Process for the production of final products

1. Process for the production of final products General procedure for production of bio-products using recombinant cells : - A single vial of the working cell bank system - Cultivation of cells in a bioreactor at different scales - Separation of cells for recovery of a target product - Purification process : purity and yield - Analysis of final product - Formulation & packaging Upstream processing : Cell culture process resulting in the initial generation of a target product Downstream processing : Actual purification of a target product and generation of finished product format ( i.e., filling into its final product containers, freeze-drying if desired, labeling and packaging)

2. Cell banking systems : To ensure essentially indefinite supply of the originally developed production cell line for manufacturing purposes - cryopreserved ampoules of cell line - Preservation of product-producing cell line - reproducible and consistent production • Preparation of cell banking system :  Initial cultivation of cell line  Aliquoted into small volume in ampoules, immersed in liquid nitrogen  cell bank system - The content of all the ampoules is identical, and the cells are effectively preserved for indefinite periods • A master cell bank : Constructed from a culture of the developed cell line  used to generate a working cell bank • A working cell bank : directly used for a single production run - Each ampoule is thawed and used for a new batch

4. Upstream processing • Initial generation of a target product - A single ampoule of the working cell bank - Inoculation for seed culture in a small volume of sterile growth medium : lab-scale starter culture of the producer cell line - Starter culture is used to inoculate several liters/ tens of liters of growth medium in a small bioreactor - Production scale starter culture is used to inoculate the production-scale bioreactor of several thousands/tens of thousands liters • Medium composition and culture conditions : optimal cell growth/ product production • Cell culture process : Industrial scale cell culture process : bioreactor configuration, oxygen transfer, mode of operation, mixing/agitation etc.. - Microbial cell culture - Animal cell culture system

5. Culture type -Batch culture: Nothing added to or removed from the fermenter during cultivation - Fed-batch: Feeding of a growth limiting nutrient substrate to a culture. The controlled addition of the nutrient directly affects the growth rate of the culture and helps to avoid overflow metabolism - Continuous type (Chemostat): Fresh medium is continuously added, while culture liquid is continuously removed to keep the culture volume constant

6. Downstream processing: Bioseparation • Most critical step for economic production • High purity and high yield - High purity is obtained at the expense of yield : ~ 1 / (yield)N • Detailed process : highly confidential • Normally undertaken under clean room conditions - Final step under Grade A laminar flow condition • First recovery step - Intracellular product : harvesting of cells using industrial scale centrifuges followed by disruption using a homogenizer or Dynomill (agitation in the presence of glass beads) - Homogenizer : combination of high shear force and pressure drop  Rupture of most microbial cells

8. - Removal of cell debris using centrifugation : generation of crude(unpurified protein product ) protein solution - Extracellular product (secretion of target protein) : recovery of cell culture medium by removal of cells using centrifugation or filtration • Concentration of crude (dilute) protein product : more convenient and faster processing - Precipitation : salts such as ammonium sulfate or solvents like ethanol - Ultrafiltration : separation based on size and shape - ultraflitration membrane with different cut-off sizes (3, 10, 30, 50, and 100 kDa) : - Molecules larger than the pore size are retained, but smaller ones are passed through the membrane, effectively concentrating the protein solution

9. - Popular method for concentration - high product recovery rate - high speed - process-scale ultrafiltration equipment is available : • High resolution chromatographic purification : - Different methods based on various physiochemical characteristics : - Gel filtration and ion-exchange chromatograph : most commonly used

10. Final product formulation • Formulation into final product format - Addition of various excipients : to stabilize and enhance the characteristics of the final product - Filtration of the final product through 0.22 um filter to generate sterile product followed by its aseptic filling - Freeze-drying if the product is marketed in a powered form • Formulation of product in liquid or powder form depends on stability of a target protein in solution

11. Factors affecting the biological activity of proteins • Loss of biological activity of protein : - Non-covalent alterations : partial/complete protein denaturation - Covalent alterations Hydrolysis, deamidation, imine formation, oxidation, sisulfide exchange, photodecomposition • Proteolytic degradation: serine, cysteine, aspartic, and metallo-proteases - Addition of protease inhibitor • Protein deamidation : - Hydrolysis of the side-chain amide group of asparagine /glutamin, yielding aspartic and glutamic acid, respectively  Accelerated at high temp and extremes of pH ex) major route by which insulin degrades

12. Oxidation and disulfide exchange - Sulfur atoms in methionine or cysteine : the most susceptible to oxidation : - Intra-chain and inter-chain disulfide exchanges : - Loss in biological activity • Alteration of glycosylation patterns of glycoprotein - Glycosylation plays a crucial role : Biological function, solubility, stability, immunogenicity - Oligosaccharide chains : D-galactose, D-mannose, L-fucose N-acetylglucosamin, N-acetylneuraminic acid( sialic acid) - O-glycosidic linkage : Sugar side chain is attached via the hydroxyl group of serine or threonine - N-glycosidic linkage : Amino group of asparagine is linked with sugar side chain

13. Stabilizing excipients • A range of various substances: added to a purified biopharmaceutical product for stabilization : - Serum albumin : - Amino acids : - Polyols : molecules having multiple hydroxyl groups : - glycerol, mannitol, sorbitol, polyethylene glycol, inositol - Surfactants : generally denaturating agents - Proteins have a tendency to aggregate at interfaces ( air-liquid or liquid-liquid)  sufficiently diluted solution : stabilizing effect by increasing the solubility of proteins

14. Final product fill - QC test to ensure its compliance with product specifications - Filtration using 0.2 um filter for sterile product • Freeze-drying - Removal of water directly from frozen products via lyophilization  Reduce the deactivation of protein by chemical/biological mechanism  Longer shelf-life • Labeling and packing

15. Analysis of the final product • Rigorous QC testing : To confirm the pre-determined specifications of the final product • Potency testing : efficacy • Safety test : Analysis of various potential contaminants - Range and medical significance of potential impurities : - Advances in analytical techniques : practical and routine QC testing • Clinical significance of protein-based impurities - Potential biological activities : deleterious effects on the product itself or the patients ex) Degradation /modification by contaminated proteases - Immunogenecity : immunological reaction against the patients ex) toxins, contaminating proteins - Administration of the product can elicit an immune response against the contaminants  sensitizing effect on the recipient against the actual protein product

16. Most of the chromatographic steps during downstream processing :  To separate the protein of interest from contaminant proteins  Particularly substantial for intracellularly produced proteins ex) Proteins produced by animal cell culture - Animal cell culture requires very complex culture media - Addition of endocucleases : to degrade the liberated DNA upon cell disruption : DNA causes increased solution viscosity • Altered forms of the protein of interest : Reduced efficacy or immunogenecity - Modified form of product : considered “ impurities” - Biologically inactive and reduces overall product potency - Biologically active, but different pharmacokinetic characteristics - Immunogenic - Generation of altered forms by various ways : -

17. Removal of altered forms of the protein product • Gel filtration chromatography : difference in size/shape of proteins - Removal of aggregated forms - Removal of proteolysed forms - Deglycosylated proteins • Ion-exchange chromatography : Difference in protein surface charge at given pH - Removal of deglycosylated proteins - Deamidation and oxidation : products with altered surface charge - Proteins with incorrect disulfide bond formation, partial denaturation, limited proteolysis : altered shape and surface charge • Hydrophobic interaction chromatography : Difference in the size and extent of hydrophobic patches on the protein surface - Similarly used as ion-exchange chromatography

18. Product potency • Final product potency specifications - Units of activity per vial of product (or per therapeutic dose, or per mg product) • Bioassays : Most widely used potency-determining assay - Directly assess the biological activity of the products in in a biological system - Applying a known quantity of the product to be assayed to a biological system : whole animals, specific organs, tissue types, cells - Comparative, and requires parallel assay of a “standard preparation” or ”control” ex) Bioassay of EPO : Mouse-based bioassay - EPO stimulates red blood cell(RBC) production, and used for treatment of certain forms of anaemia - EPO-containing sample is administered to mice, along with radioactive iron ( Fe57)

19. - Measure the incorporation rate of radioactivity into proliferating RBCs - The greater the stimulation of RBC proliferation, the more iron is incorporated for haemoglobin synthesis during a given time • Bioassay of interferons : cytopathic effect inhibition assay - Based on the ability of interferons to render animal cells resistance to viral attack - Incubation of the interferon preparation with cells that are sensitive to destruction by a specific virus - Percentage of cells that survive the viral infection : Proportional to the level of interferon present in the assay sample - Measure the percentage of cell survival - Addition of dye like neutral red - Spectrophotometric quantification of assimilatied dye - Automated assay by using a microtitre plate

20. Assay of enzyme activity - Enzyme activity : moles of substrate converted per unit time - A more practical and commonly-used value : 1 enzyme unit (EU) = 1 μmol min-1 - The specific activity : the activity of an enzyme per milligram of total protein (expressed in μmol min-1mg-1).  Measurement of the purity of the enzyme. - Types of assay - Initial rate experiments - Progress curve experiments : kinetic parameters are determined - Transient kinetics experiments : reaction behaviour is tracked during the initial fast transient  rapid mixing and measurement - Relaxation experiments : an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored.  Valid for reversible reactions

21. Mode of sampling methods - Continuous - Discontinuous • Typesof measurements - Colorimetric assays - Fluorimetric - Chemiluminescent : the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation - Radiometric assays : measure the incorporation of radioactivity into substrates or its release from substrates.  Most frequently used radioactive isotopes : 14C, 32P, 35S, Fe57 and 125I.

22. Drawbacks of bioassays - Lack of precision : complex nature of any biological system, entire animal or an individual cell, results in the responses that are largely influenced by factors like metabolic status of individual cells, sub-clinical infections, stress levels induced by human handling - Long assay time : difficult to carry out routine bioassays, and impractical to conduct a quick QC test during downstream processing - Cost : testing with whole animals are extremely expensive • Immunoassays : most popular alternative assay systems - Use of polyclonal or monoclonal antibody preparation to detect and quantify the product - Rapid, sensitive, inexpensive, straightforward - Specificity of antibody-antigen interaction : precise quantification - Radioimmunoassay (RIA) and Enzyme-Linked Immunosorbent Assay (ELISA)

23. Radio-immunoassay - Revolutionized research and clinical practice in many areas such as blood banking, diagnosis of allergies, endocrinology - Introduced in 1960 as an assay for the level of insulin in blood - First example showing that hormone levels in the blood can be detected in vitro - Yalow, RS : Nobel Prize in Medicine in 1977 for the development of RIA for insulin

24. - Principle - Preparation of standard radioactive antigen : Introduction of 125I or 131I into tyrosine residues - Mixing of known amount of radioactive antigen and respective antibody - Addition of unknown amount of antigen to be assayed - Radioactive antigen is replaced by unlabeled antigen - Antibody-bound antigen is separated - Measure the radio-activity of each fraction (bound antigen & free antigen)

25. ELISA • Widely used for assay of various proteins (antigen and antibody) Ex) EPO assay, interferons, therapeutic antibodies, antibody against HIV, Hepatitis B, C virus, - Use of enzyme-conjugated secondary antibody for signal amplification : HRP or Alkaline Phosphatase • Automated measurement using a 96 well plate • Disadvantages - Immunological reactivity is not guaranteed to correlate directly with biological activity - Relatively minor modifications of the protein product, while having a profound influence on its biological activity, may not be detected

27. Detection of protein-based product impurities - Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) : Most commonly used analytical technique - High resolution separation of proteins based on their molecular mass - Band containing as little as 100 ng protein can be visualized by staining the gel with dyes like Coomassie blue  Subsequent gel analysis by scanning laser densitometer - Use of silver-based staining : 100-fold increased sensitivity  ~ 1 ng prptein - SDS-PAGE under reducing condition : b-Marceptoethanol or dithiothreitol (DTT)  Disruption of intra- or inter- chain disulfide linkage

28. Determination of protein concentration : - Quantification of total protein in the finalproduct : standard analysis undertaken by QC - Many methods are available : - Dye-binding procedure : the most common method Ex) Bradford method - Use of Coomassie blue G-250 - Protonated in acid solution : absorbance maximum at 450 nm - Binding of the dye to a protein via ionic interactions) : shift in a maximum absorption wavelength to 595 - Quantification of protein concentration - Silver staining method : extremely sensitive method of protein detection in electrophoretic gels

29. Western blot analysis : elution of the protein bands from the electrophoretic gel onto a nitrocellulose membrane followed by probing using antibodies raised against the product • 2-D gel electrophoresis : separation of proteins based on different molecular property  Contaminants of the same molecular mass as the product can not be detected by SDS-PAGE - Utilized to determine product homogeneity - Homogeneity is best indicated by the appearance in the gel of a single protein band, exhibiting the predicted PI value - Glycoproteins with slightly different carbohydrate content : Variation in their sialic acid content  Different pI values

30. Amino acid analysis : small polypeptide ( < 10 Kda) - Determine the range and quantity of amino acids present in the product - Hydrolysis of peptides by chemical method ( 6 N HCl, 110 oC, under vacuum, 12-24 hrs) - Amino acid analyzer • Peptide mapping : Peptide fingerprint - Detection of occurrence of point mutations in the gene coding for the product during gene manipulation, transcription, translation - Full sequencing of a target protein at each production batch  Tedious and time consuming

31. - Fragmentation of a target protein into small peptides using proteases or chemical method (cyanogen bromide) - Trypsin and V8 protease produced by staphylococci : commonly used protease - Cyanogen bromide cleaves the peptide bond on the carboxyl side of methionine residue - Length of peptide is crucial for sensitive detection of alteration of any single amino acid substitution, deletion and insertion - Optimal length of fragment : 7~ 14 amino acids - Selection of the most suitable fragmentation agent based on knowledge of the full amino acid sequence of the protein ex) Human growth hormone : 20 potential trypsin cleavage sites

32. Separation of fragments by using one- or 2-D gel electrophoresis • Comparison of the peptide fingerprint between standard sample and a product • Detection of single amino acid substitutions, deletions, insertions, modifications • Monitoring batch-to-batch consistency of the product • Confirmation of the identity of the actual product • :

33. Generation of peptide map

34. N-terminal sequencing - Sequencing of the first 20 -30 amino acid residues of the protein - Popular quality control test for finished products - Advantages - Positive identification of the final product - Confirms the accuracy the amino acid sequences of at least the N-terminus of the proteins - Identifies the presence of modified forms of the product one-or more amino acids are missing from the N-terminus - Sequencing : Edman degradation in 1950s : - Automated sequencing up to the first 100 aminoacid residues • C-terminal sequencing - Use of carboxypeptidase C : sequentially removes amino acids from the C-terminus - Sequencing of the first few amino acid residues

36. Pyrogenic contaminants • Pyrogens: - Substances influence hypothalamic regulation of body temperature, resulting in fever, when they enter the blood stream - Difficult to control the pyrogen-induced fever, leading to death - A wide range of pyrogens - Chemicals, particulate matter, endotoxin - Endotoxin : Lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria which harbour 3-4 million LPS molecules on their surface, accounting for 75 % of their membrane surface area - E. coli, Haemophilus influenzae, Salmonella enterica, Klebsiella pneumoniae, Pseudomonas aeruginosa etc.. - Typical structure of LPS : - Consists of a complex polysaccharide component linked to a lipid (lipid A) moiety - Pyrogenicity : lipid A moiety

37. - Entry into the blood stream stimulates the production of interleukin(IL-1) macrophages  IL-1 directly initiates the fever response • Minimization of contamination by pyrogens - Effective implementation of GMP - Filtration of all parenteral products through 0.4 and 0.2 um filter during processing and prior to filling in final product containers - Rinsing with WFI - Contamination of the final product with endotoxins - Use of Gram negative bacterial system : source of endotoxin - Heat stability of enbdotoxin : autoclaving of process equipment will not destroy endotoxins - Effective even at dosage rates as low as 0.5 ng /kg body weight

38. Pyrogen detection • Rabbit pyrogen test : the most widely used method - Parenteral administration of the product to a group of healthy rabbits - Subsequent monitoring of rabbit temperature using rectal probes - Increased rabbit temperature above a certain point : pyrogen - European Pharmacopoeia - Initial administration of the product to three rabbits - Measure the total (summed ) increase of the temperature - Less than 1.15 oC : pass - Greater than 2.65 oC : fail - Between two limits : inconclusive, repeated test using a further batch of animals

39. Disadvantages - Expensive : requirement of animals, animal facilities, animal maintenance - False-positive results due to poor handling, sub-clinical infection, poor health of rabbits - Variation due to different rabbit colonies / breeds • in vitro assay : Limulus amoebocyte lysate (LAL) test - Certain biopharmaceuticals, (e.g., cytokines like IL-1 and TNF), induce a natural pyrogenic response - Use of rabbit-based assay for detection of exogeneous pyrogens : not valid - Endotoxin-stimulated coagulation of amoebocyte lysate obtained from horseshoe crabs : - Most widely used assay for the detection of endotoxins

40. Advantages of the LAL over the rabbit test - Sensitivity : a few picograms (pg) / mL of sample to be assayed - Cost : far less expensive - Speed : finished between 15- 60 min - Can be used for detection in WFI and in biological fluids such as serum or cerebrospinal fluid • Microbial contamination - Presence of microorganisms in the final product - Severe infection in the recipient patients - Metabolizing the final product itself, reducing its potency during storage : extracellular proteases - Release of endotoxins - Sterilization of the final product by filtration with 0.22 um filter, followed by aseptic filling into a sterile final product container - cGMP guideline

41. Viral contamination - Mostly derived from raw material sources ex) HIV or Hepatitis viruses : blood used in the manufacture of blood products • Removal of viruses - Gel filtration chromatography : size of viral particles - Repeat filtration through a 0.1 um filter - Heating at 40-60 o C for several hrs : inactivation of a broad range of viruses including blood-borne viruses - Exposure to UV radiation : no adverse effect on the product itself

42. Viral assays - Immunoassays using specific antibodies - Use of virus-specific DNA probes - Bioassays : - Incubation of the final product with cell lines sensitive to a range of viruses - Monitoring for cytopathic effects or other obvious sign of viral infection - Antibody production tests with a range of mouse, rabbit or hamster - Analysis of the serum sample from test animal for the presence of antibodies recognizing a range of viral antigens

43. Validation studies - Act of proving that any procedure, process, equipment, material, activity or system leads to the expected results (specifications) - Assure the overall safety and efficacy of the final product - All validation procedures : carefully designed, fully documented in written format, documented, and retained in the plant files