Analytical Tools for Developing Biosimilars: Part 2, Peptide Analysis

Scott Berger

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Advancements in High-Resolution Analytics for the Characterization of Innovator and Biosimilar Therapeutics

Infliximab Biosimilar: An Analytical Comparison at the Peptide Level

In the following biosimilar comparability study of infliximab performed by Waters scientists, three batches of innovator infliximab (produced in the SP2/0 murine cell line) and three batches of a candidate biosimilar infliximab (CHO cell derived) were compared using the Waters Biopharmaceutical Platform Solution with UNIFI.

Samples were analyzed at the level of the intact protein, protein subunits, protein digest, released glycan fraction, and for aggregation and charge variant profiles. In most workflows, each of the six samples was analyzed in triplicate to establish baseline analytical reproducibility.

Confirmation of the primary structure (i.e., sequence) is fundamental for establishing biosimilarity with an innovator product. This question can be indirectly addressed at the level of intact antibody mass analysis and antibody subunit mass analysis studies, but requires high-coverage peptide mapping studies to demonstrate the linear order of amino acids within the protein chains. These analyses also serve to define product variation for attributes such as glycosylation, terminal processing, and other protein modifications.

 

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In this three-part series, we’re going to review how analytical LC and MS technologies combine with software and informatics to facilitate biosimilar development at three levels:

Reduced peptide mapping

Confirmation of the primary structure (sequence) is key in verifying the true candidacy of a potential biosimilar. A standard methodology for IgG primary structure confirmation is UPLC/MSE peptide mapping on a disulfide reduced sample. Reduced UPLC/MSE peptide maps can assist in highlighting and identifying differences between protein sequences and modification variants between the candidate biosimilar and innovator. MSE is a data independent MS acquisition technique where quantitative MS profiles and MS fragmentation data are acquired on all peptides within a peptide mapping analysis.

Disulfide reduced (dithiothreitol) peptide mapping samples were alkylated and analyzed by UPLC/MSE to address the requirements for establishing protein sequence comparability against innovator, and to identify modified peptide variants and determine their relative abundance.

All peptide mapping experiments were performed on an ACQUITY UPLC Peptide BEH C18, 1.7-μm, 2.1 x 150 mm Column on the same Biopharmaceutical Platform Solution with UNIFI instrument configuration (UPLC with QTof MS) that was used for intact mass analysis. Data were automatically acquired, processed, and reported using the UNIFI Scientific Information System. Equivalent high protein coverage was obtained for both innovator and biosimilar light and heavy chains. MSE fragmentation support enabled high confidence assignments, and the analysis confirmed the sequence of terminal peptides in both biosimilar and innovator infliximab samples.

While the trypsin-based analysis established comparability at the peptide level, the regulatory requirements are to establish the comparability for the absolute sequence of amino acids in innovator and biosimilar products, excluding common processing modifications such as the C-terminal Lys on the heavy chains. The reality of this requirement is that more extensive mapping studies, typically employing other complementary digestion enzymes, are required to confirm fragmentation at every peptide bond within the protein to an acceptable level of certainty.

A typical challenge encountered in peptide mapping and the biosimilar comparability exercise is the detection, monitoring, and quantitative comparison of peptide modifications on the innovator and candidate biosimilar. In one example, HC peptide T24 contains a “hot spot” xxxDGxxx sequence with high potential for Asp Isomerization, where aspartic acid is non-enzymatically converted to isoaspartic acid. IsoAsp can potentially be immunogenic, can affect biological activity, and can alter the pharmacokinetics of therapeutic peptide and protein drugs. This isomeration product will typically resolve from unmodified peptide under UPLC mapping conditions, and the modified peptide will possess a common mass with the unmodified form.

Processed results showed that the T24 peptide in innovator infliximab contained approximately of the 0.2% IsoAsp variant, whereas the candidate biosimilar contained the variant at levels of 0.1%. This ability to reliably detect, identify, and quantify low levels of variant peptide forms is critical to arguing the case for confidence of biosimilarity.

Non-reduced peptide mapping

Protein structure does determine function, and it is important to establish that higher order, or 3D, structure is conserved in the biosimilarity exercise. This not only includes a number of physicochemical studies (CD, HDX, NMR, AUC…) beyond discussion here, but also includes an extension of the primary structure exercise: the determination of Cys-Cys disulfide bonding patterns. Disulfide bond formation plays a critical role in stabilizing the 3D-folded structure and maintaining the biological activity of therapeutic proteins.

Disulfide scrambling (mispairing) can occur either during the production of therapeutic proteins, during downstream processing steps, or when the purified biotherapeutic samples are exposed to environmental stress. Disulfide breakage and scrambling can manifest as protein misfolding and product aggregation. Thus, it is crucial to confirm the presence of expected disulfide linkages, and to prove the absence of scrambled disulfides in therapeutic proteins, to ensure drug quality and to satisfy regulatory authorities.

Disulfide bond connectivity, or binding pattern, is typically determined by non-reduced peptide mapping. Infliximab contains 16 S-S bonds (12 intra-chain, and 4 inter-chain) with molecular symmetry of an IgG1 molecule generating eight unique disulfide-bonded peptides expected within the non-reduced map. Infliximab samples were denatured, alkylated, and digested with trypsin to generate non-reduced digests for UPLC/MSE analysis.

UNIFI Software was used to automate the UPLC/MSE data acquisition, processing, and reporting of infliximab the non-reduced peptide map of the candidate biosimilar. A data filter capability available in the data review and reporting elements of UNIFI Software enables a customized view of the data, limiting data to the display of only disulfide-containing peptides.

With this filter applied to the data, all eight expected disulfide bonded peptides were confirmed in batches of innovator and biosimilar samples, all with confirmation by fragment ions from the UPLC/MSE acquisition. A subsequent search for scrambled disulfides, with more limited in-silico digestion and variable modification search criteria to limit combinatorial search requirements, did not detect evidence of scrambled disulfides in either innovator or candidate biosimilar.

See the method and data from our automated disulfide bond mapping study comparing the innovator with the infliximab biosimilar.

Next: Glycosylation, aggregation, and charge variant analysis

 

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