Regulatory Perspectives on Higher Order Structure Evaluations for Protein Products. Emily Shacter, Ph.D.

Transcription

1 Regulatory Perspectives on Higher Order Structure Evaluations for Protein Products Emily Shacter, Ph.D. Chief, Laboratory of Biochemistry U.S. Food and Drug Administration Center for Drug Evaluation and Research Office of Biotechnology Products Division of Therapeutic Proteins EU Strategy Forum, March 21, 2011, Barcelona

2 Overview What is higher order structure? Why is it important? How is it measured? Regulatory expectations for higher order structure evaluations and why? Examples

3 Regulatory Challenge How do proteins adopt and maintain a specific 3-D structure? Is knowing the amino acid sequence enough? What is the impact of variations in 3-D structure on protein products? What is important for assurance of safe, effective, consistent protein products?

4 Levels of protein structure + Random coil, Turns, Loops S-S bonds Hydrophobic, other interactions, Subunits, protein-protein interactions, aggregates

5 Many proteins have multiple domains that confer correct function under appropriate physiological conditions 3 assays vs tertiary structure Inhibitor Binding Domain (PAI) Fibrin Binding Domain (site-specific activation) Protease Domain (substrate pmgn binding & activity)

6 What do the receptor, immune system, & body see? [From Elliott et al, Nature Biotechnology 2003]

7 How do proteins fold? Amino acid sequence (Anfinson) Complex path to folding Free-energy, hydrophobic collapse Proteolytic activation Chaperones (GroEL, HSP-70/40), chaperonins (eukaryotic vs bacterial) Rate of synthesis (inclusion bodies) Host Cell

8 Primary amino acid sequence has sufficient information to direct formation of 2 e and 3 e structure. Anfinson et al. (1950s - 70s)

9 You think you know something, and then

10 The BIG questions for therapeutic proteins How do you know when: have correct structure(s)? structural variants are present? structure has changed significantly? change in structure is important (clinically)? To what extent can physicochemical and biological methods answer these questions?

11 Observations Vast knowledge about the complexities and intricacies of protein folding, 3-D structure, and function Little of this gets applied to biotechnology products on a regular and meaningful basis. Methods ARE available some amenable to QC Time to raise the bar? In which circumstances?

12 Some methods for higher order structure Circular Dichroism (near & far UV) Fluorescence (± Dyes, metal) Fourier & Raman IR Spectroscopy Differential Scanning Calorimetry Resin binding (HIC, IEX, affinity) Light Scattering Analytical Ultracentrifugation Conformational antibodies Nuclear Magnetic Resonance (± isotope label) X-ray crystallography Mass Spectrometry (H-D Exchange; ESI) Disulfide Bonding (peptide maps) Liquid Phase Partitioning Field Flow Fractionation Atomic Force Microscopy Electron Tomography

13 Why do all this? Product knowledge Product consistency (pre- & post-approval) Comparability QbD Stability Safety Conformational disorders (prion, β-amyloid) Same amino acid sequence Vastly different activity, clinical impact Aggregates (immunogenicity) Variants with different specificity or activity affect efficacy & sideeffects Formulation development

14 Prion protein conformations & TSEs

15 Why do all this? Monoclonal antibodies Intra-chain S-S bonds Ligand affinity (IgG2) Inter-chain S-S bonds Chain swapping/bi-specific antibodies (IgG4) Half molecules (IgG4) Tetramers (IgG2) all resulting in altered activity and/or specificity in vivo

16 Criticality of disulfide bonds in Antibody products Antibody monomers (bivalent) Antibody dimers (tetravalent) From J.G. Salfed Nat. Biotech. Dec Hybrid & Half Abs (heterovalent)

17 Requirements for evaluations of higher order structure in regulatory submissions Inter- and intra-chain disulfide bonding (all products) Aggregates (aberrant quaternary structure; all products) Generic polypeptide (NMR required) Proteins with potential to form amyloid Proteins for which higher order structure determines specificity (designer proteins) Proteins associated with carrier matrix Non-specific binding reflects and affects 3 e structure Binding and release characteristics Combination products e.g., osteogenic proteins & matrices

18 Higher order structure evaluations in regulatory submissions for protein products Release (rare, except for aggregates) Characterization 1 (early development) Characterization 2 (changes during development) Comparability (post-licensure) ICH Q5E: Following a manufacturing process change, manufacturers should attempt to determine that higher order structure (secondary, tertiary, and quaternary structure) is maintained in the product. If the appropriate higher order structural information cannot be obtained, a relevant biological activity assay (see biological activity below) could indicate a correct conformational structure. Stability (rare)

19 Some guiding principles Proteins are dynamic, breathing molecules Manufacturing steps, storage conditions, stressors, & formulation can alter protein folding & proteinprotein / protein-tissue interactions Variants can impact function and clinical outcomes Protein modifications associated with altered 3-D structure should be evaluated incl. glycosylation, PEGylation, oxidation, proteolysis

20 Oxidation of cofilin changes 2 e structure and causes gain of function (apoptosis) September 6, MRW native cofilin ox-cofilin 0 1. Discover oxidation (Mass Spec, proteomics) 2. Evaluate effect on function in cells and functional assays (actin, mitochondrial binding, disruption) 3. Define molecular changes (amino acid oxidation, secondary structure) Wavelength (nm)

21 Some guiding principles Protein products are usually mixtures of molecules, not a single molecular entity Spectroscopic methods measure averages o o Not sensitive to low levels of variants Not particularly useful for Mabs, which have similar spectral properties Use high resolution separation techniques coupled to 3-D assays to identify and quantify structural variants and misfolded species Stress to ensure technique is sensitive to structural changes Use state-of-the art techniques with maximal resolving power o What is sensitivity?

22 CE Applications for Biologics (from Wassim Nashabeh, GNE) Initial Publication of Zone Electrophoresis in Open Tubular Glass Capillaries in Analytical Chemistry (81), followed by a paper in Science (83) both widely credited with the launch of modern CE Increased use in academic labs and few characterization or feasibility studies in industry (often in collaboration with academic labs) First international symposium HPCE (high performance capillary electrophoresis) held in Boston with the introduction of first commercial CE instruments, indicating growing use within academic centers First conference was chaired by Prof Barry Karger Submission and approval by the FDA of two CE methods to be used as part of the control system QC release for a MAB cief (identity) and Glycan analysis present 2010 Launch of CE in the Biotech and Pharmaceutical Industry Symposium, reflecting acceptance and growing use in Pharma Symposium is currently in its 12 th year with international attendance and regulators on Organizing Committee; Also first mention of CE in ICH Q6B in appendix (c) Advances in instrumentation continued with significant expansion in applications (including CE-MS for Characterization), imaged cief and the introduction of platform methods Method becomes routine, with general chapters being developed in pharmacopeias ICH Q4B Global Harmonization of the General Chapter on CE in USP, EP, JP

23 Large modifications to proteins PEGylation - changes hydrodynamic size, biodistribution, half-life, immunogenicity Routinely measure positional isomers and gross size How do PEG variants change protein 3-D structure, function, and clinical activity? Glycosylation Routinely measure linear structures What about 3-D? What does a complex glycoprotein look like to components of the body? Conjugation to Fc how does this impact active moiety? Conjugation to nanoparticles - tissue restriction, reticuloendothelial system

24 Role of bioassays Heavy reliance on bioassays for determination of protein function appropriate, but Bioassays do not reveal some very important aspects of clinical activity Biodistribution (tissue binding, hydrodynamic size) Physiological modulators that bind to activation & inhibitory domains Activity of low level variants Potential immunogenicity What does the body see???

25 Role of bioassays Some proteins have multiple bioactivities Evaluate with multiple bioassays Support with evidence of correct and consistent structure

26 Evaluations of bioactivity and higher order structure are complementary Tests for secondary and tertiary structure not substitutes for functional bioassays, and Bioassays are not complete substitutes for evaluations of higher order structure.

27 Special thanks to: Office of Biotechnology Products Carla Lankford Ashutosh Rao Emanuela Lacana Barry Cherney Amy Rosenberg Steven Kozlowski All of the great scientists in OBP