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Studying Reversible Self-Association of Biopharmaceuticals using AUC and Light Scattering

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Hos 2014 Presentation, February 2014, John Philo

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Studying Reversible Self-Association of Biopharmaceuticals using AUC and Light Scattering

  1. 1. Studying Reversible Self-Association of Biopharmaceuticals using AUC and Light Scattering John Philo HOS2014, February 2014 © Copyright 2014 Alliance Protein Laboratories
  2. 2. Outline  Some examples of applying sedimentation equilibrium (SE) and sedimentation velocity (SV) to study reversible self-association  for each, how do we distinguish reversible from irreversible association/aggregation?  Using classical light scattering to study reversible interactions (CG-MALS)  the complex world of “crowded solutions” at ~100 mg/mL
  3. 3. 6.30 6.35 6.40 6.45 6.50 6.55 6.60 0.0 0.2 0.4 0.6 0.8 1.0 cell base Absorbance radius (cm) diffusion buoyancysedimentation meniscus The fundamentals of sedimentation equilibrium The concentration distribution depends only on molecular weight, independent of shape!  smaller sample size to reduce time to reach equilibrium 
  4. 4. Apparent mass vs. concentration plots for the soluble extracellular domain (ECD) of the atrial natriuretic peptide receptor (monomer mass 58 kDa) 0.004 0.01 0.02 0.03 0.04 0.1 0.2 0 20000 40000 60000 80000 100000 120000 Mw concentration (mg/ml)
  5. 5. Global analysis of all the data shows the ECD dimerizes with Kd = 520 +/- 20 nM (G = -8570 +/- 25 cal/mol) -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 0.0 0.3 0.6 0.9 Absorbance(280or230nm) (r 2 - r0 2 ) / 2
  6. 6. Reversible or not? This monomer-dimer-tetramer system acts as a mixture at low concentrations (? due to dilution of its cofactor) 0.005 0.010 0.020 0.050 0.100 0.200 0.500 Concentration (mg/ml) -4.0E+4 -2.0E+4 0.0E+0 2.0E+4 4.0E+4 6.0E+4 8.0E+4 1.0E+5 1.2E+5 1.4E+5 1.6E+5 1.8E+5 2.0E+5 2.2E+5 2.4E+5 2.6E+5 ApparentMass(Da) 00004.RA2 Ch A-BL 00004.RA2 Ch B 00004.RA2 Ch C 00007.RA2 Ch A-BL 00007.RA2 Ch B 00007.RA2 Ch C 00004.RA3 Ch A 00004.RA3 Ch B 00004.RA3 Ch C 00007.RA3 Ch A 00007.RA3 Ch B 00007.RA3 Ch C
  7. 7. SE data for a peptide (3-5 kDa) which strongly reversibly self-associates; above ~3 mg/mL repulsive solution non- ideality causes the apparent mass to decrease 0.3 1 10 20 0 1 2 3 4 5 Mw (#ofsubunits) concentration (mg/mL)
  8. 8. Sedimentation velocity of reversibly- associating proteins/peptides
  9. 9. First we must test for reversible association by doing a dilution series; here is such a test for a vaccine drug substance that contains a wide range of different oligomers (up to ~25-mer) 0 5 10 15 20 25 30 35 0.00 0.02 0.04 0.06 0.08 0.10 1.2 mg/mL, sw = 10.3 S 0.6 mg/mL, sw = 10.1 S 0.3 mg/mL, sw = 10.3 S normalizedg(s*)[perSvedberg] sedimentation coefficient [Svedbergs]
  10. 10. Applying separation methods to reversibly- associating proteins raises issues which don’t arise for measurements at equilibrium  Separation methods (SEC, sedimentation velocity, FFFF, electrophoresis) typically will not resolve individual oligomer species for a molecule in rapidly-reversible association equilibrium  what is detected will depend on the rates of association and dissociation compared to the speed of separation  for associations to oligomers larger than dimer, from theory multiple peaks may be seen even for infinitely-fast kinetics, but those peaks usually do not represent single oligomers  theory says it is possible to see more peaks than the number of different species that are present!  The bottom line: your so-called “dimer” or “trimer” peak may really represent a dynamic mixture of different rapidly-reversible oligomers
  11. 11. Here is a SV dilution series for a monoclonal antibody that self-associates strongly in a high ionic strength SEC mobile phase 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0.0 0.5 1.0 0.2 mg/mL; sw = 7.61 S 0.5 mg/mL; sw = 7.99 S 2.0 mg/mL; sw = 9.44 S normalizedc(s)[perSvedberg] sedimentation coefficient, s20,w [Svedbergs]
  12. 12. Studying reversible interactions using classical light scattering (CG-MALS)
  13. 13. Concentration Gradient MALS combines computerized syringes and a mixing system that flows the mixed solutions into a standard MALS detector and then on into a concentration detector concentration detector (RI or UV)
  14. 14. This is a 10-step dilution from ~1.5 mg/mL of that strongly associating mAb in its SEC mobile phase 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.73 0 0.1 0.2 0.3 0.4 0.5 0.6 Slice # 45000.000 500.0 1000 1500 2000 2500 3000 3500 4000 Det. 2 Det. 3 Plateaus UV297 AnalysisSignals
  15. 15. CG-MALS results from 3 such dilution series show that the weight- average molar mass is actually above that of dimer at 1.5 mg/mL and falls continuously as the concentration falls 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 50 100 150 200 250 300 350 400 apparentmolarmass(kDa) total protein concentration (mg/mL) combined data from three 10-step dilution experiments
  16. 16. The CG-MALS data fit fairly well to a monomer-dimer- tetramer equilibrium 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0.0 0.2 0.4 0.6 0.8 monomer dimer tetramer speciesconcentration(mg/mL) total protein concentration (mg/mL) individual species concentrations from the best-fit model 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 2 4 6 8 10 12 14 16 best fit of mAb-X CG-MALS data LSsignal(arb.units) total protein concentration (mg/mL) experiment monomer  dimer  tetramer fit
  17. 17. The next frontier---weak associations at very high concentrations (50-200 mg/mL)
  18. 18. Light scattering of monomeric HSA up to ~110 mg/mL (physiological pH, ionic strength) shows very strong non-ideality 18 ~10 mg/mL ~110 mg/mL Semi-dilute slide courtesy Mike Marlowe 2 2 3 app true 1 1 ...B c B c M M    
  19. 19. Monomeric fit with non-ideality is clearly insufficient; indicates presence of higher order species 19 Effective Hard Sphere Model Virial Coefficient Model slide courtesy Mike Marlowe
  20. 20. Weak self-association of HSA monomers into dimers starts around 30 mg/mL (~0.5 mM), and then higher oligomers also form 20 90° LS Dimer Tetramer Monomer slide courtesy Mike Marlowe
  21. 21. Some remaining challenges 1. None of the analytical methods are good at handling samples containing both reversible and irreversible oligomers (which is often the real situation) 2. For these samples at very high concentrations can we really quantitatively account for the non-ideality well enough to reliably measure weak reversible interactions? How can we cross-check such results to prove they are right? 3. CG-MALS requires ~5-10 mL of the solution at high concentration, which is likely prohibitive except for late stage development  can sedimentation equilibrium get us the same information with much less material?

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