
o OPENING OF CONTAINER
Open the sample containers using vial opener/ampoule cutter place each container in vertical
position in L.F.H (as per SOP).
o SAMPLE PREPARATION
Transfer the required quantity of the product from each container into the flask containing
sterile peptone water and allow dissolving the sample. In case of antibiotic powder samples add
Penicillinase enzyme (ß – lactamase) in sterile peptone water as neutralizer before adding
antibiotic powder for sample solution preparation. If Penicillinase enzyme is not available
dilute the antibiotic powder much (in 200ml peptone water and after filtration wash the
membrane with again 400ml peptone water) to undo the effects of antibiotic powder in sample
preparation.
o TECHNIQUE
Sterilize the filtration assembly wrapped in Butter Paper/Aluminum Foil in an autoclaved at

121°C for not less than 20 minutes. Remove the paper under L.F.H. Place a membrane filter (0.45
μm) aseptically on the filter holder. Place the funnel on the filter holder and clamp it. Connect
the filtration assembly flask to the vacuum pump
Open the sample flask, flame the mouth of the flask and pour the solution into the funnel.
Start filtration at 15 – 20 PSI. Rinse twice with peptone sterile water 100 ml each. Remove the
funnel. Cut the filter into two parts by using forceps and scissors. Transfer aseptically one
portion of the filter into fluid Thioglycolate medium (FTM) and the other into the Tryptic Soy
Broth (TSB). And also for Negative Control (for the conformation of media sterilization, weather
media is sterilized or not) same procedure follow but just filter the peptone water through the
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membrane filter (not the sample), then Cut the filter into two parts, Transfer aseptically one
portion of the filter into FTM and the other into the TSB. And Incubate Thioglycolate medium at
32.5ºC ± 2.5°C and Tryptic Soy Broth at 22.5°C ± 2.5°C for 14 days. Observe the tubes at intervals
for any sign of growth.
 INTERPRETATION OF STERILITY TEST RESULTS
1. FIRST STAGE
At the prescribed intervals during and at the conclusion of the incubation period, examine
the contents of all the vessels for evidence of microbial growth, such as the development of
turbidity or surface growth. If no growth is observed the article test meets the requirements
of the test for sterility. If microbial growth is found, a review should be made in the sterility
testing facility, material used, testing procedure, negative controls and aseptic technique
used in the test. The first stage declared invalid and may be reported. If microbial growth is
observed but there is no evidence invalidating the first stage of the test, proceed to the
second stage.
2. SECOND STAGE
The minimum number of specimens selected is double the number tested in the first stage.
The minimum volumes tested from each specimen, the media and incubation periods are
the same as those for the first stage. If no microbial growth is found, the article tested meets
the requirements of the test for sterility. If however, it can be demonstrated that the second
stage was invalid because of faulty or inadequate aseptic technique in the performance of
the test, the second stage may be repeated. If the sample fails on repeat test and no fault is
observed in second stage of repeat test, an investigation report for positive sterility test
should be held that can result into the rejection of the material.
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VALIDATION OF STERILIZATION PROCESS

 BACTERIOSTATIC AND FUNGISTATIC ACTIVITY
The validation of the sterility test also known as Bacteriostasis and

Fungistasis Test (B&F) must be conducted for each Pharmacopeial article.
This test in necessary to demonstrate that the product is free of inhibiting
factors eliminating the occurrence of false negative results.
Prepare dilute cultures of bacteria and fungi. Inoculate the sterility test media
with 10-100 viable microorganisms/ml. Add the specified portion of the article
to half the number of the containers already containing inoculate in each
culture medium. Incubate the container at the appropriate conditions for not
less than 7 days. If growth of the test organisms in the article medium mixture
is comparable to that in the control vessels, use the amounts of article and
medium regularly. If product is bacteriostatic or fungi static, when tested use-
neutralizing amount of medium (containing β-lactamase enzyme) to be used
and decrease the amount of product, if β-lactamase enzyme not available,
rinse the membrane in each case with two 200 ml portions of the diluting
fluid.
The samples are observed for evidence of microbial growth daily and media
turbidity must be observed within 5 days.
Depyrogenation and sterilization process
Introduction.
Sterilization can be defined as any process that effectively kills or eliminates
transmissible agents (such as fungi, bacteria, viruses and prions) from a surface,
equipment, foods, medications, or biological culture medium. In practice sterility is
achieved by exposure of the object to be sterilized to chemical or physical agent for a
specified time. Various agents used as steriliants are: elevated temperature, ionizing
radiation, chemical liquids or gases etc. The success of the process depends upon
the choice of the method adopted for sterilization.
Pharmaceutical Importance of Sterilization
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• Dry heat sterilization can only be used for thermo stable, moisture sensitive or
moisture impermeable pharmaceutical and medicinal. These include products like;
Dry powdered drugs, Suspensions of drug in non aqueous solvents, Oils, fats waxes,
soft hard paraffin silicone, Oily injections, implants, ophthalmic ointments and
ointment bases etc.
Moist heat sterilization is the most efficient biocidal agent. In the pharmaceutical
industry it is used for: Surgical dressings, Sheets, Surgical and diagnostic
equipment, Containers, Closures, Aqueous injections, Ophthalmic preparations and
Irrigation fluids etc
DEPYROGENATION
Heat Sterilization
Heat sterilization is the most widely used and reliable method of sterilization, involving
destruction of enzymes and other essential cell constituents. The process is more effective in
hydrated state where under conditions of high humidity, hydrolysis and denaturation occur,
thus lower heat input is required. Under dry state, oxidative changes take place, and higher
heat input is required.
This method of sterilization can be applied only to the thermos table products, but it can be
used for moisture-sensitive materials for which dry heat (160-180 0C) sterilization, and for
moisture-resistant materials for which moist heat (121-134 0C) sterilization is used.
The efficiency with which heat is able to inactivate microorganisms is dependent upon the
degree of heat, the exposure time and the presence of water. The action of heat will be due to
induction of lethal chemical events mediated through the action of water and oxygen. In the
presence of water much lower temperature time exposures are required to kill microbe than
in the absence of water. In this processes both dry and moist heat are used for sterilization.
a. Dry Heat Sterilization:

Operation
 Open the door of oven keeping the door to the sterile area closed.
 Load the chamber with the items to be sterilized.
 Close the door tightly.
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 Connect the power supply socket.

 Make the main supply to oven “ON”.
 Adjust the temperature to required figure (2500C for 4hours).
 Adjust the “Sterilization time” and “cooling time”.
 After sterilization is completed (as indicated by the indicator light and
heater OFF) let the items to be cooled.
 Collect the sterilized and cooled items in sterile area, (keeping the door on
non-sterile area closed) close the oven doors both sides.
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STERILIZATION
b. Moist Heat Sterilization: Moist heat may be used in three forms to achieve
microbial inactivation
1. Dry saturated steam – Autoclaving
2. Boiling water/ steam at atmospheric pressure
3. Hot water below boiling point
Moist heat sterilization involves the use of steam in the range of 121-134 0C. Steam
under pressure is used to generate high temperature needed for sterilization.
Saturated steam (steam in thermal equilibrium with water from which it is derived)
acts as an effective sterilizing agent. Steam for sterilization can be either wet
saturated steam (containing entrained water droplets) or dry saturated steam (no
entrained water droplets).
Autoclaves use pressurized steam to destroy microorganisms, and are the most
dependable systems available for the decontamination of laboratory waste and the
sterilization of laboratory glassware, media, and reagents. For efficient heat transfer,
steam must flush the air out of the autoclave chamber. Before using the autoclave,
check the drain screen at the bottom of the chamber and clean if blocked. If the
sieve is blocked with debris, a layer of air may form at the bottom of the autoclave,
preventing efficient operation. Autoclaves should be tested periodically with
biological indicators like cultures of Bacillus stearothermophilus to ensure proper
function. This method of sterilization works well for many metal and glass items but
is not acceptable for rubber, plastics, and equipment that would be damaged by high
temperatures.
Autoclaves, or steam sterilizers essentially consist of following:
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i) A cylindrical or rectangular chamber, with capacities ranging from 400 to 800 liters.
ii) Water heating system or steam generating system
iii) Steam outlet and inlet valves
iv) Single or double doors with locking mechanism.
v) Thermometer or temperature gauge
vi) Pressure gauges
Operation
 Before opening the door, check that the door on other side is closed.
 Clean the Autoclave from Inside with duster (Not shedding the fiber’s)
 The Stuff to be autoclaved is arranged in the S.S boxes.
 Close the Autoclave door tightly.
 Open the main valve for filling water into the S.S tank.
 Close the steam valve.
 Switch on the Electric panel.
 Adjust the time and temperature required for sterilization by adjusting the
concerned timers.
 When the temperature is attained upto 100 °C .Close the main valve and
opens the steam valve.
 The steam will enter into the S.S jacketed autoclave. On reaching the
required temperature (121°C) and pressure (15 lb) close the steam valve and
maintain this temperature and pressure for required time (required for the
sterilization of specific item).
 The decrease or increase in temperature and pressure can be adjusted by
closing or opening the steam valve and pressure release gauge.
 Make entry after every 10. Minutes in autoclave Monitoring sheet until the
required time is reached.
 After attaining the required time Switch off the Panel.
129
Pakistan
 Open the drain valve and let the autoclave cool down.
 Open the door of the other side (towards sterile area) and take out the
required item for use.
S.# TOPICS
1. Form“5”
2. Under taking of stability study data for proposed shelf life and storage
conditions of drugs
3. Under taking of change of brand name in case of resemblance
4. Proposed master formulations
5. Complete description of manufacturing method
6. Identification and description of critical steps and intermediate
7. Recommended Clinical Use and dosage
8. Details of international availability of applied drug
9. Pharmacological group of applied drug.
10. Raw material and finished product specification
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Pakistan
11. Sterility testing, sterilization, depyrogenation and release criteria
12. Microbiological program, environmental monitoring of sterile area
13. SOP for cleaning Validation
14. List of equipments in production department
15. List of equipments in quality control department
16. List of technical staff in production area
17. Specification of packaging Materials
18. In-process quality control checks
19. Environmental control processing
20. Water processing facility schematic presentation of double pass R.O plant
21. Process validation
22. Stability study Protocol
23. Validation of Cleaning Method
24. Performance Qualification Protocol
25. Last inspection report
26.
131
Pakistan
STABILITY STUDIES REPORT

ACCELERATED
132
Pakistan
STABILITY STUDIES REPORT

REAL TIME
133

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HYGEIA PHARMACEUTICALS:

Plot No. 295, Industrial Triangle, Kahuta Road, Islamabad,

APPLICATION FOR REGISTRATION OF A DRUG FOR LOCAL MANUFACTURE.

Date ……………… Signed:

Quality Control Manager Production Manager

ENCLOSURES OF THE APPLICATION FOR REGISTRATION OF A DRUG FOR

Quality Control Manager Production Manager

14. Name(s) of company(s) manufacturing in Pakistan. Rehmat pharma, Lahore

19. Full descriptions of the specifications and analytical methods Annexure -G

Quality Control Manager Production Manager

22. Labeling and prescribing information ( to be mentioned on the N/A

23. Facility of water processing with specifications. Annexure –I

Production Manager Quality Control Manager

Quality Control Manager Production Manager

LIST OF EQUIPMENTS IN QUALITY CONTROL LAB:

Quality Control Manager Production Manager

9 Analytical Weighing Balance 02

The label will be submitted at the time of Manufacturing.

Quality Control Manager Production Manager

Quality Control Manager Production Manager

Quality Control Manager Production Manager

2. PATIENT INFORMATION LEAFLETS:

Description. Printing quality /Color scheme:

Description. Printing quality /Color scheme:

Quality Control Manager Production Manager

2nd Pass R.O Water

Pump Column Water

Multiple Effects Still

Environmental Control processing

Quality Control Manager Production Manager

QUALITY CONTROL WASTES:

1. There is no chemical of extreme hazard used in objectionable quantities.

TEMPERATURE AND HUMIDITY CONTROL:

PEST CONTROL AND DISINFECTION PLAN

Following is the pest control and disinfection plan:

Quality Control Manager Production Manager

For Pesticide – Malathion

Quality Control Manager Production Manager

Author Reviewed By: Approved By:

Signature Signature Signature

Quality Control Manager Production Manager

b. Quality Control Manager

iii. To arrange samples to Quality Control for testing purpose.

5.0 Environment, Safety and Health Precautions

Quality Control Manager Production Manager

1.0 6.0 Procedure

Process validation is to be performed as mentioned in schedule of validation Master plan (will be

Quality Control Manager Production Manager

The validation report comprises into following:

i. Product Name, Batch No’s, Mfg. & Exp. Date

7.0 Change Control Procedure

Document No. Description

9.0 Distribution List

Copy No. Designation Issued To

Quality Control Manager Production Manager

2. Quality Control Manager

Name of product: ---------------------Batch#:------------------Batch size: ----------------

Quality Control Manager Production Manager

Storage Condition : _____Temp: _ RH____

Analyst: _______ Date:

Analyst: ___________ Date: _

Analyst : ___________ Date: _

Analyst : ___________ Date: _

Reviewed By: ____ Date: _

Prepared By: ________ Date: _

Reviewed By: ________Date: _

Reviewed By: ___________Date: _

Reviewed By: ___________Date: _