Regulatory Consideration for the Characterization of HOS in Biotechnology Products Maria Teresa Gutierrez Lugo, Ph.D. OBP/CDER/FDA 5 th International Symposium on Higher Order Structure of Protein Therapeutics April 11 13, Long Beach, CA
Considerations for the characterization of Higher Order Structure (HOS) New biological entity Biosimilar Examples of methods used for HOS characterization Biosimilar applications Advantages and limitations of common methods used for HOS characterization Considerations for method selection Challenges Overview 2
Characterization of biotecnology products Structural Primary structure Higher order structure including aggregates Molecular weight Degree of heterogeneity (derived from enzymatic, unintended and intentional modifications) Functional Biological activity (i.e. potency) Functional domain Enzyme kinetics Receptor binding Protein target binding Fc effector function Impurity profiles Product related impurities Inactive protein variants generated during manufacture or storage Process related impurities Host cell proteins & DNA Media components Column leachates (e.g., protein A) Stability Degradation profiles under accelerated, stress (high temperature, freeze thaw, light exposure, agitation), forced conditions Collectively, these quality attributes can be used to define identity, purity, potency, and stability of the products, and if critical, they correlate with safety and efficacy 3
Characterization during product development 351(a), New Biological Entity 351(k), Biosimilar IND enabling, Initial Clinical Studies Additional Clinical Studies Licensure EXTENT OF CHARACTERIZATION Preliminary product characterization Extensive product characterization Sufficient data to support proposed product is similar to the reference product Strong evidence to support that proposed product is highly similar to the reference product. LOW DEGREE OF UNCERTAINTY
Hierarchy of protein structure Primary Secondary Tertiary Quaternary Asp His Ala Iso Val Gln Tyr Leu All need to be evaluated as part of the characterization of the product The three dimensional conformation of a protein is critical for its biological activity, specificity and stability 5
Correct protein conformation versus disordered aggregates Tyedmers J. et al (2010). Nature Reviews Molecular Cell Biology 11, 777 788. 6
Characterization of HOS for NBEs Early in development, characterization of HOS is desirable (e.g. phase I) but it is not required. Functional studies (e.g. biological activity, target binding) could be used to support proper HOS Later in development, full characterization of HOS using orthogonal methods is expected. HOS is an integral part of product characterization. Assessment of aggregates is expected at all stages of development along with a risk assessment of their potential impact on safety and efficacy. 7
Characterization of HOS for biosimilars Characterization HOS is expected as part of the data supporting that the proposed biosimilar product is similar to the reference product. HOS can be affected by environmental conditions (e.g. formulation, container closure system). Some of these conditions may differ between the proposed biosimilar product and the reference product. Any differences in HOS between a proposed biosimilar and reference product should be evaluated in terms of a potential effect on protein function. Thus, functional assays are also critical tools for evaluating the integrity of the HOS. Guidance for Industry Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product. February 2012 8
Characterization of HOS for biosimilars Evaluation of higher order structure adds to the body of information supporting biosimilarity The more comprehensive and robust the comparative structural and functional characterization, the stronger the scientific justification for a selective and targeted approach to animal and/or clinical testing to support a demonstration of biosimilarity Guidance for Industry Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product. February 2012 9
Common methods 1 used for characterization of HOS Circular dichroism Far UV Near UV Fourier transform infrared spectroscopy Fluorescence spectroscopy Intrinsic Extrinsic (fluorescent probes) Nuclear magnetic resonance ID and 2D Disulfide bonding (MS) H/D exchange MS Differential scanning calorimetry Light scattering Analytical Ultracentrifugation Raman spectroscopy X ray crystallography 1 Methods used in regulatory applications 10
HOS methods used/proposed in biosimilar applications 1 Functional assays were also used to support higher order structure Most applications include assessment of disulfide bonds (MS) and free cysteine 1 Survey as of March 2016 11
Advantages and limitations CD Method Advantages Disadvantages Small amount of protein Amenable to quantitative analysis Short acquisition and data analysis Small and large proteins Thermal stability and conformational flexibility Measure average of populations Difficult to determine the structural origin of changes Quality of deconvoluted data depends on the quality of the database used Artifacts associated with light scattering Fluorescence FT IR High signal to noise ratio Small amount of protein Fast acquisition time (suitable for folding/conformation studies) Sensitive to immediate environment of the probe (eg. Trp) which changes drastically upon unfolding Small and large proteins Able to detect proteins in dynamic equilibrium Thermal stability and conformational flexibility Measure average of populations Difficult to determine the structural origin of changes (it reports on the environment surrounding the probe) Does not provide information concerning the relative positioning of individual functional groups 12
Advantages and limitations Method Advantages Disadvantages DSC NMR Small amount of protein Suitable for conformational studies (stability) Sensitive to changes Sensitive to protein conformation changes 1 H NMR does not require labeling Suitable for structural fingerprinting analysis Provide information concerning the relative positioning of individual functional groups Complete assignment of 3D structure across the entire molecule in solution at atomic level resolution May be able to detect protein mixtures (?) Protein may denature at high or low temperature May not distinguish between different populations Limited to proteins < 20 30 kda (?) Sensitivity depends on protein concentration Require large amount of protein (relative to the methods) Time consuming (not labeled proteins, 2D NMR). Resonance assignments are time consuming (if assignments have not been done previously) Expensive instrumentation HDX MS Small and large proteins Small amount of sample (nmol) Identify specific sites in the molecule Can be automated Difficult data analysis Interference of some formulation component (e.g. detergents) Day to day drift 13
Considerations for method selection Nature of protein being characterized MW, heterogeneity, solubility, etc. Knowledge regarding the structure Type, amount and quality of information needed E.g. Higher order structure characterization or understanding of conformational stability Small change in all molecules of a population (batch) Change in a small percentage of the population 14
Considerations for method selection Use of orthogonal methods, including functional assays Use high resolution separation methods to identify population of proteins with different conformations In analyzing results consider manipulations of the samples 15
Considerations for method selection Test used to characterize the products do not necessarily need to be validated for routine quality control purposes but should be: Scientifically sound Fit for the intended purpose Produce results that are reproducible and reliable Capable of identifying product differences Qualitative Quantitative Sensitive Analysis of stress or spiked samples 16
Challenges Assessment of HOS in highly heterogeneous or complex products (e.g. glycosylated products and large proteins) Evaluation of a small change (e.g. change of conformation in a specific domain) in all molecules of a population (batch) vs a change in a small percentage of the population (e.g. low level of incorrectly folded protein) Establish the link between HOS, functional activity and potential impact on safety and efficacy 17
Thank you for your attention 18