Antivenoms’ development

Analytical method
TITLE: CODE: PAGE:
DETERMINATION OF THE NEUTRALIZING M-AV-002
POTENCY OF THE FINISHED PRODUCT AGAINST 1 / 11
REVISION: SUBSTITUTES:
THE NORTH AMERICAN VIPERIDAE SNAKES Does not apply
FAMILY VENOMS THROUGH THE SPEARMAN- A
KÄRBER METHOD
REFERENCE: ELABORATION DATE:
Internal
August 01st, 2016

1. BASIS
The method is designed to determine the capacity of the fabotherapic finished product to neutralize
the lethal effect of the venom in a murine model. It consists in the dispense of different doses of
fabotherapic (antivenom) challenged against the equivalent to 3 LD 50 of a venom, by intravenous route
and, after 48 hours, the survival percentage is recorded in the different assessed doses. The objective
is to determine the dose in which 50% of the population survives. This antivenom quantity is known as
Median effective Dose 50 (ED50). The calculation of this value can be performed by different
mathematical methods. The one suggested in the Pharmacopoeia of the United Mexican States
(known in Spanish as FEUM), is the Spearman-Kärber method.

2. REACH
This method applies to the determination of the neutralizing potency of the finished product against the
species: Crotalus atrox, Crotalus adamanteus y Crotalus scutulatus scutulatus type A.

3. ABBREVIATIONS
AV Antivenom
ED50 Median effective dose
LD50 Median lethal dose
mcg Micrograms
μl Microliter
ISS Isotonic saline solution

4. REAGENTS

● Sodium chloride, ≥ 99.8 % purity.

5. MATERIALS

● Magnetic stir bar

● Centrifuge

● Adhesive tape

● Disposable gloves and masks

● 3ml sterile syringes with needle 21 G x 32 mm

 Antivenom Development
Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

● Calibrated 10, 100, 200 and 1000 mcL calibrated micropipettes

● 1L volumetric flask

● 10-200 μl micropipette tips

● 1000 μl micropipette tips

● Spatulas

● 0.6 mL, 1.5 mL, 2 mL Eppendorf type polypropylene pipes

● 15 mL and 50 mL Falcon type conical plastic tubes with screw cap

● 1 L beaker

● Rodent food

● Drinking fountains

● Table lamp with 40-70 watt spotlight

● Mice boxes

● Bed shavings for mice

6. BIOLOGICAL MATERIAL

● Mice Weighing in 18 to 20 grams, indistinct sex, Strain BALB/c

● Rat poison. Venoms of the species Crotalus atrox, Crotalus adamanteus and Crotalus
scutulatus scutulatus type A
● Finished product to be evaluated: Antivipmyn ® or Anavip ®.

7. EQUIPMENT

● Granatary balance
● Analytical balance
● -94 °F ± 37.4 °F Ultrafreezer
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Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

● 41 °F ± 37.4 °F Refrigerator
● Laminar flow hood

8. SAFETY AND HYGIENE GUIDELINES

8.1. Wear a closed lab coat and latex gloves during the whole analysis
8.2. For venom handling, wear a closed lab coat, gloves, dust mask, and safety glasses.
8.3. Put sharp materials in rigid containers intended for the disposal of infectious biological
materials. Have a container available for the disposal of infectious biological material, such as
micropipette tips, tubes, absorbent towels, etc.

9. METHODOLOGY

9.1. SOLUTIONS PREPARATION

9.1.1. 0.9% isotonic saline solution (0.154 M NaCl) (ISS)

Weigh 9g of Sodium Chloride (NaCl) and dissolve it in 800mL of water (filtered by the Milli Q
system). Transfer to a 1L volumetric flask and calibrate with water. Transfer to sterilizable screw-
capped glass bottles.
Sterilize the solution by humid heat at 249.8 °F, 15 lb pressure for 20 minutes.
Allow the solution to cool and make aliquots in 50 mL sterile polypropylene tubes inside a laminar
flow hood. Store at room temperature.

9.1.2. Venom preparation

Prepare the venom solution and store in aliquots at -94 °F ± 50 °F.
The recommended C. scutulatus venom stock concentration range is 1 mg/mL to 5 mg/mL, and for
C. adamanteus and C. atrox is 10 to 15 mg/mL.

Prepare the volume of venom stock necessary for the execution of the method considering the
number of neutralizing potency trials to be performed.

The concentration of the venom (mg/mL) must ensure a lethal potency of the stock greater
than the equivalent of 60 LD50/mL. Refer to the M-AV-001 method: “DETERMINATION OF THE
LETHAL POTENCY OF THE NORTH AMERICAN VIPERIDAE SNAKES FAMILY VENOMS
THROUGH THE SPEARMAN-KÄRBER METHOD” for the determination of the lethal potency of
venom stock.

9.2. DETERMINATION OF LETHAL POTENCY OF THE VENOMS STORED IN ALIQUOTS

 Antivenom Development
Analytic Method
TITLE: CODE: SUBSTITUTES: PAGE:
DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

The determination of neutralizing potency requires prior calculation of the lethal potency of the shock
(LD50/mL) of each rat poison. Conduct this determination based on the M-AV-001 method.
“DETERMINATION OF THE LETHAL POTENCY OF THE NORTH AMERICAN VIPERIDAE SNAKES
FAMILY VENOMS THROUGH THE SPEARMAN-KÄRBER METHOD”.

9.3. SELECTION OF THE ANTIVENOM DOSES TO EVALUATE AND COMPOSITION CALCULUS OF

THE INOCULUM

The inoculum or venom-antivenom mixture must contain the equivalent of 3 LD50 of venom per mouse and
different antivenom doses. The objective is to construct a dose-response relationship, in which the
relationship is the percentage of survivorship in the mice population.
The selection of antivenom (AV) doses should be such that when carrying out the dose-response curve at
least one dose of antivenom that generates 0% survival and at least one dose that generates 100%
survival is obtained. Consider the following steps:

9.3.1. Consider a previous value of neutralizing potency (in neutralized LD50/vial) for the type of
product and venom to be used as the theoretical neutralizing potency of the product to be
evaluated.
9.3.2. For the following steps use as a support the Neutralizing Potency determination spreadsheet
(Annex I). Enter the data concerning the analyst, the sample and the challenge venom in the
corresponding cells in the section “A. GENERAL DATA” (cells with double bottom border).
9.3.3. Enter the theoretical neutralizing potency data of the product (neutralized LD50/vial) in the
corresponding cell in section A of Annex I.The neutralizing potency in neutralized LD50/mL of the
product reconstituted in ISS diluent will be automatically calculated.

9.3.4. The theoretical effective dose 50 (theoretical ED50) will also be calculated in mcl per mouse,
which will correspond to the volume in microliters of antivenom per mouse that in theory will allow
the survival of 50% of the challenged population with 3 LD50 of venom per mouse. This data will
correspond to the central dose of antivenom of at least five to evaluate.
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Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

9.3.5. Enter in section A the data concerning the challenge venom (marked cells with double
bottom edge) in the specified units. Write down the lethal potency value of the poison stock in
LD50/mL of previously determined stock. (numeral 8.2).

9.3.6. Prepare a venom solution containing 60 LD 50/mL according to indicated volumes in section B
of Annex I. This preparation of this solution is calculated automatically based on the lethal potency
of the poison stock, using the following equations:

9.3.7. In section C, SPREADTABLE, "Antivenom dose" column, the antivenom dose to be

evaluated will be automatically generated, calculated from the central dose and keeping between
them a constant dilution factor of 1.5.

9.3.8. For every dose, the inoculation of a group of 6 mice (plus 2 excess mice) and an inoculation
volume of 500 μl per mouse is considered. Therefore, the composition of each inoculum:

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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

A
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Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

Venom solution volume at 60 DL50/mL for each inoculum, to have a venom quantity in the inoculum
equivalent to 6 DL50/mL or 3 DL50 /mouse:

9.4. INOCULA´S PREPARATION

Ubicate the “INOCULUM´S PREPARATION CHART”, that is found in section “D” within Annex I. In it the |
antivenom´s volumes are found, venom´s solution at 60 DL 50/mL and ISS to mix for the inocula´s
preparation with each test dosage.

9.4.1. Label five 15 mL type Falcon tubes with data from each test dosage. Transfer to each tube the
corresponding volume of ISS, indicated on section D.

9.4.2. Transfer to each tube the corresponding volume of antivenom´s stock indicated on section D.

9.4.3. Finally add the venom´s solution volume (same volume for all the inocula.)

9.4.4. Cover the tubes and mix smoothly by inversion.

9.4.5. Incubate inocula at room temperature for at least 30 min before inoculating mice.

9.5. INOCULATION OF MICE

9.5.1. Identify the boxes for the mice (one for each dose) with the same data you labeled the inocula and
the date the assay was run.

9.5.2. Place a 40 to 75 watts lightbulb over the grille that covers the box where the total number of mice
that will be used for the inoculation of the test doses are. Turn on the lightbulb for at least 5 minutes
before beginning the inoculation, in order to favor the dilatation of the caudal vein and facilitate the
inoculation.

9.5.3. Inject each mouse with 500 μL of the corresponding inoculum by IV (6 mice for each inoculum),
trying to inject within a time of 5 to 10 seconds. As you inoculate each mouse, place it inside of the box
identified with the data of the corresponding inoculum.
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Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

9.5.4. Keep the mice in observation for 48 hours. Provide food and water ad libitum.

9.5.5. During the observation time, remove mice as they die to avoid cannibalism. DO NOT wait at the
end of the assay to remove them.

9.5.6. At the end of the observation time, register the number of surviving animals for each group.

9.5.7. Enter the data in section C of Annex I, column: "# alive". The spreadsheet will automatically
perform the corresponding operations according to the following equations:

Survival percentage:

9.5.8. Verify compliance with the first two acceptance criteria (numeral 9). If so, proceed to the
neutralizing power calculation.

9.5.9. Once the survival data have been entered, "Annex I" calculate the logarithm of DE50, using the
simplified Spearman-Kärber equation for cases where there is a constant dilution factor and equal
number of test units (number of mice):

Where:
k = k-th dose lower which obtains the 100 % of survival.
xi = Log of the i-th dose
ri = number of survivors to the i-th dose
n = number of mice per group
f = the amplitude of an interval for xi equally spaced

The f value is calculated by the next equation:

The ED50 value in mcL of antivenom per-mouse (mcL antivenom/mouse) is obtained through
the antilogarithm of M:

The variance estimation of M when there is the same number of test sets is:
 Antivenom Development
Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

The confidence interval (CI) at 95 per-cent for the real value of M is:

The lower and higher value of the confidence interval of ED50 is determined obtaining the
antilogarithm for each limit:

The neutralizing power of antivenom stock in volume units (DL50 neutralized/mL) is calculated
through:

The neutralizing power per vial of finished product (DL50 neutralized/vial) is calculated through:

The lower and upper bounds of CI for the neutralizing power in units of volume are calculated with
the following equations:

The below and top values of CI for neutralizing power per vial:
 Antivenom Development
Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

The calculation of CI (%) is made through the equation:

Where CI ED50 = confidence Interval at 95 % for ED50.

10. ANALYSIS REPORT

Generate an analysis report (Annex II) “Determination of Neutralizing Power" and attach the Annex I.

The data of the analysis report of Annex II are generated automatically based on the Annex I. Enter in
Annex II the following information: analyst’s data, data of who’s verifying, the observations regarding the
trial (if there are no observations, write: “None”) and the issue date of the report.

11. CRITERIA OF ACCEPTANCE.

The test is deemed as valid if:

● There are ends of 0% and 100% survival.
● The CI (%) is within the range from 70% to 130% with regard to the outcome of the SD50 or, in
case that the I.C. cannot be calculated, the variance (S2) equals zero.

12. RESPONSIBLE FOR THE DOCUMENT

Preparation of the document

CHEMICAL IN BIOLOGICAL TESTS _______________________________________
Dr. Arlette Mena Arizmendi
(Signature and date)

Revisions of the document

CHEMIST SPECIALIZED IN
 Antivenom Development
Analytic Method
TITLE: CODE: SUBSTITUTES: PAGE:
DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

IMMUNOCHEMISTRY/BIOASSAYS _______________________________________
Dr. Dulce Aurora Frausto Del Río
(Signature and date)

CHEMICAL ANALYST _______________________________________

CPB. Carlos Gerardo Cisneros Livera

13. ANNEXES
Annex I. Spreadsheet for the determination of Neutralizing Power.
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Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION:

ANNEX II. Analysis Report of Neutralizing Power

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Analytic Method
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DETERMINATION OF THE NEUTRALIZING POTENCY
Non
OF THE FINISHED PRODUCT AGAINST THE NORTH M-
applicable
AMERICAN VIPERIDAE SNAKES FAMILY VENOMS AV-002
THROUGH THE SPEARMAN-KÄRBER METHOD 11/11
REVISION: