protein stability and formulation bioseparation engineering
TRANSCRIPT
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Protein Stability and Formulation
Bioseparation Engineering
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Protein Formulation/Stability Test
Formulation:
→ Storage stability before use (1.5 ~ 2 years)
→ Add stabilizer and bulking agent
→ 0.22 μ filter (for sterilization)
→ Packing , or
→ Freeze drying (lyophilization) → powder packing
Stable Protein → liquid–form product
Unstable Protein → solid–form product
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Protein Formulation/Stability Test
Stabilizer:→ human serum albumin
→ amino acid
lowers glass wall attachment
• lowers lysozyme attachment to glass wall• lowers globulin aggregation
→ polyol (sorbitol, glycerol, mannitol)use for lyophilization
→ antioxidant, salt and surfactant
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Protein Stability: Model
N ↔ U → Iunfoldinginactivation
N – native (folded)U – unfoldedI – inactivated
where:
reversibleirreversible
Thermodynamic (conformational) stability
Long-term (kinetic) stability
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Protein Stability: Thermodynamics
fuu GGG Gibb’s Free Energy
relatively stable, when ∆Gu is big.
0mutantwild uu GGG
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Folding Stability Measurement
Optical
Aggregation
UVFluorescence
CD (circular dichroism)
Molecular Size Change
Net Charge Change
viscositylight scatteringturbidity
gel electrophoresisHPLC
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Stability: Experiment
Assume: A ↔ B (linear)
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Stability: ExperimentN ↔ U Equilibirum constant
N
UK f
ff KRTG ln
ΔG in the absence of denaturant
Can be estimated by molecular modeling
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Case study
Human Growth Hormone Ref : “Directed expression in Escherichia coli of a
DNA sequence coding for human growth hor-
mone”, Goeddel, D.V. et al., Nature 281:544 (1979)
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Structure
Tertiary structure of hGH 3D structure of pGH
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Characterization
Spectroscopy - UV absorption - CD (Circular dichroism) - Fluorescence
Electrophoresis - SDS-PAGE - IEF (Isoelectric focusing) gel electrophoresis
Immunoassays
Bioassays
Chromatographic methods - Reversed – phase HPLC - Size – exclusion chromatography - Ion - exchange chromatography
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Degradation
Deamidation :
Conversion of the side chain in aspargine and glutamine residues
to the carboxylate groups of aspartate and glutamate, respectively
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Degradation
Oxidation : Methionine, tryptophan, histidine and tyrosine residues
corresponding sulfoxide in methionine
Reduction / Interchange of disulfide bonds
Aggregation
Proteolysis / Hydrolysis
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Stability
Solution stability
Plot of the first – order rate constants in days for deamidation of hGH
in solution as a function of pH at 250C(•), 400C(■).
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Stability
Stability in solid state
Plot of the percent dimer, as measured by a size-exclusion HPLC assay, for freeze-dried samples of hGH, as a function of storage time at 400C
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Case study
Orthoclone OKT3 Therapeutic Monoclonal Antibody Ref : “Stability and Characterization of Protein
and Peptide Drugs : Case Histories”, edited by Wang and Pearlman, Plenum Press, New York (1993)
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Background
Orthoclone OKT3 : Marketed since 1986 for reveral of human kidney graft rejection
- Murine monoclonal antibody directed to a component of the antigen
receptor present on all mature, human T cells
- First mouse monoclonal antibody approved by FDA for human therapy
- Formulated for intravenous use as a 1mg/ml sterile solution in pH 7.0
phosphate buffered saline containing 0.02% polysorbate 80.
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Schematic diagram of mouse IgG2a with amino aicd numbering from OKT3
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Degradation
- Shift in isoelectric focussing (IEF) pattern
( Slight alterations in the charge of OKT3 as it aged ; acidic shift)
- Alteration in HPLC – IEC retention times
- Protein chain alteration detected by SDS-PAGE
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Degradation mechanism
* Multiple mechanism of degradation
- Acidic shift : deamidation of amino acids (glutamate, asparagine)
- Smaller molecular weight fragment : hydrolysis of peptide bonds
- Oxidation of labile amino acids
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Mechanism study
- Oxidative degradation : 5 methionine residues
- Storage at 50C both mechanism occur
Storage at elevated temperature more IEF changes (deamidation)
- Deamidation ( Asn – Gly, Asn – Ser segments)
- OKT3 is filled in ampules under nitrogen
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Chromatography clean-ing validation
Seoul National Uni-versitySchool of Chemical and Biological EngineeringJin Min
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INDEX
• Necessity of cleaning column• Contaminants• Removal of Impurities• Cleaning Validation• Analytic Methods
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Necessity of cleaning column
• Loss in capacity may occur due to non-specific bindings between col-umn packing and impurities
• Accumulation of contaminants can affect to the purity of products– affect column performance– contaminate subsequent runs– cause denaturation
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• Cleaning after each cycle prevents and minimizes fouling, and extends the lifetime of the medium
• Cleaning and sanitization helps en-sure the process produces a repro-ducible product of specified quality
• Suitable cleaning program should begin at the start of the development
Necessity of cleaning column
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Contaminants
• Unnecessary proteins, nucleic acids, and lipids
• Viruses• Bacteria• Yeast• Fungi• Prion• Endotoxin
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Removal of Impurities
• NaOH – virus, endotoxin, nucleic acids, proteins– O.5M NaOH for 30 min, at RT
• NaCl – nucleic acids, proteins– 3M NaCl
• Detergent – lipids, hydrophobic pro-teins
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Removal of Impurities
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Cleaning Validation
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Analytic Methods
• UV-Vis– Commonly used for detection of small
molecule active pharmaseutical ingredi-ents (APIs) or detergent residues
• (common UV wavelength – 210nm, 254nm)
– Benefits; not limited to water, quantita-tive results, fast spectral acquisition
– Drawbacks; lacks of peak separation, requires chromophore for specificity
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Analytic Methods
• Total Organic Carbon (TOC)– Specific to organic compounds and theoret-
ically measures all the covalently bonded carbon in water
– Benefits; acceptable way to detect residues of contaminants
– Drawbaks; considered a “worst case” anal-ysis (incorporates all organic molecules in solution and represents surface area), sam-ples must be water soluble, excellent water quality needed, lacks of specificity
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Analytic Methods
• GC / MS– Used for detection of detergent residue– Benefits; improved peak shape due to
capillary column usage, provides sepa-ration, identification, and quantitation of results
– Drawbacks; samples require vaporiza-tion
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Analytic Methods
• HPLC (High Performance Liquid Chro-matography)– Used for detection of APIs or detergent
residues– Benefits; not limited to water, possibility
of identification of specific peaks of inter-est and quantitative results, multiple de-tection options (UV, fluorescence, etc.)
– Drawbacks; requires more time and in-formation about the excipients, expensive
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Analytic Methods
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Provide some economic data on a technique that effectively separates relatively
large amounts of monoclonal antibodies.Recovery of therapeutic-grade of antibodies: Protein A and ion exchange chromatography
(Duffy et al, 1989)
Example 8.7
2011. 11. 23Choi Wonji
Bioseparation Engineering2011-2 Prof. Young Je Yoo
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Ion exchange chromatography
Use charge-charge interaction
S-sepharose : cation exchange chromatography
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Protein A chromatgraphy
Bead: CNBr-activated sepharose
IgGSepharose
Protein A: Staphylocuccus aureus Protein, binding affinity for Fc region of IgG monoclonal IgG, polyclonal IgG subclasses, serum proteins
Elusion: acidic buffer, denature the proteins
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General procedure
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Experimental details
• After pretreatment by the ultrafiltration unit the ioad of the antibody to the column was close to 100mg
Feed material greater than 1000 L feed
“Pre-concentration” step; need to minimize the denaturation of the antibodies
Ultrafiltration system; permitted a 50- to 100- fold concentra-
tion of the feed material
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Results and Conclusion
Ion-exchange Chromatography Protein A Chromatography
53 $/gram 217 $/gram
Does not co-purify other Ig Fails in removing some contaminants
IEC technique is more cheaper than the protein A chromatography technique
IEC is free of contaminating immunoglobulins may cause undesirable reations
But IEC very specific method to each protein, needs to be developed in each application
Whereas, protein A chromatography is generic method & applied to a wide variety of Abs.
The cost of 10 cycles1/4
Various application immunoadsorption in biomedical area even though high cost