Protein Formulation/Stability Test. Formulation: Storage

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Bioseparation Engineering
Protein Stability and Formulation
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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 ? liquidform product
Unstable Protein ? solidform product
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Protein Formulation/Stability Test
Stabilizer
? human serum albumin
lowers glass wall attachment
? amino acid

• 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
unfolding
inactivation
N ? U ? I
reversible
irreversible
Thermodynamic (conformational) stability
Long-term (kinetic) stability
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Protein Stability Thermodynamics
Gibbs Free Energy
relatively stable, when ?Gu is big.
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Folding Stability Measurement
UV
Optical
Fluorescence
CD (circular dichroism)
viscosity
Molecular Size Change
light scattering
turbidity
Aggregation
Net Charge Change
gel electrophoresis
HPLC
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Stability Experiment
Assume A ? B (linear)
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Stability Experiment
N ? U
Equilibirum constant
?G 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
  hormone,
• Goeddel, D.V. et al., Nature
  281544 (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
songvic_at_snu.ac.kr
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Chromatography cleaning validation
Seoul National University School of Chemical and
Biological Engineering Jin 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 column 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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Necessity of cleaning column

• Cleaning after each cycle prevents and minimizes
  fouling, and extends the lifetime of the medium
• Cleaning and sanitization helps ensure the
  process produces a reproducible product of
  specified quality
• Suitable cleaning program should begin at the
  start of the development

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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 proteins

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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 ingredients (APIs) or
  detergent residues
• (common UV wavelength 210nm, 254nm)
• Benefits not limited to water, quantitative
  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 theoretically
  measures all the covalently bonded carbon in
  water
• Benefits acceptable way to detect residues of
  contaminants
• Drawbaks considered a worst case analysis
  (incorporates all organic molecules in solution
  and represents surface area), samples 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 separation,
  identification, and quantitation of results
• Drawbacks samples require vaporization

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Analytic Methods

• HPLC (High Performance Liquid Chromatography)
• Used for detection of APIs or detergent residues
• Benefits not limited to water, possibility of
  identification of specific peaks of interest and
  quantitative results, multiple detection options
  (UV, fluorescence, etc.)
• Drawbacks requires more time and information
  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)
Bioseparation Engineering
2011-2 Prof. Young Je Yoo
Example 8.7
2011. 11. 23 Choi Wonji
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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

• 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

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Results and Conclusion

• 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.