BIOPHARMCEUTICALS MANUFACTURING

To develop a robust, reproducible and cost effective process that results in safe and efficacious biopharmaceuticals

MANUFACTURING BY FERMENTATION/EXTRACTION

PROCESS AIDS

RAW MATERIALS
AIR STEAM WATER INOCULUM

HEAT

FERMENTOR

DOWNSTREAM PROCESSING

PROCESS CONTROL

WASTE

BIOPHARMACEUTICAL PROCESS DESIGN

1. PRODUCT DEFINITION
PRODUCT SPECIFICATIONS DEFINES ANALYTICAL NEEDS MARKET SIZE

2.
3. 4.

SELECT SYNTHETIC TECHNOLOGY

CREATE PROCESS FLOW DIAGRAM MATERIAL AND ENERGY BALANCES TO CALCULATE COST
MATERIALS (REAGENTS AND CONSUMABLES)

EQUIPMENT
UTILITIES LABOR

5. 6. 7. 8.

ASSESS ASSUMPTIONS AND UNCERTAINTY IDENTIFY QUALITY HOT SPOTS ASSESS PROFITABILITY AND RISK CREATE THE R/D AGENDA

PRODUCT DEFINITION

PRODUCT SPECIFICATIONS

R+D pipeline
ANALYTICAL NEEDS

Pharmacopea
Martindale. The Complete Drug Reference Physicochemical properties Absortion and fate Uses and app. mode of administration

Adverse/side effects
MARKET SIZE Suitable dosage levels

Martindale Structure Drug Monographs

The order of sections is usually as follows: Nomenclature and Pharmacy Adverse Effects Treatment of Adverse Effects

Precautions
Interactions Pharmacokinetics

Uses and Administration

http://www.pharmpress.com/assets/docs/martindale%20structure%20pdf.pdf

Pharmacopoeia: INSULIN, HUMAN

PRODUCTION Enzymatic modification of pig insulin (change of Ala for Thr at position B30) Recombimant REQUIREMENTS ANALYSIS Details of properties: Martindale. The Complete Drug Reference At least 171 million people worldwide have diabetes; this figure is likely to be more than double by 2030 (90% type II)

India, China, USA, Indonesia, Japan, Pakistan, Russia, Brazil, Italy and Bangladesh
Dose 50 IU/day; 1 IU of insulin 0,0347 mg 1,75 mg /day (600 mg/year)

Spanish market: 250 kg/year

BIOPHARMCEUTICAL PRODUCTION 2006

SELECT SYNTHETIC TECHNOLOGY

EXPRESSION SYSTEMS

Bacterial (doubles in 20 -30 min)

Insect (doubles in 18-24 hr)

Yeast (doubles in 90 min) (oldest) Mammalian cell lines (doubles in 24 hr) Transgenic Animal Plant

SELECT SYNTHETIC TECHNOLOGY

EXPRESSION SYSTEMS

Postranslational modifications
Glycosilation (attachment of carbohydrates to the protein) OK for all types of cells except for E. Coli N-glycosilation (amide N of the Asn side chain) (yeast only adds mannose) O-gycosylation (hydroxyl groups of Ser or Thr) Phosphorylation (esterification by fosforic acid at the hydroxyl group: Ser, Thr, Tyr) OK for all types of cells except for E. Coli Acetylation (amide formation by acetic acid at the Lys side chain amine) OK for all types of cells except for E. Coli

g-carboxylation (proteins in blood clothing and Ca metabolism contain the residue Gla: g-carboxyglutamic acid. only perfomormed in mammalian cells

SELECT SYNTHETIC TECHNOLOGY

EXPRESSION SYSTEMS

Downstream process
Refolding is neccessary when using E.Coli. Sometimes in yeast

E. Coli does not secrete proteins

Expression level of foreign proteins in E. Coli is usually high Cost for culturing E. Coli and yeast are low Synthesis of mAb needs expression of several genes: mammalian cells are desirable to carry out cell fusion Constraints like high growth rate, good fermentation properties and cultivation requirements, expression level of foreign genes, and genetic stability limited the number cell lines available

SELECT SYNTHETIC TECHNOLOGY

EXPRESSION SYSTEMS (SUMMARY) Property Bacteria Yeast Insect/mammal

Growth
Cost of media Nutrient demand Product yield Secretion Glycosilation Association Folding SS bridges

Fast
Low Minimal High No no Inclusion bodies Misfolded Limitation

Fast
Low Minimal High Yes/no Possible Correct May be required No limitation

Slow
high Complex low yes possible correct correct No limitation

Risk retroviral
Risk pyrogens Scalability Process robustness

Low
High Good Good

Low
No Good Good

high
no low low

MANUFACTURING BY FERMENTATION/EXTRACTION

PROCESS AIDS

RAW MATERIALS
AIR STEAM WATER INOCULUM

HEAT

FERMENTOR

DOWNSTREAM PROCESSING

PROCESS CONTROL

WASTE

INOCOLUM

Genetic engineering or Gene Splicing

UPSTREAM PROCESS
RAW MATERIALS

Nutritional requirements
Elemental requirements Specific nutrients

FERMENTOR
AIR

STEAM
WATER

INOCULUM

PROCESS CONTROL

Environmental requirements
pH profile Temperature profile Dissolved oxygen Catabolite repression Physiological constraints

MEDIUM PREPARATION
COMMON REQUIREMENTS
NITROGEN SOURCE: Ammonium sulfate;/protein hydrolysates CARBON SOURCE: Glucose:; lactose; sacarose; maltose; dextrins

Cellulose; stach; molasses; corn sugar

Fatty acids ; triglycerides Fats/oils: soybean oli; cottonseed oil Yeast extract; tryptone

SALTS (NaCl; KCl; CaCl2)

SPECIFIC REQUIREMENTS
TRACE ELEMENTS (FE, Mn, Cu, Co, Zn) VITAMINS (Ascorbic acid; thiamine; ribofalvine; folic acid;) HORMONES (growth factors) ANTIBIOTICS SUPLEMENTAL SERUM (Albumin; transferin/fetal calf) BUFFERING AGENT (carbonates, phosphates,..) WATER FOR INJECTION

FERMENTOR REQUIREMENTS LIST

Keep cells uniformly distributed in the reactor volume Keep temperature constant and homogeneous Minimize nurint gradients Prevent precipitation and floculation

Allow gas diffusion to the speed necessary for the process

Keep fermentaion clean Keep aspetic atmosphere Maximaize yield and production Minimize expenditure and production cost Reduce production time

BIOREACTORS

Suspension culture
STR
Air

Medium Medium Cells

high matter/gas transfer 15,000 L PROBLEMS: Shear due to mechanical agitation

Damage due to gas sparging

ALR 2,000 L
Air

Stirred tank

Airlift

BIOREACTORS
Adherent culture: Immobilized cells

Product

Product

Spent Medium

microcarriers
Product

Product

Fresh Medium Fresh Medium Packed bed

Fresh Medium
Hollow fiber

Fluidized bed

Large-Scale Bioreactor 25,000 liters

Lab scale reactor 3L

Feed bioreactors- Wave Technology

S20 n 20L Bag n 2-10L Working Volume

S200 n 2 X 100L Bags n 10-100L Working Volume

21

4-8 L 5000 -10000L 300 L 800 L-2000L 300 L

Monitoring Growth Conditions

pH Often drops as cells grow and divide, if the culture doesnt get enough oxygen so that glucose is broken down by glycolysis into lactic acid which crosses the cell membrane enters the media and creates an acid environment. If there is plenty of oxygen, glucose is broken down into pyruvic acid which enters the mitochondria producing H2O, CO2, and energy (ATP and heat). Bacteria: 6,5-7,5; yeast: 4-5; mammalian cells: 6-8 Analyate analysis - Glucose concentration measurements using an analyate analyzer such as a Biolyzer or a Nova, allows us to determine when glucose has been used up and therefore when to start feeding methanol for protein production or to determine when lactate is being produced, a sign of anerobic respiration Temperature Each cell type needs different temperature. Psychrophile: 15-18C; mesophille: 30-45C; thermophile: 45-75C Dissolved Oxygen Oxygen is needed to accept protons from the NADH hydrogen atoms in the mitochondrial electron transport chain.

PROCESS CONTROL
Head Plate Stirrer motor mount (motor controls the agitation rate) Condenser (outlet) Inoculation port (pump seed reactor contents here) pH probe (measures pH of media; feedback loop adds acid or base) Thermo well port (put temperature probe in here to measure temperature; feedback loop cools or heats) Sparger (bubbles gasses into media) Feed bottle (to add glucose, acid or base, methanol) DO probe (measures dissolved oxygen in the media; feedback loops control agitation, air and oxygen) Harvest port (for harvesting batch) Impeller (like a propeller moves fluid and propelled by motor) Computer Controller

1. Sample bottle assembly 2. Head plate assembly 3. Stirrer motor mount 4. Condenser air outlet 5. Condenser water outlet (from) 6. Inoculation port 7. Condenser water inlet (to) 8. CO2 overlay port 9. pH probe 10. Thermowell port 11. Mill fastener 12. Sparger 13. Feed bottle 14. Blind stopper 15. DO probe 16. 3 Feed ports 17. Harvest tube

OPERATION MODE
BATCH
Inoculum and nutrients added once

FED-BATCH Replenishing the growth-limiting nutrient CONTINOUS PRODUCTION Fresh medium is added continously. Culture is removed at the same time PERFUSION Number of cells is kept constant Fresh medium is added continously Harvesting consist in removing supernadant