Protein Stability and Formulation Bioseparation Engineering.

Protein Stability and Formulation

Bioseparation Engineering

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

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

Protein Stability: Model

N ↔ U → Iunfoldinginactivation

N – native (folded)U – unfoldedI – inactivated

where:

reversibleirreversible

Thermodynamic (conformational) stability

Long-term (kinetic) stability

Protein Stability: Thermodynamics

fuu GGG Gibb’s Free Energy

relatively stable, when ∆Gu is big.

0mutantwild uu GGG

Folding Stability Measurement

Optical

Aggregation

UVFluorescence

CD (circular dichroism)

Molecular Size Change

Net Charge Change

viscositylight scatteringturbidity

gel electrophoresisHPLC

Stability: Experiment

Assume: A ↔ B (linear)

Stability: ExperimentN ↔ U Equilibirum constant

N

UK f

ff KRTG ln

ΔG in the absence of denaturant

Can be estimated by molecular modeling

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)

Structure

Tertiary structure of hGH 3D structure of pGH

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

Degradation

Deamidation :

Conversion of the side chain in aspargine and glutamine residues

to the carboxylate groups of aspartate and glutamate, respectively

Degradation

Oxidation : Methionine, tryptophan, histidine and tyrosine residues

corresponding sulfoxide in methionine

Reduction / Interchange of disulfide bonds

Aggregation

Proteolysis / Hydrolysis

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(■).

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

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)

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.

Schematic diagram of mouse IgG2a with amino aicd numbering from OKT3

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

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

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

INDEX

• Necessity of cleaning column• Contaminants• Removal of Impurities• Cleaning Validation• Analytic Methods

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

• 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

Contaminants

• Unnecessary proteins, nucleic acids, and lipids

• Viruses• Bacteria• Yeast• Fungi• Prion• Endotoxin

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

Removal of Impurities

Cleaning Validation

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

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

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

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

Analytic Methods

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

Ion exchange chromatography

Use charge-charge interaction

S-sepharose : cation exchange chromatography

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

General procedure

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

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