Transcript Production of final product

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 processing
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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
- Harvest of the crude product
 Downstream processing
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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, fedbatch, 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 processing
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Most 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 steps
Detailed process : highly confidential
Normally 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
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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
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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 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 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 excipients
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A 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 product
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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 :
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 product
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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 :
- Methods used to characterize protein biopharmaceuticals :
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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
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Gel filtration chromatography : difference in size/shape of proteins
- Removal of aggregated, proteolysed and deglycosylated forms of proteins
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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
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Hydrophobic interaction chromatography : Difference in the size and
extent of hydrophobic patches on the protein surface
- Similarly used as ion-exchange chromatography
Product potency
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Finished 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)
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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 μmol min-1
- 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.
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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
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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
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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 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
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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)
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A central analytical tool for assessing the purity of low molecular mass
pharmaceutical substances as well as proteins
Typical 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 ionexchange 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 product’s 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
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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 product
- Confirmation 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 contaminants
• Pyrogens:
- 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
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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 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 :
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