Bioseparation of final product General procedure for production of biopharmaceuticals using recombinant prokaryotic or eukaryotic system - 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 for final product : purity and yield - Analysis of final product (QC) - Formulation & packaging Upstream processing : Cell cultivation processes resulting in the initial generation of a target product Downstream processing :Cell harvesting, purification of a target product, and generation of finished product format ( i.e., filling into its final product containers, freeze-drying if if a dried product format is required), followed by sealing of the final product containers

Cell banking systems : Ensure essentially indefinite supply of the originally developed production cell line for manufacturing purposes - cryo-preserved ampoules of constructed cell line - reproducible and consistent production

Preparation of cell banking system : initial cultivation of production cell line The resulting cell line is aliquoted into small volume in ampoules, immersed in liquid nitrogen - The content of all the ampoules is identical, and the cells are effectively preserved for indefinite periods

A master cell bank : - Constructed first from a culture of the newly constructed production cell line several hundred individually stored ampoules used to generate a working cell bank

A working cell bank : - A working cell bank is constructed from a single ampoule from a master cell bank - Directly used for a single production run - Each ampoule is thawed and used for a new batch - When all the vials of first working cell bank are exhausted, a second vial of the master cell bank is used to generate a second working cell bank

Upstream processingInitial 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 - Harvest of the crude product Downstream processing Choice of media - Exact nutrient requirements of producer cell line to maximize cell growth and product - Economics (total media cost) - Extracellular or intracellular nature of product In the case of intracellular product, the less complex media composition, the better for subsequent product purification process

Optimal culture conditions : temp, pH, Dissolved oxygen level etc.

Cell culture process : - Industrial scale microbial culture process : Bioreactor configuration, oxygen transfer, mode of operation(batch, fed-batch, continuous), heat removal (microbial metabolism and mechanical friction), mixing/agitation etc.. - Mammalian cell culture system - More technically complex and expensive - Long culture time due to slow growth rate - Only used for production of therapeutic proteins requiring extensive post-translational modifications like glycosylation

Outline of the upstream processing stages involved in the production of a single batch of product : - Gradual scale-up : 100 mL 5 L 30 L 1,000L

Downstream processingMost critical step for economic production Feasibility High purity and high yield - High purity is obtained at the expense of yield : ~ 1 / (yield at each step )N N: number of separation stepsDetailed process : highly confidentialNormally undertaken under clean room conditions - Final step under Grade A laminar flow condition First recovery step : Intra- or extra-cellular product - 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 A suspension of cells is forced under high pressure through an orifice of narrow internal diameter High shear force High pressure drop at the outlet to atmospheric pressure Rupture of most microbial cells

- 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 under applied pressure

- Popular method for concentration - High product recovery rate - High speed - Process-scale ultra-filtration equipment is available

High resolution chromatographic purification after concentration : - Different methods based on various physiochemical characteristics : - Gel filtration, ion-exchange, and affinity chromatograph (immuno-affinity) : most commonly used - Hydrophobic interaction chromatograph - Yields around 98-99 % purity of final target protein

Final product formulationFormulation 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

Factors affecting the biological activity of proteinsLoss of biological activity of protein by various molecular alterations: - Non-covalent alterations : partial/complete protein denaturation - Covalent alterations Hydrolysis, deamidation, imine formation, oxidation, disulfide 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

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 - Micro-heterogeniety in glycoproteins : Variation in the composition and structure of carbohydrates : Visualized by iso-electric focusing

Stabilizing excipientsA range of various substances: added to a purified biopharmaceutical product for stabilization : - Addition of human serum albumin : cf) Blood-born pathogens or virus - Addition of amino acids : - Addition of 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) Surfactant reduces the surface tension of the aqueous solutions Increases the solubility of proteins Reduces the rate of protein denaturation at interfaces

Final product fill - Final product undergoes QC testing to ensure its compliance with product specifications - Filtration using 0.2 um filter for sterile product - aseptically filled into pre-sterilized final product containers

Freeze-drying - removal of water directly from frozen products via lyophilization powdered product reduce the deactivation of protein by chemical/biological mechanism longer shelf-life

Labeling and packing

Analysis of the final productRigorous QC testing : To confirm the pre-determined specifications of the final productPotency testing : efficacySafety test : Analysis of various potential contaminants - Range and medical significance of potential impurities : Microorganisms, viral particles, pyrogenic substances, DNA, proteins - 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

Removal of altered forms of the protein productAltered 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 : - Methods used to characterize protein biopharmaceuticals :

Removal using 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

Gel filtration chromatography : difference in size/shape of proteins - Removal of aggregated, proteolysed and deglycosylated forms of 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

Product potencyFinished biopharmaceuticals should meet product potency specifications : Internationally accepted standard preparations - WHO (world health organization) or US pharmacopoeia - Expressed in terms of units of activity per vial of product (or per therapeutic dose, or per mg product)

Bioassays : the most widely used potency-determining assay - Directly assess the biological activity of the products 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)

- 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 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 - Viable cells can assimilate certain dyes like neutral red - Spectrophotometric quantification of assimilated dye after its addition - Assay in a single well of a microtitre plate Facilitate automated assay of a large number of samples with ease

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 mol min-1 - Specific activity : the activity of an enzyme per milligram of total protein (expressed in mol min-1mg-1). Measurement of the purity of the enzyme.

Types of assay - Initial rate measurement - Progress curve experiments : kinetic parameters are determined - Transient kinetics experiments : reaction behavior 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

Mode of sampling methods - Continuous - Discontinuous

Types of measurements - Colorimetric assays - Fluorimetric - Chemiluminescent : 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.

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 : the most popular alternative assay systems instead of bioassay - 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)

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

- 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)

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 PhosphataseAutomated measurement using a 96 well plate

Disadvantages of EIA and ELISA - 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

Determination of protein concentration : - Quantification of total protein in the final product : standard analysis undertaken by QC - Many methods are available : Biuret method, Lowry method, Bradford method - 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

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 - the 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

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 Binding of the antibody to the contaminant bands Existence of product variants (differently glycosylated, proteolytic fragments etc..)

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

ex) Glycoproteins with slightly different carbohydrate content : Variation in their sialic acid content different pI values Analysis of protein variant with a different property like surface charge cf) pI : pH at which the protein exhibits no overall net charge

High-pressure liquid chromatography (HPLC)Fast Protein Liquid Chromatography(FPLC)

A central analytical tool for assessing the purity of low molecular mass pharmaceutical substances as well as proteinsTypical features - Excellent fractionation speeds under high pressure - Superior peak resolution - High degree of automation - Commercial availability of various sophisticated systems

Types of HPLC - Reverse-phase HPLC(RP- HPLC) : Separates proteins on the basis of differences in their surface hydrophobicity - Stationary phase : silica or polymeric support with hydrophobic arms ( usually alkyl chains like butyl, octyl, or octadecyl groups) - Mobile phase : a mixture of various solvents including water - Very powerful analytical techniques, capable of separating very similar molecules with only minor differences in hydrophobicity

- Analysis of protein with a single amino acid substitution or damidation Ex) analysis of modified forms or insulin polymers - Despite the analytical usefulness, the highly hydrophobic stationary phase can induce irreversible protein denaturation

Size-exclusion HPLC : Separation of proteins on the basis of size and shape - Stationary phase : Silica-based supports and cross-linked agarose of defined pore size - Most often used to analyze product for the presence of higher molecular mass aggregates or proteolysed fragments - Used for determination of molecular mass

Ion-exchange HPLC (both cation and anion): Separates proteins on the basis of surface charge of protein - Analysis of protein impurities and deamidated forms

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 : separates the constituent amino acids using ion-exchange chromatography - Comparison with the expected (theoretical) values

Peptide mapping : Peptide fingerprint - A major concern relating to therapeutic proteins in recombinant cells : Occurrence of point mutations in the products gene during gene manipulation, transcription, translation - Detection of occurrence of point mutations in the gene Full sequencing of a target protein at each production batch Tedious and time consuming

- Most commonly used method to detect alterations in amino acid sequence

Fragmentation of a target protein into small peptides : Critical to the success of this process 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 - Generation of a few very large peptides : difficult to detect amino acid alterations - Generation of a large number of very short peptides : difficult to resolve all the peptides - 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

Generation of peptide map : Separation of fragments by using one- or 2-D gel electrophoresis 2D separation resolves each peptide fragment from the others

Comparison of the peptide fingerprint between standardized product preparation and each batch of product

Detection of single amino acid substitutions, deletions, insertions, modifications

Monitoring batch-to-batch consistency of the productConfirmation of the identity of the actual product

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 - Advanced, automated sequencing up to the first 100 amino acid residues

C-terminal sequencing - Use of carboxypeptidase C : sequentially removes amino acids from the C-terminus - Sequencing of the first few amino acid residues

Pyrogenic contaminantsPyrogens: - Substances influencing 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 - Constitutes the major structural component of the outer membrane - Consists of a complex polysaccharide (~ 50 monosaccharide) component linked to a lipid (lipid A) moiety - Pyrogenicity : lipid A moiety

- 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 is difficult to control because : - the use of Gram negative bacterial system for the production of biopharmaceuticals : source of endotoxin - heat stability of enbdotoxins : autoclaving of process equipment will not destroy endotoxins - effective even at dosage rates as low as 0.5 ng /kg body weight

Pyrogen detectionRabbit 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

- 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 : The presence of endotoxin causes stepwise sequential activation of various clotting factors within the amoebocyres, resulting in the generation of the polypeptide fragment coagulin, which polymerizes , forming a gel or clot : - Currently the most widely used assay for the detection of endotoxins

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 - the presence of microorganisms in the final product - a 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 - GMP guideline

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

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

Validation studies

- the act of proving that any procedure, process, equipment, material, activity or system leads to the expected results (specifications) - routine inspection of a entire manufacturing process and facility - Routine and adequate validation studies : core principle of GMP to 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 - All old and new items of equipments - Periodic validation of clean room - Aseptic filling validation - Contaminant-clearance validation