Transcript Production of final product

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)
• 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
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 : labscale 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
• 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
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
- 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
- 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
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
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
• 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
•
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
• 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
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
• 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 :
-
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
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)
- 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
• 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
• Mode of sampling methods
- Continuous
- Discontinuous
• Types of 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.
• 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)
• 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 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
•
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
• Determination of protein concentration :
- Quantification of total protein in the final product : 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
• 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
• 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, 1224 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
- 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
-
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
-
:
Generation of peptide map
• 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 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 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
- 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
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
• 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
•
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
• 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
- 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