Demonstrating Biosimilarity of a Therapeutic Protein Product to a Reference Product ICH Guidance

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2 Demonstrating Biosimilarity of a Therapeutic Protein Product to a Reference Product ICH Guidance Derek Bradley M.Sc. BioClin Research Laboratories 13 th May 2016

3 Agenda Brief overview of biosimilars ICH Q6B Guidance Structural characterisation strategies Analysis of physiochemical properties Impurity profiling

4 What are biosimilars? Biosimilars are reverse engineered innovator biologics Innovator development Biosimilar development Drug discovery research Biosimilar product Cell line development Comparability testing Process development Cell line and process development Innovator product Innovator product analysed

5 Why biosimilar and not same? Vast complexity compared to conventional drugs Different production cell to innovator No access to proprietary production process Differences in raw materials Impact of excipients and formulation Cloning of gene into viral or non-viral vector Transfer into host cell for expression Cell expansion Protein recovery Protein purification Formulation Protein characterisation and stability Sources of differences between innovator and biosimilar Use of different vector Use of different host cell Different media or method of cell expansion Different method or recovery conditions Different binding and/or elution conditions Different formulations Different methods

6 ICH Q6B Guidance Document Setting and justification of a uniform set of specifications Critical quality standards proposed by the manufacturer, agreed by regulatory authorities as conditions of approval Specifications list of tests, analytical procedures, and appropriate acceptance criteria (numerical limits, ranges or other criteria) for the tests described

7 Structural characterisation Amino acid composition Protein is acid hydrolysed to release constituent amino acids Amino acids are derivitised to enhance detection E.g. PITC for UV detection, or AQC (6-aminoquinolyl-nhydroxysuccinimidyl carbamate) for fluorometric detection Amino acid derivatives are separated by RP-HPLC and quantified Results compared to gene sequence and for biosimilars, to reference biologic Contamination big issue!

8 Structural characterisation Amino acid composition

9 Structural characterisation Peptide mapping/peptide mass fingerprinting Enzymatic and chemical cleavage agents

10 Structural characterisation Peptide mapping/peptide mass fingerprinting Disulfide bonds reduced using DTT or ME Free thiols alkylated with IAA prevents S-S bonds reforming Proteolytic enzymes or chemical cleavage agents added to cleave protein into distinct fragments Fragments separated via HPLC Fragments detected using UV or MS and compared

11 Structural characterisation Peptide mapping/peptide mass fingerprinting In-silico digestion of protein

12 Structural characterisation N- and C-terminal sequencing Procise protein sequencer based on Edman degradation

13 Structural characterisation N- and C-terminal sequence Sequencing via peptide mapping and ESI mass spectrometry

14 Structural characterisation PTM analysis Top down analysis: Intact protein is analysed Fragmented via MS/MS to reveal site and nature of PTMs Typically requires TOF mass spectrometry Can provide complete coverage of PTM s in the intact protein Bottom up analysis: Proteins first broken down into smaller fragments Fragments analysed independently MS/MS fragmentation of peptides can reveal site and nature of PTM Typically only partial coverage can be obtained

15 Structural characterisation Sulfhydryl groups and disulfide bridges Only relevant where cysteine residues are present Peptide mapping under both reducing and non-reducing conditions Data sets from both analyses compared in order to determine the position of disulfide bonds Free thiol groups can be determined spectrophotometrically using Ellman s reagent, or fluorimetrically using malemide or bimane reagent.

16 Structural characterisation Carbohydrate structure Many factors can alter carbohydrate structure Altered carbohydrate structure can lead to biologics that are less efficacious; less stable; more immunogenic Amino acid sequence can be predicted from gene sequence no such template for glycosylation poorly understood Glycan structure can be highly branched, and many structures are isobaric complicates analysis

17 Structural characterisation Carbohydrate structure Glycan analysis typically involves enzymes that cleave sugar moieties sequentially Specific enzymes for cleaving the commonly encountered sugars: Glucose Galactose Mannose N-acetylglucosamine Fucose N-acetylneuraminic acid (sialic acid) Strategy: release glycans using PNGase F/PNGase A, then sequentially cleave sugars and analyse (e.g. HPLC) Rebuild glycan structure by combining data sets

18 Physiochemical properties Size or molecular weight SDS-PAGE Resolution of approximately 0.5 kda Molecular mass standard required Size-exclusion chromatography Lower resolution Can reveal presence of aggregates under non-reducing conditions Mass spectrometry Resolution to the sub-dalton range Reference standard not required

19 Physiochemical properties Size or molecular weight Electrospray ionisation mass spectrometry Y ESI mass spectrometers range = m/z Proteins are typically greater than 30,000 Da Therefore, protein mass needs to be calculated: X M = molecular weight of protein z = charge state of protein X and Y differ in z by 1 X = (M + z)/z Y = (M + z + 1)/(z + 1) To calculate charge state of X, (z X ) z X = (Y - 1)/(X - Y) = ( )/( ) = (rounded to 29) Therefore, X has a charge state of 29+, which implies that Y has a charge state of 30+ To calculate protein mass: M = (X * z X ) z X = ( * 29) 29 = 34,591.2 Da

20 Physiochemical properties Size or molecular weight Estimation of molecular mass via SDS-PAGE Molecular mass interpolated from the standard curve

21 Physiochemical properties Protein content and extinction coefficient Many options available Bradford assay Folin-Lowry assay Biuret assay Bicinchoninic acid (BCA) assay UV determination (A280 nm) Quantitative amino acid analysis When protein concentration is accurately known, the extinction coefficient (molar absorptivity) can be determined

22 Physiochemical properties Electrophoretic patterns Determine molecular weight and purity of a protein using SDS-PAGE Isoelectric point and charge heterogeneity due to PTM s, such as glycosylation or deamidation Assess product-related impurities, such as aggregates with native PAGE Study cellular protein expression, resolving tens to hundreds of proteins using twodimensional PAGE High resolution pi determination using cief

23 Physiochemical properties Chromatographic patterns Size Exclusion Chromatography (SEC) Ion Exchange Chromatography (IEX) Reverse-Phase Chromatography (RP- HPLC) Hydrophilic interaction liquid chromatography (HILIC) Identity, homogeneity and purity Many analytical techniques include chromatographic element

24 Physiochemical properties Spectroscopic profiles Circular dichroism (CD) spectroscopy difference in absorbance of circularly polarised light information on relative amounts of secondary structural elements (α-helices and β-sheets) Fourier-transform infra-red spectroscopy information on certain functional groups provides a fingerprint for a molecule that can be used for identification Nuclear magnetic resonance (NMR) spectroscopy determination of spatial arrangement of atoms in a molecule

25 Physiochemical properties Spectroscopic profiles Normalised CD spectra of Non-recombinant, Recombinant, Lyophilised asparaginase

26 Physiochemical properties Spectroscopic profiles Functional groups and amide bands typical of protein FT-IR

27 Process-related impurities Cell substrate-derived impurities: HCP s derived from the host organism which can co-purify with the therapeutic protein, and may also include nucleic acids Cell culture-related impurities: May include inducers, antibiotics, serum, and other media components that are deliberately added during the production processes Downstream processing-related impurities: Enzymes, processing reagents (e.g. oxidising and reducing agents), inorganic salts (which may also introduce heavy metals), solvents, ligands, and extractables/leachables

28 Product-related impurities Truncated forms: Hydrolytic enzymes (from the host cell used for expression for instance) may catalyse the cleavage of peptide bonds Other modified forms: Post-translational modifications (PTM s) such as deamination, oxidation, isomerization, glycosylation, and phosphorylation Aggregates: Dimers and higher multiples of the desired product may not have the same pharmacokinetic and pharmacodynamic properties of the intended structure, and so must be screened for in finished products.

29 Acknowledgements Dr. James J. Roche and Dr. Alessia Stocca Biosciences Research Institute Athlone Institute of Technology Mary Burke and Roisin Browne Directors BioClin Research Laboratories

30 Thank you! Questions/comments?