The Present State and Future Outlook for Characterizing the Higher Order Structure (HOS) of Protein Drugs in the Biopharmaceutical Industry

Transcription

1 The Present State and Future Outlook for Characterizing the Higher Order Structure (HOS) of Protein Drugs in the Biopharmaceutical Industry Steven Berkowitz, Senior Principal Scientist Biogen Idec 2 nd International Symposium on the Higher Order Structure (HOS) of Protein Therapeutics Feb. 11, 2013

2 Assessing the HOS in the Biopharmaceutical Industry HOS = secondary, tertiary & quaternary structure Structure = spatial (3-D) & temporal (dynamics/flexibility) Lecture slide from Dr, Randall Mauldin Department of Biochemistry and Biophysics, UNC Medicine 2

3 Points to Consider in Discussing the HOS of Protein Biopharmaceutical 1. Protein biopharmaceuticals are made using a living system (cell) 2. Chemically heterogeneous due to post-translational modification (PTMs), both in vivo & in vitro (degradation) 3. Production lots of a biopharmaceutical "Cannot be made so they are identical 4. Rather they are made Highly Similar in terms of key attributes: structure, purity, efficacy and safety 5. Most of the HOS of a protein is held together th by secondary (weak) bonds 3

4 In the Biopharmaceutical Industry What Do We Want Know in Characterizing the HOS of Protein Drugs (Research, Process Development & Manufacturing) 1. Impact of primary structure changes on the HOS of the protein drug (variants forms of the protein drug) 2. Detection & impact of silent changes on the HOS of protein drug 3. An understanding of the physico-chemical properties (stability) of a protein drug by stressing it and studying changes to its HOS formulation 4. Consistency of manufacturing (lot-to-lot), via consistency of the protein drug s HOS (Internal) t Comparability studies 5. Impact of process/site changes on the HOS of the protein drug (internal) Comparability studies 6. Biosimilar HOS vs Innovator HOS (External) Comparability studies 4

5 Ideal Attributes of a Biophysical Tool/Method for Assessing the HOS in the Biopharmaceutical Industry 1. High precision & accuracy 2. Large response per unit change (high sensitivity) 3. Probes all the parts of the protein drug molecule 4. High resolution information (detect small change in a molecule) 5. Minimal sample processing/handling minimal matrix effects 6. Robustness, easy to use and level of expertise/training required not high 7. High sample throughput (measurement completed in a short time and automated measurement & analysis) 8. Multiple vendors, not expensive 5

6 Basic Biophysical Toolbox Used Presently to Assess the HOS of Protein Drugs in the Biopharmaceutical Industry Spectroscopic: UV-Vis aromatic residues Fluorescence aromatic residues CD amide bond (polypeptide backbone) & aromatic residues FTIR amide bond (polypeptide backbone) Hydrodynamic (size): SEC aggregation AUC aggregation, S m, concentration-dependent aggregation LS (SLS & DLS) aggregation Thermodynamic: DSC domain T m, thermogram Native Chromatography & electrophoresis IEC, HIC surface properties IEF charge Binding: Via Biacore SPR, ITC, Fluorescence drug target, co-factor (metal, etc.), dyes Biological cell assay 6

7 Understanding the Uncertainty of Biophysical Measurements in Order to Assess Comparability Far-UV CD

8 Understanding the Uncertainty of Biophysical Measurements in Order to Assess Comparability DSC 8

9 Present Routine Spectroscopic Tools Used to Assess Biophysical Comparability UV VIS Fluorescence RS RECD RECD ODnm-OD380)/(OD280-OD380) d 2 (ODnm)/d(nm) RECD RS Wavelength, (nm) Fluore rescence Intensity (Vnm) RECD RECD RS RECD RECD RS Wavelength, (nm) Wavelength (nm) (Far UV) Circular Dichroism (Near UV) MRE, ( nm) RECD RECD RS MRE, ( nm) 50 RECD RECD RS Wavelength, (nm) -30 Wavelength, (nm) 9

10 Impact of Overlapping Signals UV-Vis Spectroscopy Circular Dichroism 10

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12 High Resolution CZE Under Conditions that Maintain the Native Structure of the Protein Biopharmaceutical 27cm 73 cm 12

13 1D 1 H NMR Spectrum of Lysozyme (14kDa) Lecture slide from Dr, Randall Mauldin Department of Biochemistry and Biophysics, UNC Medicine 13

14 Going From 1D 1 H to 2D 15 N- 1 H NMR 1D NMR 2D NMR Lecture slide from Dr, Randall Mauldin Department of Biochemistry and Biophysics, UNC Medicine 14

15 1D 1 H NMR Spectrum of a Pegylated IFN (~23 kda Protein) PEG-IFN Aliphatics PEG Buffer intens sity unit Aromatics & peptide bonds 150 H 2 O H chemical shift (ppm)

16 Assessing Quantitative HOS Comparability (Pre- vs Postchange) Using 1D 1 H NMR: Local vs Global Linear Correlation Coefficient (Using Pearson correlation coefficient) Aromatics & peptide bonds Aliphatics R X,Y = covariance (X,Y)/([standard deviation of X]*[standard deviation of Y]) = Σ[(X(δ) - <X>)*(Y(δ) - <Y>)] ({Σ(X(δ) - <X>) 2 } 0.5 )*({Σ(Y(δ) - <Y>) 2 } 0.5 ) 16

17 Assessing Quantitative HOS Comparability (Lot to Lot) Using 1D 1 HNMR:Difference NMR with 99% Confidence Limits 0.06 <(pifn-pur-c10-02) - (pifn-pur-c10-03)> Exp 99% CL Exp 99% CL 0.04 normalize ed intensity unit H chemical shift (ppm)

18 (Amide) H/DX-MS: Continuous Labeling Experiment D 2 O (1:10 to 1:20) A) Take samples at different times & stop (slow) exchange by lowering temp. & ph B) Measure mass change via MS H s at backbone amide positions H s & D s at backbone amide positions Engen & Smith (2001). Anal. Chem. 73, 256A-265A. Wales & Engen (2006). Mass Spectrom. Rev. 25,

19 Interferon Beta 1a (IFN) Met 117 Met 62 Met 36 Met 1 Cys 17 19

20 Local, Bottom-Up, H/DX-MS (Pepsin Peptide Pattern for Interferon β-1a) MSYNLLGFLQ RSSNFQCQKL LWQLNGRLEY CLKDRMNFDI PEEIKQLQQF QKEDAALTIY EMLQNIFAIF RQDSSSTGWN ETIVENLLAN VYHQINHLKT VLEEKLEKED FTRGKLMSSL HLKRYYGRIL HYLKAKEYSH CAWTIVRVEI LRNFYFINRL TGYLRN MW ~23kD 1 N-linked glycan 1 free SH, 4 Met Sequence coverage > 94% 167 residues 20

21 Displaying & Evaluating H/DX-MS (Using Relative Mass Change vs Time Plots) 21

22 H/DX-MS Comparison of 2 Different IFN Lots Made Using Different Culture Media (Growth Conditions) Standard Culture Media New Culture Media T = 0.17 min T = 1 min T = 10 min T = 60 min T = 240 min DI(1) = 0 DI(2) = 0 22

23 H/DX-MS: Comparison of IFN vs Oxidized (H 2 O 2 ) IFN T = 0.17 min T = 1 min T = 10 min T = 60 min T = 240 min DI(1) = 187 DI(2) =

24 Effect of Ca 2+ on rfix & FIX region of rfix-fc (Fusion Protein) rfix: with vs without Ca 2 a) Difference (Da Gla EGF Catalytic 8 6 FIX region of rfix-fc: with vs without Ca 2+ Difference (D Da) Peptide Number, i 24

25 rfix vs FIX region of rfix-fc With & Without Ca 2+ Diffe erence (D a) Without Ca 2+ : rfix vs. FIX region of rfix-fc Peptide Number, i DI(1) = 0 DI(2) = 0 Diff ference (Da) With Ca 2+ : rfix vs. FIX region of rfix-fc DI(1) = 0 DI(2) = Peptide Number, i FIX sequence coverage > 90% 25

26 Sequence Coverage of the FVIII Region of rfviii-fc (Fusion Protein) 416 peptides covering 94% of the FVIII sequence region in rfviii Fc 26

27 Relative Fractional H/DX Comparison Plot rfviii Region of rfviii-fc (top) vs rfviii (bottom) onal Exchange ive Fractio Relati Peptide Number, i 27

28 H/DX Difference Plot rfviii Region of rfviii-fc vs rfviii nce (Da) Differen DI(1) = 0 DI(2) = 1 Peptide Number, i 28

29 Where are We and Where are We Going in Terms of Characterizing the HOS Protein Biopharmaceutical 1. The present biophysical tool box consists of low resolution techniques 2. Nevertheless the tools have a fair amount of orthogonality measure different physical attributes 3. Need sensitive, high precision and high resolution biophysical tools that are robust and easy to use 4. Key find biophysical tools capable of high dispersion so signal output consists of non-overlapping bands/peaks that provide meaningful Fingerprint Output 5. Small difference can/will be seen by these techniques remember protein biopharmaceuticals are not made Identical rather Highly Similar 6. When differences are seen, are they important? 7. All drug approvals are based on the Totality of the Evidence 8. Balanced and transparent in managing & dealing with Risk & Economics 9. Do a better job in finding & developing these better biophysical tools 10. What is the role of industry (biopharmaceutical & instrument companies) /academia/government in doing 9! 29

30 Speaker Would Like to Thank and Acknowledge the Following People: Damian Houde, Julie Wei & George Bou- Assaf Dr. John Engen and members of his group (Northeastern Univ.) Staff at Waters Dr. Igor Kaltashov and members of his group (UMass, Amherst) Hemophilia Group at Biogen Idec Special Thanks to Drs. Rohin Mhatre & Helena Madden (Biogen Idec) 30