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INTRODUCTION Any manufacturer seeking to develop and market a biopharmaceutical product requires comprehensive physicochemical structural characterization of the (glyco)protein. For biosimilar products, this task is magnified and forms the basis for further comparability. In line with various regulatory guidelines (see below), characterization should be performed at distinct stages of development. Initially, batches of the target originator molecule should be studied to de- termine the exact sequence of the target protein, the post-translational modifications and the variability of quality attributes in batches over time. Then, once the biosimilar product is manufactured, characterization needs to be performed to confirm it’s structure. Finally, manufacturers must provide com- parative data for the biosimilar side-by-side with the originator molecule. Analyses must determine if proteins have the same biochemical, biophysical and physiological attributes. Analytical strategies should include a battery of physicochemical techniques for primary and higher order structure (Table1) with appropriate sensitivity to detect product related variants and impurities. The complexities of biomanufacturing make exact replication of the originators molecule impossible. Instead, biosimilars may be granted approval on the basis of analytical, pre-clinical and clini - cal data which show they are highly similar to the reference product at 3 levels: • Quality • Safety • Efficacy Table 2 describes the common step-by-step approach to take in a biosimilar analytical development program. BIOSIMILAR DEFINITIONS “A biosimilar is a copy version of an already authorized biological medicinal product (reference product) with demonstrated similarity in physicochemical characteristics, efficacy and safety, based on a comprehensive comparability exercise” 1 COPY BIOLOGICS • “True” biosimilars – also called similar biotherapeutic products, follow-on protein products, subsequent entry protein products, subsequent entry biologics, similar biologics etc. depending on regulatory region are compared to originator. Copy biologic products not developed and assessed in a scientific comparability program vs. a reference product are referred to as non- comparable biologics (NCB). • Biobetters are an improvement of an existing drug by e.g pegylation. Treated as new molecule, not a biosimilar. REGULATORY BACKGROUND OBJECTIVE: STRUCTURAL CHARACTERIZATION OF BIOSIMILARS The first step in development is the determination of the exact sequence of the originator protein. This involves de-novo MS/MS sequencing with careful interpretation and assignment of levels of post-translational modifications in multiple batches. The objective is to define the “Quality Target Product Profile (QTPP), representing the desired specifications for the final product. It is es- sential at this stage that the correct AA sequence is deduced. Figure 1 illustrates a case in which sequence differed from that anticipated from published data. During initial cell-line development to produce the desired biosimilar, screening characterization can also be used to aid selection of appropriate clones, such as those expressing the desired glycosylation profile. Once the biosimilar product has been manufactured, extensive side-by-side comparison is performed. There are many variables to consider such as: • choice of reference material – where it is manufactured • number of batches of biosimilar vs number of batches of originator • changes in originator product over time. Some regulatory guidelines mention “fingerprinting” of quality attributes to establish biosimilarity. Figures 2- show examples of many of the techniques regularly used to generate these data in an antibody case study. METHODS Analytical strategies include a battery of physicochemical techniques for primary and higher order structure with appropriate sensitivity to detect product related variants and impurities. Techniques include MALDI-TOF MS, ESI-MS, ESI-MS/MS, GC-MS, HPAEC-PAD, cIEF, CD and AUC which can be used to establish comparative analysis on glycoprotein products. ANALYTICAL CHARACTERIZATION OF BIOSIMILAR PRODUCTS TO ESTABLISH BIOSIMILARITY DR. FIONA M GREER, GLOBAL DIRECTOR, BIOPHARMA SERVICES DEVELOPMENT, SGS M-SCAN TABLE 1: WHAT REGULATIONS COVER PHYSICOCHEMICAL- CHARACTERIZATION? CONCLUSIONS • Advances in MS instrumentation and Proteomic/Glycomic strategies enable rapid identification of protein products and their PostTranslational Modifications, including glycosylation. MS techniques are applicable to characterization at all stages of development, but essential for determination of originator sequence. • MS techniques alone are not enough for full physicochemical characterization and comparison of biosimilar with reference products for regulatory purposes. Other orthogonal methods should also be included. • Strategies for comparability must include assessment of both primary and higher order structure. Batch-to-batch variation of the product should be determined for both the biosimilar and the originator. In essence, an analytical strategy will follow closely the requirements of the ICH guideline Q6B. • This extensive comparability is carried out prior to any clinical assessment of the biosimilar product. REFERENCES 1. Weise, M et al., Nature Biotechnology 29, 690-693 (2011). 2. Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ UCM291134.pdf. 3. Scientific Considerations in Demonstrating Biosimilarity to a Reference Product. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291128. pdf. 4. Biosimilars: Questions and Answers Regarding Implementation of the Biologics Price Competition and Innovation Act of 2009. http://www.fda.gov/downloads/Drugs/GuidanceCompliance- RegulatoryInformation/Guidances/UCM273001.pdf. 5. Guidance for Industry Formal Meetings Between the FDA and Biosimilar Biological Product Sponsors orApplicantshttp://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryIn- formation/Guidances/UCM345649.pdf. 6. EMA Multidisciplinary:Biosimilars. http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000408.jsp&mid=WC0b01ac058002958c 7. ICH HarmonisedTripartite Guideline,Topic Q6B. Specifications:Test Procedures and Acceptance Criteria for Biotechnological/ Biological products. Step 4, Consensus Guideline, March 1999 (CPMP/ICH/365/96). SAMPLE % α-HELIX % OTHER HELIX % β-SHEET % TURNS % ‘OTHER’ A 70 13 0 6 12 B 70 11 0 9 14 C 68 12 0 8 13 D 67 12 0 8 13 E 68 12 0 8 13 -10 -5 0 5 10 15 20 190 200 210 220 230 240 250 260 Wavelength (nm) dE mol-1 dm3 cm-1 Sample A Sample B Sample C Sample D Sample E Far-UV Circular dichroism analysis of various batches of originator molecule and biosimilar. UNITED STATES • Biologics Price Competition and Innovation Act (BPCIA) signed March 2010. New pathway- 351(k) introduced into the PHS Act. • Feb 2012, FDA issued draft Guidance docu- ments (two plus Q&A) to accompany legal acts, March 2013, fourth guidance issued 2-5 EUROPE • 2005, EMEA issued guidelines on “Biosimi- lars” and continues to issue and update including product specific guidances 6 • Approved first Biosimilar in 2006 and now has 14 REST OF WORLD • Brazil, Australia,Turkey, India,Taiwan, Malaysia, Argentina, Mexico, Japan, Canada, SA. have some form of pathway. • Some adopted EMA guides, others wrote their own. • Oct 2009, WHO “Guideline on Evaluation of Similar Biotherapeutic Products TABLE 2: BIOSIMILAR ANALYTICAL DEVELOPMENT - A STEP-BY-STEP APPROACH In essence, an analytical strategy will follow the requirements of ICHTopic Q6B 7 “Specifications:Test Procedures and Acceptance Criteria for Biotechnological/Biological Products” FIGURE 1: ESTABLISHING THE QTPP During initial assessment of a target molecule, peptide mass mapping was carried out to ob- tain confirmation of expected structure. However, a particular peptide stretch was missing from the Mass MAP ESQXXXXXXXR and ESQXXXXXXXRXXXXK A careful search for expected y” ions within one of the MS/MS channels was successful. However the molecular weight of the parent peptide was 10 amu higher than anticipated. The found mass corresponding to the missing peptide was +10amu higher. From MS/MS interpretation, the mass difference was attributed to site of the second residue, possibly Serine to Proline change: ESQXXXXXXXRXXXXK to EPQXXXXXXXRXXXXK FIGURE 2: CASE STUDY: ANTIBODY CHARACTERIZATION In the case of antibodies, the size and complexity of the molecule requires LC/MS/ MS approaches. The comparability program includes: • Mass spectrometry of intact protein and released L &H chains • Amino Acid Composition Analysis • N-terminal sequencing • Peptide “MAPPING” Analysis (Sequence coverage: 100% LC and 100% HC) • Monosaccharide and sialic acid analysis • Oligosaccharide population analysis • SDS-PAGE analysis • Circular Dichroism • Analytical Ultracentrifugation FIGURE 3A: INTACT MASS MEASUREMENT FIGURE 3B: INTACT MASS MEASUREMENT G0F Mass shift = +1444 Da G1F Mass shift = +162 Da G2F Mass shift = +324 Da Mab +2 x G0F Mab +1 x G0F + 1 x G1F Mab +2 x G1F Mab +1 x G1F + 1 x G2F IgG -> N-Linked biantennary core fucosylated with varying number of galactose residues FIGURE 4: MAPPING WORKFLOW FIGURE 5: ANALYSIS OF GLYCOSYLATION FIGURE 6: COMPARABILITY ON BASIS OF CHARGE FIGURE 7: SECONDARY STRUCTURE ANALYSIS (FAR UV CD ANALYSIS 260-190NM) Imaging cIEF comparability assessment of 3 batches of intact Monoclonal Antibody products N-Glycans O-Glycans Intact Mass by MALDI or ES MS Monosaccharide Composition Analysis (LC & MS) Monosaccharide Composition Glycan Population Screening Glycan Antennary Profile Glycosylation Site Linkage Analysis Permethylation MALDI, Nanospray-MS/MS & Linkage analysis LC & MS methods Reduction Carboxymethylation Specific Protease Digest PNGase F Sep-pak Reductive elimination ORIGINATOR STAGE 1 • Regulatory Consultancy and Advice • Formulation Development • Quality Control, Batch Consistency, Batch Release • Microbiology, Mycoplasma, Mycobacteria, Sterility • Virology (bulk harvest, bulk purified, final product) • Stability Storage (ICH) and Testing • Process-Related Impurities • Product-Related Impurities (Host Cell Protein, Host Cell DNA) • ContainerTesting (extractables/leachables) STAGE 2 STAGE 3 STAGE 4 SIDE-BY-SIDE COMPARISON PRE & POST-COMPARABILITY • Chemistry testing of raw materials – Purity, identity & stability – Virus & microbial detection – Pre-MCB screening – MCB, WCB, bulk characterization – EPC – Genetic stability • Primary and higher order structure • ICH Q6B analytical regime • Qualitative and Quantitative asses- sment of Quality At- tributes of multiple batches • Functional/potency: – In-vitro assays – Bioassays, ADCC – Cell-based assays – Immunogenicity and biomarkers • Safety - Immunology - Virology • Clinical trials • Comparative PK/PD studies in healthy and target populations • Clinical efficacy in randomized parallel trials in sensitive populations • Regulatory Affairs • Bioanalysis CELL LINE & PROCESS DEVELOPMENT PHYSICOCHEMICAL BIOLOGICAL ACTIVITY PRE-CLINICAL CLINICAL COMPARISON & BIOANALYSIS • Determination of Exact Sequence & Structure • MS/MS de-novo sequencing • Protein/Glycoprotein sequencing • Determination of PTMs • Quality Target Pro- duct Profile defined T2144 STRUCTURAL CHARACTERIZATION AND CONFIRMATION 1. Amino acid sequence 2. Amino acid composition 3. Terminal amino acid sequence 4. Peptide map 5. Sulfhydryl group(s) and disulfide bridges 6. Carbohydrate structure PHYSICOCHEMICAL PROPERTIES 1. Molecular weight or size 2. Isoform pattern 3. Extinction coefficient 4. Electrophoretic pattern 5. Liquid Chromatographic pattern 6. Spectroscopic profiles Fuc Asn - GlcNAc-GlcNAc- Man Man – GlcNAc - Gal Man - GlcNAc - Gal
