High Throughput Biophysical Approaches to Preformulation Characterization and Comparability Analysis of Protein Drugs and Macromolecular Vaccines

Transcription

1 High Throughput Biophysical Approaches to Preformulation Characterization and Comparability Analysis of Protein Drugs and Macromolecular Vaccines David B. Volkin, Ph.D. Higuchi Distinguished Professor Department of Pharmaceutical Chemistry Macromolecule l and Vaccine Stabilization ti Center WCBP January 2012

2 Introduction Outline of Presentation Analytical challenge: improved analysis of higher-order protein structure for formulation development and comparability Case Study #1 High-Throughput Analysis of Protein Solubility Case Study #2 High-Throughput Analysis of Protein Conformational Stability Future Directions Applicability to Comparability?

3 Formulation Development Approaches and Analytical Challenges Empirical approach ( strategic experimentation ) Stability studies to discover problems Use previous experience to add excipients Trial and error; fire-fighting Design approach ( rational design ) Determine protein s structure under conditions of interest Select formulations based on known effects on structure Define design space (structure vs. environmental stress) Analytical challenge: How to gather more data under time and cost pressures? How to better monitor structural changes leading to aggregation? How to combine diverse data from different analytical methods into a comprehensive picture?

4 Biopharmaceutical Comparability Approaches and Analytical Challenges Biochemical and biophysical testing: QC analytical tests Analytical characterization tests for structure or activity Stability profile and degradation profile Biological and animal testing: Biological assays that are linked to mechanism of action Animal pharmacology & toxicology studies if appropriate Clinical testing: Human PK studies where good correlates with clinical activity are known In the event all of the above are inconclusive, human efficacy and/or safety studies may be needed Analytical challenge: Need for improved methodologies to examine higher-order structural integrity and conformational stability

5 Need for New Analytical Approaches to Evaluate Higher Order Structure and Stability 1. One approach is to use higher-resolution biophysical techniques: X-ray Crystallography NMR Ion Mobility Mass Spectrometry HD-Exchange Mass Spectrometry 2. Are there alternative approaches to obtain better biophysical characterization data with protein drugs??? Large amounts of lower-resolution data High throughput screening Data analysis and visualization

6 High Throughput Biophysical Measurements: Protein Solubility Case Study DOE Dispense Assay Centrifuge Measure Supernatant A 280 Gibson TJ, McCarty K, Mcfadyen IJ, Cash E, DalMonte P, Hinds KD, Dinerman AA, Alvarez JC, Volkin DB Application of a high-throughput screening gprocedure with PEGinduced precipitation to compare relative protein solubility during formulation development with IgG1 monoclonal antibodies J Pharm Sci 100, (2011)

7 Relative Solubility of mab-a in Different Formulation Buffers

8 Relative Solubility of Four Different mabs Across Different Formulation Buffers mab-a mab-b Acetate Citrate Histidine Phosphate EG midpoint % P ph % PEG midpoint mab-c ph Acetate Citrate Histidine Phosphate %PEG midpoint mab-d Acetate Citrate Histidine Phosphate ph Tris

9 Relative Solubility of mab-a Made from Two Different Cell Lines Cell Line A in Citrate % PEG Mid dpoint Cell Line B in Citrate Cell Line A in Acetate Cell Line B in Acetate ph

10 High Throughput Biophysical Measurements: Conformational Stability 1. Use numerous lower-resolution biophysical techniques 2. Use wide variety stress conditions (temperature, ph, etc.) 3. Use high through-put instrumentation as much as possible 4. Evaluate large data sets for patterns Secondary Structure Far UV CD FTIR Size/ Aggregation Dynamic Light Scattering Optical Density SE HPLC and AUC MFI and Nanosight Tertiary Structure Fluorescence - Intrinsic and Extrinsic probes Derivative UV Absorbance Near UV CD Protein Dynamics High resolution ultrasonic spectroscopy py Pressure perturbation calorimetry Red-edge excitation shift fluorescence spectroscopy

11 Empirical Phase Diagrams- General Approach to Visualize Large Amounts of Biophysical Data Relative helix content Relative ME at 222 nm 1.1 A D Relative ANS Fl. peak intensity Relative Fl. peak intensity 1.1 B C ANS Fl. peak position (nm) Fl. peak position (nm) E F Experimental Data Visualization 0.6 G 1.0 H 16 I Optical density at 350 nm Relative Fl. light scattering Excess MHC Relative ME at 222 nm A Relative Fl. peak intensity B Figure 1 Fl. peak position (nm) 100 C Interpretation Re elative helix content D Relative e ANS Fl. peak intensity E ANS Fl. peak position (nm) F G H I Optical density at 350 nm Relative Fl. light scattering Excess MHC Figure 2

12 Typical Procedure for Constructing EPD Techniques: Fluorescence Emission Circular Dichroism UV Absorbance Buffer Subtraction Buffer Subtraction Buffer Subtraction Peak Select one or two 2 nd Derivative / Preprocessing: Picking/Tracing Wavelength(s) Smoothing Average Multiple Runs Normalization Average Multiple Runs Normalization Peak Picking/Tracing Average Multiple Runs Normalization Input Matrix Preparation EPD Processing: Singular Value Decomposition Color Mapping Empirical Phase Diagram

13 Protein and Vaccine Formulation Development Using EPD Approach Temp T Ricin -- ANS Int., Intrinsic Int., & CD P3 P2 P1 ph ph High-throughput excipient screening assay development: ph Temperature Biophysical Technique Usually involves a 96-well plate reader GRAS library of excipients Optimal Solution Conditions: Optimized i Formulation (ph, ionic strength) Candidates

14 Empirical Phase Diagrams and Formulation Development: Summary of Different Proteins and Vaccines Target Techniques Search Space Figure Ref. Measles virus CD, DLS, SLS, EF ph, T a Human respiratory syncytial virus CD, UVAS, OD 350, UV-IF ph, T 25 Live atten. Ty21a bacterial typhoid vaccine CD, EF ph, T 29 Adenovirus type 5 (Ad5) UVAS, DLS, UV-IF, EF ph, T 2.32, Recombinant ricin toxin A-Chain vaccine CD, UF-IF, EF ph, T 2.20, Adenovirus type 2 (Ad2) CD, UVAS, OD 350, DLS... ph, T Hepatitis C virus envelope glycoprotein E1 CD, DLS, UV-IF, EF ph, T, S a Clostridium difficile toxins and toxoids CD, OD 350, UV-IF, EF ph, T 8 16 Type III secretion system tip proteins CD, UVAS, UV-IF, EF ph, T 14 Type III secretion system needle proteins CD, UVAS, EF ph, T 17 Malaria antigen EBA-175 RII-NG CD, UV-IF, EF ph, T 15 H1N1 influenza virus-like particles CD, DLS, EF ph, T Norwalk virus-like particles CD, UVAS, UV-IF, EF ph, T 9 23 Nonviral gene delivery complexes CD, DLS, EF ph, I a Human Inteferon-β-1a CD, UVAS, UV-IF, EF ph, T 2.12, Bovine granulocyte colony stim. factor UVAS ph, T Immunoglobulin-G (IgG) CD, EF, PPC, HRUS, TRFS... ph, T 1 4 Pramlintide (antihyperglycemic peptide) CD, UVAS, OD a 350, UV-IF ph, T, C 7 7 Monoclonal antibodies CD, UVAS, OD 350, UV-IF T, C Clostridium botulinum type A neurotoxin CD, UV-IF, EF ph, T 9 Molecular chaperones Hsc70 and gp96 CD, UVAS ph, T 10 Human fibroblast growth factor 1 (FGF-1). CD, UVAS, UV-IF, EF ph, T, S Fibroblast growth factor 20 (FGF-20) CD, UVAS, UV-IF ph, T 12 rpa of B. anthracis CD, UV-IF, EF ph, T 2.31, Recombinant vault particles CD, UV-IF, EF ph, T Recombinant human gelatins CD, UV-IF, UVAS ph, T EC5 domain of E-Cadherin CD, UV-IF, UVAS ph, T, N/R a a T = Temperature, I = Ionic Strength, C = Concentration, S = Stabilizer, N/R = Native/Reduced Maddux NR, Joshi SB, Volkin DB, Ralston JP, Middaugh CR, Multidimensional Methods for the Formulation of Biopharmaceuticals and Vaccines, J. Pharm. Sci. 100, (2011)

15 Recent Example with IgG1 Monoclonal Antibody: (1 and 100 mg/ml) Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions Bhambhani A, Kissmann JM, Joshi SB, Volkin DB, Kashi RS, Middaugh CR. J Pharm Sci. 101, (2012)

16 Recent Example of Improved Automation Examined four model proteins EPD for BSA: Chirascan-plus Biophysical data from near-uv CD, far-uv CD, optical density, and intrinsic fluorescence spectroscopy Hu L, Olsen C, Maddux M, Joshi SB, Volkin DB, Middaugh CR, Investigation of Protein Conformational Stability Employing a Multimodal Spectrometer Anal. Chem. 83, (2011) EPD for BSA: separate instruments

17 Are Empirical Phase Diagrams Useful for Comparability Assessments?

18 Model Protein: Fibroblast Growth Factor (FGF1) 16 kda, heparin-binding protein Multifunctional protein involved in angiogenesis, wound healing, embryonic development Possible therapeutic agent for the treatment of ischemic diseases Collaboration with Michael Blaber, Florida State University Bernett MJ, Somasundaram T, Blaber M, Proteins: Structure, Function, and Bioinformatics 57, (2004)

19 Mutants of Fibroblast Growth Factor (FGF1) FGF1 is unstable near physiological conditions!! Requires heparin as a stabilizer, but animal-derived excipient which has its own pharmacological activity. Stabilize FGF1 through h site directed d mutagenesis approach (Michael Blaber, Florida State Univ.) EPD approach to analyze stability of mutants WT, WT + Heparin Ten Different Mutants without Heparin Focus on WT and 2 mutants for today s talk Alsenaidy MA, Wang T et al, EPD Approach to Investigate Conformational Stability of Functional Mutants of FGF1 Protein Science, epub online (Nov 2011)

20 The Empirical Phase Diagram (EPD) Summarizes and Visualizes Biophysical Data ANS Fluorescence ph 3 ph 4 ph 5 ph 6 ph 7 ph 8 Empirical Phase Diagram of FGF1 Wild Type Intrinsic Fluorescence Light Scattering Tempera ature ( C) Circular Dichroism ph ph Spectroscopic data: ph 3-8, C, FGF1- WT

21 90 WT FGF1 Empirical Phase Diagram of WT WT FGF1 + Heparin perature( o C) Tem ph Empirical FGF1- Phase Diagram Mutant of K12V-P134V-C117VH FGF1- Empirical Phase Mutant Diagram of SYM6 J Temperature( o C) Temperature( o C) ph ph

22 Future Work: Possible Use of EPDs for Biopharmaceutical Comparability? How to define structural regions more quantitatively? Similar color regions represent similar conformational behaviors Clustering analysis helps identify regions computationally Temp ( C) ph Temp ( C) ph Currently assessing EPDs using an IgG1 mab with varying glycosylation patterns

23 Introduction Summary of Presentation Analytical challenge: improved analysis of higher-order protein structure for formulation development and comparability Case Study #1 High-Throughput Analysis of Protein Solubility Case Study #2 High-Throughput Analysis of Protein Conformational Stability Future Directions Applicability to Comparability?

24 Acknowledgements High Throughput Solubility Studies: Co-authors on published paper presented Centocor R&D, TransForm Pharmaceuticals High Throughput Conformational Stability Studies: Russ Middaugh and Sangeeta Joshi at KU Macromolecule and Vaccine Stabilization Center Mike Blaber, Florida State University Co-authors on published papers presented EPD Slides: Jae Kim and Mohammad Alsenaidy

25 KU Macromolecule and Vaccine Stabilization Center Macromolecular and Vaccine Stabilization Center at KU: Unique and innovative center specializing in the characterization and stabilization of vaccines as well as protein and DNA based pharmaceuticals.

26 Thank you for your attention! Questions?