High Pressure Disaggregation and Refolding of Proteins

1. High Pressure Disaggregation and Refolding of Proteins Ted Randolph, Matt Seefeldt, Jon Webb, Rick St. John, Yongsung Kim, Ryan Crisman, Amber Haynes, John Carpenter Center for Pharmaceutical Biotechnology Department of Chemical and Biological Engineering University of Colorado

2. Protein Therapeutics • 125 biotechnology-based medicines on the market* Out of more than 2700 drugs in clinical or later development*: • 418 new biotechnology-based medicines are currently in testing*, most of which are proteins *Pharmaceutical Research and Manufacturers of America, 2006

3. Therapeutic Proteins: Ripe for Engineering Progress • Offer remarkable new treatments for cancer, AIDS/HIV, autoimmune disorders, digestive disorders, blood disorders… • But there are number of important challenges that need to be addressed to allow more widespread use- many of which require engineering solutions.

4. Some Challenges of Protein Therapeutics • Cost • Example: human growth hormone • Retail price: $50/mg (yes, that’s $50M/kg!!!) • Typical dose size: 0.3 mg/kg/week- for a 20 kg pediatric patient a year’s treatment retails at $20,000 • Cost to develop new drug: $802,000,000* • Cost to develop a new protein drug $1,240,000,000 • Safety • Immune response • Other adverse effects • Regulatory • Regulatory environment lead to conservative approach to process changes/improvements *J. A. DiMasi, R. W. Hansen and H. G. Grabowski, Journal of Health Economics 22 (2003): 151-185.

5. Some contributors to high costs: • Process yields are low • Processes are inefficient • Products are unstable • Testing is expensive • Regulatory burden is high • Long development times

6. To become a therapeutic product… • Protein must be produced in a form that is chemically pure • Protein must be produced in a form that is conformationally pure (properly folded) • Protein must be produced in the correct assembly state (monomeric, dimeric, etc.) • Protein must remain so for duration of its labeled shelf life (typically two years)

7. An unfortunate start • Proteins are synthesized within cells as linear polymers • Polymers must “fold” to achieve correct 3D structure that imparts biological activity • Incorrect folds typically show (greatly) reduced biological activity, and may be toxic • Human proteins synthesized in lower organisms frequently misfold and aggregate

8. Protein Folding- A Bottleneck Early in the Process To become functionally active, proteins must fold correctly form a disordered state to the highly ordered native state If all goes well, unfolded protein molecules fold through a biased walk as they concomitantly lower their free energy and reduce the number of available conformations- “sliding down the folding funnel” (Dill et al, Proteins, 1998)

9. But… Partially folded protein intermediate states are often very “sticky” These intermediates may assemble in “off-pathway” reactions to form aggregates Aggregates are biologically inactive, and must be disaggregated and then folded to become active • Radford S., “Protein folding: Progress made and promises ahead”, Trends in Biochemical Sciences, V25, 611-618, 2000.

10. Traditional Chaotrope-Based Refolding Methods • Aggregates are dissolved in large amounts of chaotropic solvents • Chaotropes removed by diafiltration • Low protein concentrations used to favor folding over re-aggregation • Overall yields often 10-50% • Multi-day process Collect, wash, concentrate aggregates fermentation Add Guanidinium HCl Dissolve aggregates in chaotrope Buffer exchange by dilution, Ultrafiltration/Diafiltration to effect refolding

11. Unfortunately, most of our valuable product ends up as useless aggregate

12. Disadvantages of Current Methods • Low Yields • Capital cost • Guandine incompatible with 316SS • Guanidine interferes with Ion Exchange Chromatography- extensive dialysis required • Product dilute • Waste handling costs • Slow

13. Intermediates on Folding Pathway • Under atmospheric conditions, folding intermediates: • Exhibit attractive protein-protein interactions- “sticky” • Self-associate to form aggregated species • Slow down folding • Reduce yields

14. What drives protein aggregation? • Non-native conformations of proteins such as partially unfolded molecules more reactive • Hydrophobic effect causes protein-protein interactions to be attractive

15. To understand this, we’ll have to dive into thermodynamics

16. But it’s really not that unfriendly

17. Second osmotic virial coefficient describes protein-protein interactions B22 characterizes the overall two-body interactions between proteins where U(r) is the overall protein-protein interaction potential: Hard sphere - excluded volume Electrostatic - charge-charge van der Waals - charge-dipole, dipole-dipole, dispersion Osmotic - ion excluded volume Association - interaction to account for protein association Solvation - hydration and hydrophobic forces B22 > 0, repulsive interactions B22< 0, attractive interactions We anticipate that systems with negative (attractive) B22 values will be more prone to assemble into aggregates than those with positive B22 values

18. In the presence of ~1-2 M GuanidineHCl, B22 values for lysozyme show a minimum, causing the protein to aggregate during refolding Data at 1 bar: Liu, W., T. Cellmer, et al. (2005). Biotechnology and Bioengineering90(4): 482-490.

19. Is there a way around this? • One way of influencing hydrophobic effects is by manipulating the system pressure • Studies dating back nearly 100 years have shown that high pressures can so drastically alter hydrophobic effects as to cause proteins to unfold

20. P-T Stability Boundaries

21. Protein Unfolding in P-T space Hawley, 1971, Biochemistry 10, 2436-2442 (chymotrypsinogen)

22. Protein Folding “Pressure Window” • Multimeric proteins dissociate @1-3 kbar • Monomeric proteins unfold @ >5 kbar • Aggregates may be thought of as ill-defined “multimers” • In “window” between ca. 1-5 kbar, pressure should dissociate aggregated state, while still favoring native conformation for monomers

23. Pressure Window

24. A new process for folding proteins • Take aggregated protein, pressurize to dissolve aggregates • Reduce pressure to point where native conformation is favored, but aggregation is disfavored • Allow to refold, then reduce pressure

25. The experiment • Boil egg for 14 minutes • Remove aliquots of polymerized egg white • Refold under pressure • Aggregated protein at 2 mg/ml • Add Disulfide-Shuffling Agents: 4mM glutathione, 2 mM dithiothreitol • Pressurize at 400 MPa, 25°C • Depressurize • Test for Lysozyme Activity, measure soluble protein (size exclusion chromatography) • Compare with “conventional” refolding • Solubilize 2 mg/ml protein in 6M guanidine, 4mM glutathione, 2 mM dithiothreitol • Dilute to 0.5M guanidine • Test for Lysozyme Activity, measure soluble protein (size exclusion chromatography) http://www.aeb.org/recipes/basics/hard-cooked_eggs.htm

26. The result- an egg unboiled! • High-Pressure Process: • 25 % of starting protein recovered as soluble protein • Lysozyme activity recovered • Conventional process: • Negligible protein soluble • Negligible lysozyme activity recovered

27. Example I Human Growth Hormone • Monomeric protein • Aggregates easily, especially at surfaces • High thermodynamic stability of native conformation • Strategy: Single high pressure step for aggregate dissolution, protein refolding

28. Agitation-Induced Aggregation of rhGH • rhGH aggregates nearly quantitatively after 24 hours of mild agitation • Aggregates are irreversible at 1 atm, 25 C • Aggregates formed by agitation in citrate buffer or citrate buffer with 0.75 M guanidine

29. Structure of agitation-induced rhGH aggregates (FTIR)

30. 4th derivative UV @284 nm shows nativestate of rhGH is stable to >4500 bar

31. rhGH Fluorescence as Function of Pressure Shows Native State is Stable to >6500 bar

32. Refolding of human growth hormone from agitation-induced aggregates: aggregated states destabilized under pressure

33. Protein refolding is independent of protein concentration

34. Kinetics of rhGH aggregate dissolution at 2000 bar Dissolution time constants 4.8 and 10 hours

35. Kinetics of rhGH refolding @ 2000 bar Refolding time constant = 3.2 hours

36. Example: Disaggregation and Folding from Aggregates of Interferon-g (IFN-g) • Protein is dimeric in its native state • Aggregates easily • Strategy: High pressure to dissolve aggregates; moderate pressure to refold → Choose operating points based on equilibrium unfolding as f(P)

37. Pressure Effects on Equilibria

38. IFN-g Dissociation

39. IFN-g UV Spectra as f(P)

40. 4th Derivative UV Spectra of IFN-g as f(Pressure) Convert UV data to fraction protein folded as f(P) Calculate folding equilibrium constants Calculate

41. Partial Molar Volume Change of IFN-g Dissociation, 40oC

42. IFN-g: Elliptical stability diagram generated from pressure-induced dissociated data used to choose process operating points AggregateDissolution Conditions Refolding Conditions

43. Aggregate Dissolution at 2500 bar

44. Refolding Rate at 100 MPa

45. Monomer-Dimer Equilibrium Re-established from γ-Interferon Aggregates Aggregates ~650kDa Dimers Monomers Red – Size distribution before pressure Black – after high pressure treatment

46. At high pressures, aggregation is suppressed- why? • High pressures generally conformationally destabilize proteins, leading to higher populations of molecules with non-native conformations. Why doesn’t this accelerate aggregation? • How does pressure affect protein-protein interactions? Aim: Explore the interplay between conformational and colloidal stability as a function of pressure- what causes the “pressure window”?

47. Hydrophilic Surface, Low P Hydrophobic Surface, Low P Hydrophilic Surface, 2 kbar Hydrophobic Surface, 2 kbar Giovambattista, Debenedetti1, and Rossky, J. Phys. Chem. B.

48. 5 ) 2 4 (ml mol/g 3 1000 bar 2 Liu et al. 3 *10 1 (atmospheric) 22 B 0 -1 0 2 4 6 GdnHCl Concentration [M] At 1 kbar, protein-protein interactions for HEW lysozyme are repulsive during folding- in contrast to folding at atmospheric pressure! Data at 1 bar: Liu, W., T. Cellmer, et al. (2005). Biotechnology and Bioengineering90(4): 482-490.

49. Model system: T4 lysozyme variants exhibit widely varying conformational stabilities, but nearly identical folds (see Matthews et al., Sathish, et al.)

50. Pressure makes intermolecular interactions more repulsive T4 Lysozyme L99A/A130S at 1 bar (solid symbols) and 1kbar (open symbols)