1. Protein Stability and Formulation Bioseparation Engineering

2. 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

3. 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

4. Protein Stability: Model N ↔ U → I unfoldinginactivation N – native (folded) U – unfolded I – inactivated where: reversible irreversible Thermodynamic (conformational) stability Long-term (kinetic) stability

5. Protein Stability: Thermodynamics Gibb’s Free Energy relatively stable, when ∆G u is big.

6. Folding Stability Measurement Optical Aggregation UV Fluorescence CD (circular dichroism) Molecular Size Change Net Charge Change viscosity light scattering turbidity gel electrophoresis HPLC

7. Stability: Experiment Assume: A ↔ B (linear)

8. Stability: Experiment N ↔ U Equilibirum constant ΔG in the absence of denaturant Can be estimated by molecular modeling

9. 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 281:544 (1979)

10. Structure Tertiary structure of hGH 3D structure of pGH

11. 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

12. Degradation  Deamidation : Conversion of the side chain in aspargine and glutamine residues to the carboxylate groups of aspartate and glutamate, respectively

13. Degradation  Oxidation : Methionine, tryptophan, histidine and tyrosine residues  corresponding sulfoxide in methionine  Reduction / Interchange of disulfide bonds  Aggregation  Proteolysis / Hydrolysis

14. Stability  Solution stability Plot of the first – order rate constants in days for deamidation of hGH in solution as a function of pH at 25 0 C( ), 40 0 C( ■ ).

15. 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 40 0 C

17. 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)

18. 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.

19. Schematic diagram of mouse IgG2a with amino aicd numbering from OKT3

20. 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

21. 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

22. Mechanism study - Oxidative degradation : 5 methionine residues - Storage at 5 0 C  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@snu.ac.kr

23. Chromatography cleaning validation Seoul National University School of Chemical and Biological Engineering Jin Min

24. INDEX Necessity of cleaning column Contaminants Removal of Impurities Cleaning Validation Analytic Methods

25. 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

26. 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 Necessity of cleaning column

28. Contaminants Unnecessary proteins, nucleic acids, and lipids Viruses Bacteria Yeast Fungi Prion Endotoxin

29. 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

30. Removal of Impurities

31. Cleaning Validation

33. 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

34. 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

35. 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

36. 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

37. Analytic Methods

38. 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. 23 Choi Wonji Bioseparation Engineering 2011-2 Prof. Young Je Yoo

39. Ion exchange chromatography  Use charge-charge interaction  S-sepharose : cation exchange chromatography

40. Protein A chromatgraphy  Bead: CNBr-activated sepharose IgG 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

41. General procedure

42. 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 concentration of the feed material

43. Results and Conclusion Ion-exchange ChromatographyProtein A Chromatography 53 $/gram 217 $/gram Does not co-purify other IgFails 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 cycles 1/4 Various application immunoadsorption in biomedical area even though high cost