Analytical Tools for Developing Biosimilars: Part 3, Glycosylation, Aggregation, and Charge Variants

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

Infliximab Biosimilar: An Analytical Comparison of Glycosylation, Aggregation, and Charge Variants

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 for 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.

In this three-part series, we’re reviewing how analytical UPLC and QTof-MS technologies, that have been purposefully designed for biopharmaceutical characterization, combine with software and informatics to facilitate biosimilar development at three levels:

• Intact protein and subunit analysis
• Peptide mapping, reduced and non-reduced
• Glycosylation, aggregation, and charge variant analysis

Glycosylation analysis

In all antibodies and many commercialized biotherapeutics, glycosylation state has a direct and pronounced effect of the structure, stability, serum half-life, immunogeneicity and bioactivity of the molecule, and constitutes a critical quality attribute (CQA).

Characterizing and comparing the glycosylation profile of infliximab began by examining the glycosylation profiles from intact and reduced subunit mass data. Through these analyses it became clear that the innovator and candidate biosimilar shared many common glycoforms, but that measurable differences in glycoform levels between the samples would be expected between the products. Analysis at the glycopeptide and released glycan level enabled us to address these differences in a more sensitive and targeted analysis.

UPLC/MS^E peptide maps revealed that both innovator and biosimilar infliximab samples were glycosylated on heavy chain tryptic peptide T26. Within the peptide map, we can follow the abundances of individual glycoforms of this peptide detected as modified peptides within the mapping study. In one application of this approach, we showed that the G1F glycoform abundance varied among three batches each of the innovator and biosimilar.

The percentage of the G1 glycoform, automatically calculated by UNIFI Software, showed biosimilar infliximab levels around 18%, outside the range of the innovator batches (around 25 to 28%) that were tested. As would be expected from properly executed studies, similar abundance trends were observed the G1F glycoform when examining the heavy chain subunit intact mass data.

Glycopeptide trend line plots were automatically generated within UNIFI Software to highlight T26 glycopeptide glycovariation across all batches and replicates tested. Glycopeptide analysis is useful as orthogonal results to intact mass profiling and released glycan analysis (below), but can be particularly important when a biotherapeutic contains multiple distinct glycosylation sites.

N-linked glycans were enzymatically (PNGase F) released from the batches of innovator and candidate biosimilar, were labeled with a 2-AB fluorescent tag (FLR), and analyzed by hydrophilic interaction liquid chromatography (HILIC) coupled to fluorescence and accurate mass detection. This sample preparation and cleanup was accomplished using a Waters GlycoWorks Kit, with analysis by the Glycan Application Solution, which includes access to the Waters GU Glycan Library; this is an available option with the Waters Biopharmaceutical Platform with UNIFI. Collected data from the FLR detection channel is used for peak identification and quantification, and accurate mass data is used for confirmation of peaks first assigned by retention. (Our study was done prior to the introduction of our GlycoWorks RapiFluor-MS Kit for labeling and releasing N-glycans, which optimizes that process.)

In order to produce glycan profiles that are easily reproducible day-to-day and instrument-to-instrument, the use of a normalized/calibrated retention time is part of the standard methodology utilized for this analysis.

Glycan retention is calibrated using a labeled dextran ladder (poly-glucose) that is run bracketing the unknown samples, generating a plot of glucose ladder length vs. retention time. The recalibration of peaks into GU or glucose unit retention values overcomes many common sources of experimental variability in released glycan analysis, and enables GU-based peak assignments from a search of the Waters GU Glycan Library for 2AB-labeled glycans. UNIFI Software automated the GU calibration, peak assignments from the GU database, mass confirmation of those assignments, and quantification from the FLR data channel.

In the case of infliximab, there were considerable differences between the innovator and biosimilar products with respect to glycosylation. This was not surprising since the innovator’s infliximab was expressed from an SP2/0 mouse cell line, while the biosimilar infliximab was expressed from a CHO cell line. 24 glycan species were identified in the innovator sample and 18 identified in the biosimilar. This greater variety of N-glycans detected in the innovator was primarily due to the detection of two classes of sialic acid containing glycans (NeuAc and NeuGc) versus only the NeuAc forms observed in the CHO derived biosimilar candidate.

A consistently low (~1%) level of 1,3 alpha-Galactose (potentially immunogenic) species were also detected uniquely in the innovator. Glycosylation represented the clearest primary structural difference between the innovator and this set of candidate biosimilar samples, and would likely require the biosimilar sponsor to establish that the observed differences did not negatively impact the efficacy, stability, and safety profiles established by the innovator company.

See how we’ve updated the method and data for comparing the innovator and biosimilar of infliximab to use RapiFluor-MS label along with its corresponding GU Glycan Library.

Aggregate analysis

Higher order structure is another critical attribute in biotherapeutics, including aggregates that are implicated in enhancing product immunogenicity negatively affecting product safety and efficacy (potency, clearance). Size exclusion chromatography (SEC) is commonly used for aggregate analysis of mAbs and other biotherapeutic products.

Overlaid SEC chromatograms of the innovator and candidate biosimilar established that dimers are present at higher levels within the candidate biosimilar.

Automated batch-to-batch comparison of the samples showed that 4-6% of the protein was dimerized within the biosimilar batches, a level nearly 10-fold higher than for the innovator product. There was also considerable variability in dimer content among the biosimilar batches, suggesting that downstream process improvements or formulation adjustments would likely be necessary to reduce aggregate to levels more typical of a marketed mAb product.

Charge variant analysis

Protein charge variants reflect amino acid modifications, glycan composition, and structural variants of a biotherapeutic. Monitoring the profile of acidic and basic variant peaks flanking the main product peaks is commonly employed as a test of identity, product quality, and process stability in a purified biotherapeutic. A pH gradient as well as a salt gradient may be used to achieve optimal separation selectivity for charge variant analysis of infliximab. The use of Carboxypeptidease B to remove the C-terminal lysine of the heavy chains can simplify the charge variant profile, raise signal for lower abundance forms above detection limits, and enable monitoring of more charge variants within the analysis.

Waters’ Biopharmaceutical Platform Solution with UNIFI supports Auto•Blend Plus Technology, which automates the generation of mobile phases from reservoirs of simple concentrated stock solutions of buffers, salt and water, simplifying buffer preparation and reducing the risk of human error in the preparation of IEX buffers. The Auto•Blend Plus gradient method ensures that the desired pH and ionic strength are reproducibly delivered to the IEX column for charge variant separations. Charge variants can be optimally resolved by application of a salt gradient, pH gradient, or combination of both. Auto•Blend Plus enables the scouting of ideal conditions in method development, and simplifies execution of those methods when optimized.

Ion exchange chromatography (IEX) with UV detection analysis of intact infliximab and LC/MS analysis of deglycosylated intact infliximab were used to compare lysine variation in innovator and biosimilar infliximab batches. IEX Lys variant data were well correlated with results from intact mAb and heavy chain intact mass results. This was demonstrated using the example of the null-lysine mAb variant.

See how Auto•Blend Plus helps future-proof the biopharma QC lab by automating mobile phase delivery, improving pH consistency in SEC methods, or for IEX method development.

Summary of Biosimilar Characterization Studies

Automation of analytical techniques can minimize error in method execution as well as increase productivity. In this studies discussed here, we have shown how Waters’ analytical technologies and informatics tools for biotherapeutics have enabled efficient characterization of both innovator and candidate biosimilar infliximab. The workflow assessed similarity of primary structure (sequence), post-translational modifications (PTMs), glycosylation, disulfide linkages pattern, aggregation levels, and charge variant profiles, all using the Biopharmaceutical Platform Solution with UNIFI.

The capabilities of UNIFI Scientific Information System, used in conjunction with Waters’ UPLC, optical detection, and MS technologies, enable researchers to readily generate data, automate processing, and effectively communicate results throughout an organization. This enables biotherapeutic and biosimilar developers to streamline their processes, share their methods, and reduce time to market, ensuring profitability and patient access to modern biotherapeutic drugs.

Review:

• Read our introduction on biosimilar development and our analytical focus on how to compare biosimilars at the intact protein level
• Read our chapter on peptide mapping (reduced and non-reduced) in biosimilar development

Related reading:

• Download the application note: Combining RapiFluor-MS and UNIFI Scientific Information System for a Total N-linked Glycan Solution for Innovator vs. Biosimilar Infliximab Comparisons
• Download a pdf of this white paper
• See additional application examples of our biopharmaceutical characterization technology platform
• About the Biopharmaceutical Platform Solution with UNIFI
• About our solutions for biosimilars