Bioseparation and analysis with upstreaming and down streaming process abcdefgh

1. 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

2. 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

3. • 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

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
- 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

5. • 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

6. Downstream processing
• 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

7. - 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

8. - 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

9. 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

10. 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

11. • 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

12. Stabilizing excipients
• 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

13. • 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

14. 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 :
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

15. Removal of altered forms of the protein product
• 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 :
- 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

16. • 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

17. Product potency
• 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)
• 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)

18. - 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

19. • 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.
• 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

20. • 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.

21. • 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)

22. • 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

23. - 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)

24. • 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 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

26. • 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

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
- 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

28. • 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

29. 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 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

30. - 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

31. • 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 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

32. • 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

33. - 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

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
- 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

35. 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

36. - 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

37. 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

38. - 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

39. • 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

40. • 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

41. • 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

42. • 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