Starting with a fighting chance: Happy protein Shane Seabrook PEW - PowerPoint PPT Presentation

Starting with a fighting chance: Happy protein Shane Seabrook – PEW 2014 #PEW2014 @CSIROC3 @CSIROnews

CSIRO FMF Biophysics Group  Structural Biology Campaigns  Small Molecule Therapy Development  Biotherapy Formulation Development  Antibody Screening  Mechanistic Biology+Nano Particle Interactions  Bio-remediation, bio-cataysis & bio-fuels

Topics • Protein Formulation • Stability • Aggregation

Why are you so unhappy , Protein? 1. Expression 2. Purification 3. Crystallisation 4. Structure Protein Production Protein Crystallization

Challenge: Happy Protein 1. Obtaining a population of identical protein – Same shape (fold) – Same activity 2. Keeping that population stable (folded/active)

Goal: Uniformity

Building insight 1. Use a different construct. 2. Change the expression/purification process. OR 3a. Alter the solvent surrounding your protein. 3b. Use ‘binders’ to tighten the structure. ...providing armour and weaponry; giving it a fighting chance!

Formulations

What is a Formulation • A mixture of two of more chemicals – Buffer – Salt – Additive  Subtle changes in the formulation chemistry can have a significant effect on the behaviour / stability of your protein Learn the chemistry that makes your protein happy

Protein stability ƒ (formulation) ...strongly affected by the formulation... Seabrook, S. A.; Newman, J., High-Throughput Thermal Scanning for Protein Stability: Making a Good Technique More Robust. ACS Combinatorial Science 2013. 1. Crowther, G. J.; He, P.; Rodenbough, P. P.; Thomas, A. P.; Kovzun, K. V.; Leibly, D. J.; Bhandari, J.; Castaneda, L. J.; Hol, W. G. J.; Gelb, M. H.; Napuli, A. J.; Van Voorhis, W. C., Use of thermal melt curves to assess the quality of enzyme preparations. Analytical Biochemistry 2010, 399 (2), 268-275. 2. Crowther, G. J.; Napuli, A. J.; Thomas, A. P.; Chung, D. J.; Kovzun, K. V.; Leibly, D. J.; Castaneda, L. J.; Bhandari, J.; Damman, C. J.; Hui, R.; Hol, W. G. J.; Buckner, F. S.; Verlinde, C.; Zhang, Z. S.; Fan, E. K.; Van Voorhis, W. C., Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands. Journal of Biomolecular Screening 2009, 14 (6), 700-707. 3. Dupeux, F.; Rower, M.; Seroul, G.; Blot, D.; Marquez, J. A., A thermal stability assay can help to estimate the crystallization likelihood of biological samples. Acta Crystallographica Section D 2011, 67 (11), 915-919. 4. Ericsson, U. B.; Hallberg, B. M.; DeTitta, G. T.; Dekker, N.; Nordlund, P., Thermofluor-based high-throughput stability optimization of proteins for structural studies. Analytical Biochemistry 2006, 357 (2), 289-298. 5. Falconer, R. J.; Marangon, M.; Van Sluyter, S. C.; Neilson, K. A.; Chan, C.; Waters, E. J., Thermal Stability of Thaumatin-Like Protein, Chitinase, and Invertase Isolated from Sauvignon blanc and Semillon Juice and Their Role in Haze Formation in Wine. Journal of Agricultural and Food Chemistry 2009, 58 (2), 975-980. 6. Froese, D. S.; Healy, S.; McDonald, M.; Kochan, G.; Oppermann, U.; Niesen, F. H.; Gravel, R. A., Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder. Molecular Genetics and Metabolism 2010, 100 (1), 29-36. 7. Gast, K.; Damaschun, G.; Misselwitz, R.; Zirwer, D., Application of dynamic light scattering to studies of protein folding kinetics. European Biophysics Journal 1992, 21 (5), 357-362. 8. Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R. M. M., Hydrogen Ion Buffers for Biological Research*. Biochemistry 1966, 5 (2), 467-477. 9. He, F.; Becker, G. W.; Litowski, J. R.; Narhi, L. O.; Brems, D. N.; Razinkov, V. I., High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions. Analytical Biochemistry 2010, 399 (1), 141-143. 10.He, F.; Hogan, S.; Latypov, R. F.; Narhi, L. O.; Razinkov, V. I., High Throughput Thermostability Screening of Monoclonal Antibody Formulations. Journal of Pharmaceutical Sciences 2010, 99 (4), 1707-1720. 11.Layton, C. J.; Hellinga, H. W., Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes. Biochemistry 2010, 49 (51), 10831-10841. 12.Niesen, F. H.; Berglund, H.; Vedadi, M., The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols 2007, 2 (9), 2212-2221. 13.Pantoliano, M. W.; Petrella, E. C.; Kwasnoski, J. D.; Lobanov, V. S.; Myslik, J.; Graf, E.; Carver, T.; Asel, E.; Springer, B. A.; Lane, P.; Salemme, F. R., High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery. Journal of Biomolecular Screening 2001, 6 (6), 429-440. 14.Parkins, D. A.; Lashmar, U. T., The formulation of biopharmaceutical products. Pharmaceutical Science and Technology Today 2000, 3 (4), 129-137. 15.Phillips, K.; de la Pena, A. H., The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Curr Protoc Mol Biol 2011, Chapter 10, Unit10 28. 16.Privé, G. G., Detergents for the stabilization and crystallization of membrane proteins. Methods 2007, 41 (4), 388-397. 17.Santoro, M. M.; Bolen, D. W., Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl .alpha.-chymotrypsin using different denaturants. Biochemistry 1988, 27 (21), 8063-8068. 18.Santoro, M. M.; Liu, Y.; Khan, S. M. A.; Hou, L. X.; Bolen, D. W., Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry 1992, 31 (23), 5278-5283. 19.Senisterra, G. A.; Finerty, P. J., High throughput methods of assessing protein stability and aggregation. Molecular Biosystems 2009, 5 (3), 217-223. 20.Senisterra, G. A.; Ghanei, H.; Khutoreskaya, G.; Dobrovetsky, E.; Edwards, A. M.; Prive, G. G.; Vedadi, M., Assessing the Stability of Membrane Proteins to Detect Ligand Binding Using Differential Static Light Scattering. Journal of Biomolecular Screening 2010, 15 (3), 314-320. 21.Senisterra, G. A.; Hong, B. S.; Park, H. W.; Vedadi, M., Application of high-throughput isothermal denaturation to assess protein stability and screen for ligands. Journal of Biomolecular Screening 2008, 13 (5), 337-342. 22.Senisterra, G. A.; Markin, E.; Yamazaki, K.; Hui, R.; Vedadi, M.; Awrey, D. E., Screening for ligands using a generic and high-throughput light-scattering-based assay. J Biomol Screen 2006, 11 (8), 940-8. 23.Vedadi, M.; Arrowsmith, C. H.; Allali-Hassani, A.; Senisterra, G.; Wasney, G. A., Biophysical characterization of recombinant proteins: A key to higher structural genomics success. Journal of Structural Biology 2010, 172 (1), 107-119. 24.Vedadi, M.; Niesen, F. H.; Allali-Hassani, A.; Fedorov, O. Y.; Finerty, P. J., Jr.; Wasney, G. A.; Yeung, R.; Arrowsmith, C.; Ball, L. J.; Berglund, H.; Hui, R.; Marsden, B. D.; Nordlund, P.; Sundstrom, M.; Weigelt, J.; Edwards, A. M., Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci U S A 2006, 103 (43), 15835-40.

Formulation Workflow Formulation(s) Common initial strategy is to take a punt on a combination of the following: Base condition: Tris, PO4, HEPES (cheap, readily available) Assays 50mM to 1M NaCl Additives: 0 to 10% polyol (> solubility of an unhappy protein) Reducing agent (DTT, TCEP, bME) Optimization Co-factors (divalent cations, organics) Other stuff – sufactants, aa’s , sugars(polyols)

A CHEMICAL DEGUSTATION

Formulation Workflow (HTP) Screen HTP Screen Production HTP Assay Optimization Aggregation / Activity

Systematic Screen Design Crystallisation friendly buffer screen: • Diverse pH, variable [NaCl] • Duplicate pH with different buffers • Controls ( is the result real? ) pH 5.0 pH 7.0 pH 9.0 All buffers at 50mM with 50mM and 200mM NaCl Water (milli Q) pH 7.0 – imidazole pH 5.0 – sodium acetate pH 7.0 – MOPS (3-(N-morpholino)propanesulfonic acid) pH 5.5 – piperazine pH 7.5 – HEPES pH 6.0 – MES (N-morpholino)ethanesulfonic acid) pH 7.5 – Na2H/KH2 PO4 pH 6.0 – citric acid pH – 8.0 tris chloride pH 6.5 – bis-tris pH 8.5 – glycyl-glycine pH 6.5 – ADA (N-(2-Acetamido)iminodiacetic Acid) pH 9.0 – CHES (N-Cyclohexyl-2-aminoethanesulfonic acid)

Additive/Ligand Screening • Find a good ‘base condition’, then – Hampton Research Solubility and Stability Screen – (conditions available on c6.csiro.au) • Or be specific: – Family of known co-factors and/or inhibitors – Specific chemical groups (e.g. Polyols, Surfactants)

Summary - Formulations I. Look at a wide range of chemical space, your protein could be unhappy by only 0.5 pH unit, or 50mM NaCl II. Think about ways to assess the formulations in HTP (we will look at an accessible method next) III. Make a list of potential chemical agents that could enhance stability – co-factors, surfactants, polyols IV. Think about controls – be sure you’re improving the stability and not just complicating the process

Thermal Stability

Assumption! ...thermal stability is a proxy for overall stability The more energy (heat) required to unfold a protein in a given chemical condition = the less prone it will be to denaturation in that chemical condition over time... ...this means you will have more of the same (uniform) protein to use for your campaign of interest.