Automated Chemical Denaturation as a Tool to Evaluate Protein Stability and Optimize the Formulation of Biologics

Transcription

1 Automated Chemical Denaturation as a Tool to Evaluate Protein Stability and Optimize the Formulation of Biologics Ernesto Freire Johns Hopkins University Baltimore, MD ef@jhu.edu

2 AVIA 2304 Automated Protein Denaturation System

3 Globular Proteins Proteins Fold into Well-Packed Globular Structures Secondary Structure (α-helix, β-sheet) Hydrophobic Residues Mostly Inside Polar/Charged Residues Mostly at Surface

4 Native and Denatured States - ph - Ligands K - Salt Concentration

5 Stability and Aggregation are Inversely Correlated E. Y. Chi, et al

6 Strategies for Optimizing Conformational and Colloidal Stability - ph - Ligands K - Salt Concentration Minimize Population Solubilize

7 Stability of Native State K K = e G RT - The stability of the native state is measured by its resistance to: - Physical perturbations (temperature, pressure). - Chemical denaturants (urea, GuHCl, ph).

8 Important Practical Quantity: Denatured Fraction Fraction Denatured ΔG kcal/mol K Native K Denatured F D 0.4 K = [Denatured] [Native] F D = K (1 + K) K

9 Temperature Denaturation of HIV-1 Envelope Glycoprotein gp120 Reversible Tm = 61.1ºC ΔH = 110 kcal/mol ΔS = 331 cal/k mol ΔCp = 4 kcal/k mol ΔCp

10 G cal/mol Temperature Denaturation of HIV-1 Envelope Glycoprotein gp120 G = H T T S(T) G = H Tr T S Tr + C p T Tr T C p ln T Tr T m Stable Denatured Temp deg C

11 DSC of Proteins for Over Forty Years P.L. Privalov, N.N. Khechinashvili (1974) J.Mol. Biol. 86,

12 T m and Low Temperature Stability P.L. Privalov, N.N. Khechinashvili (1974) J.Mol. Biol. 86,

13 T m degc T m and Low Temperature Stability 110 Are Not Correlated G kcal/mol

14 T m degc T m and Low Temperature Stability 110 Are Not Correlated It is erroneous to use Tm to predict 80 protein stability at lower temperatures G kcal/mol

15 The situation is even worse because temperature denaturation is often irreversible, thus invalidating any thermodynamic analysis

16 The Alternative is Chemical Denaturation K K = e G RT G = G o mc

17 G cal/mol G Decreases Linearly with Denaturant Concentration G = G o mc G o C 1/ slope = m Urea M

18 AVIA 2304 Automated Protein Denaturation System - Instrument automatically prepares buffers, formulations and protein solutions from stock solutions. - Up to point denaturation curves. - Entire fluorescence spectrum for each data point. - Average protein consumption per denaturation curve is ~ 10 µg

19 Fluorescence Intensity Four different samples of cytochrome c at ph 7.0. Analysis of the data yields the following thermodynamic parameters: ΔG o = ± 0.12 kcal/mol; m = 1.4 ± 0.08 kcal/mol*m; C 1/2 = 6.3 ± 0.07 M. Urea denaturation of cytochrome c at ph 7 G = G o mc 2 K C 1/2 = Go m [Urea] M

20 Antibodies and Fusion Proteins

21 G protein-coupled receptors GPCR

22 From Very Low (0.01mg/mL) to Very High (100 mg/ml) Protein Concentrations. Trypsinogen 92mg/mL

23 ΔG Measured by DSC or Chemical Denaturation y = x R= G chemical G dsc Ramprakash et al Anal Biochem. 374 (2008)

24 Temperature and urea denaturation of an anti- EGFR monoclonal antibody

25 Automated Protein Denaturation & Renaturation The Chemical Denaturation is Totally Reversible Denaturant Concentration Denaturant Concentration

26 Chemical Denaturation Allows Evaluation of the Effects of: - ph - Salt Concentration - Sugars - Detergents - Metal Ions - Ligands - Excipients

27 Effect of Salt Concentration on Conformational Stability

28 G kcal/mol Effect of Salt Concentration on Conformational Stability 10.5 y = x R= [NaCl] mm

29 Urea denaturation of HIV-1 envelope glycoprotein gp120 as a function of ph

30 C 1/2 G kcal/mol ph dependence of structural stability of HIV-1 envelope glycoprotein gp ph ph

31 Effect of Amino Acids on Conformational Stability Glycine L-Arginine L-Proline

32 Effect of Sugars on Conformational Stability Glucose Maltose Sucrose

33 Ligands Preferentially Stabilize Conformations To Which They Bind Better - ph - Ligands K - Salt Concentration G L o = G o + RTln 1 + L K d,n RTln 1 + L K d,d

34 Contrary to Thermal Denaturation, Chemical Denaturation Allows Determination Binding Constants

35 Determination of Binding Constants (Kd) Carbonic Anhydrase and TFMSA [TFMSA] 5 µm 20 µm ΔC½ ITC (1) ITC (2) K D (nm) ΔG (kcal/mol)

36 K d other nm K d from Chemical Denaturation versus K d 10 4 from Other Methods K d chemical denaturation nm

37 K d other nm K d from Chemical Denaturation versus K d 10 4 from Other Methods Six Orders of Magnitude in K d K d chemical denaturation nm

38 Thank You!