Biosimilar Monoclonal Antibodies Research Articles

Revisions to the requirement of the Japanese clinical study data for biosimilar developments in Japan

ABSTRACT Background The ‘Questions and Answers (Q&A)’ document regarding Japanese biosimilar guideline elucidated that Japanese participant enrollment in at least one comparative clinical study was required for the marketing authorization application (MAA) of biosimilars in Japan. Research design and methods To discuss the requirement of Japanese clinical study data for biosimilar development, the trend in comparative clinical studies conducted for approved biosimilars of monoclonal antibodies and fusion proteins was analyzed, and the consistency of the results between the overall population and the Japanese population according to the publicly available information was reviewed. Results The number of comparative clinical studies enrolling Japanese participants was 25 cases, and the type and percentage were 13 (52%) and 12 (48%) cases of comparative pharmacokinetic study and comparative efficacy study, respectively. In all comparative clinical studies, consistent results between the overall population and the Japanese population were shown. Conclusions Our study indicated that Japanese participant enrollment in comparative clinical studies may not always be necessary for biosimilar development when certain conditions are satisfied. This has been described in the revised Q&A document published by the Ministry of Health, Labour and Welfare in January 2024.

Biomass specific perfusion rate as a control lever for the continuous manufacturing of biosimilar monoclonal antibodies from CHO cell cultures.

Continuous manufacturing enables high volumetric productivities of biologics such as monoclonal antibodies. However, it is challenging to maintain both high viable cell densities and productivities at the same time for long culture durations. One of the key controls in a perfusion process is the perfusion rate which determines the nutrient availability and potentially controls the cell metabolism. Cell Specific Perfusion Rate (CSPR) is a feed rate proportional to the viable cell density while Biomass Specific Perfusion Rate (BSPR) is a feed rate proportional to the biomass (cell volume multiply bycell density). In this study, perfusion cultures were run at three BSPRs in the production phase. Low BSPR favored a growth arresting state that led to gradual increase in cell volume, which in turn led to an increase in net perfusion rate proportional to the increase in cell volume. Consequently, at low BSPR, while the cell viability and cell density decreased, high specific productivity of 55 pg per cell per day was achieved. In contrast, the specific productivity was lower in bioreactors operating at a high BSPR. The ability to modulate the cell metabolism by using BSPR was confirmed when the specific productivity increased after lowering the BSPR in one of the bioreactors that was initially operating at a high BSPR. This study demonstrated that BSPR significantly influenced cell growth, metabolism, and productivity in cultures with variable cell volumes.

Safety of marketed biosimilar monoclonal antibody cancer treatments in the US: a disproportionality analysis using the food and drug administration adverse event reporting system (FAERS) database

ABSTRACT Background By 31 December 2022, the United States Food and Drug Administration (FDA) has approved 12 biosimilar monoclonal antibody cancer treatments. This study detected disproportionate adverse event (AE) reporting signals and compared safety profile of individual biosimilars to their originator biologics and between each pair of biosimilars. Research design and methods The FDA Adverse Event Reporting System data (6/1/2018–12/31/2022) were used to identify AE reports for rituximab, bevacizumab, trastuzumab, and their marketed biosimilars. Reporting odds ratios and empirical Bayesian geometric mean were computed to detect reporting disproportionality in serious, death, and specific AEs between studied biologics/biosimilars and all other drugs. Results Significant AE reporting signals were identified: 1) death for biological rituximab, pruritus for biosimilar rituximab-pvvr, and infusion-related reactions for biological rituximab and biosimilar rituximab-pvvr (significantly higher ROR for rituximab-pvvr than biological rituximab, p < .0001); 2) death for biological bevacizumab and biosimilar bevacizumab-bvzr (significantly higher ROR for bevacizumab-bvzr than biological bevacizumab, p < .0001), hypertension, platelet count decreased (PCD), and proteinuria for biological bevacizumab and biosimilar bevacizumab-awwb (significantly higher ROR of PCD for bevacizumab-awwb than originator bevacizumab, p = .001); and 3) rash for biosimilar trastuzumab-anns. Conclusions Findings call for large, longitudinal studies to examine causality of certain AEs with rituximab-pvvr and bevacizumab biosimilars.

Effects of thermal treatment on quality of biosimilar and originator monoclonal antibodies

Investigating products under stress conditions provides valuable information for assessing stability, biosimilarity, and degradation behaviors during monoclonal antibody (mAb) development. Proper sample preparation is crucial for accurately evaluating the biosimilarity and effects of stress conditions in comparability assessment, where these studies guide biosimilar mAb development steps. Enzymatic and chemical treatments applied during sample preparation of mAbs generally require treatment of samples in temperatures higher than the storage temperatures of antibodies.In this study, samples of a TNF-α inhibitor IgG1 biosimilar (BIO) and its originator (OR) were treated for 7 days at commonly used temperatures during sample preparation. Alterations in the intact IgG, size variants, charge variants, binding kinetics, and post-translational modifications (PTMs) were investigated with CE-SDS, SE-UPLC, icIEF, SPR, and LC-MS/MS, respectively. Samples treated at 50 °C exhibited significant degradation, while minor differences were observed in samples treated at 37 °C. Monomer and intact IgG levels were decreased to levels below 97 % and 94 %, respectively, after 7 days of thermal treatment at 50 °C for both BIO and OR samples. Similar rates of degradation were observed between the treated biosimilar and originator samples. The percent monomer degradation rate between the biosimilar and the originators was similar at 50 °C (p = 0.32). Thermal treatment increased acidic variant levels in the products of the BIO (23.10 %) and OR (23.16 %). During post-translational modification monitoring, an increase in pyroglutamic acid formation and a decrease in C-terminal lysine were observed after thermal treatments. Acidic variant alterations were associated with asparagine deamidation and N-terminal pyroglutamic acid formation. Post-translational modifications were mainly located at the Fc domain, with methionine oxidation and asparagine deamidation as the main modifications occurring at the Fab domain.In conclusion, these results revealed that prolonged thermal treatment under elevated temperatures induces molecular alterations, thereby facilitating the degradation of IgG1. In addition, our findings indicate that both BIO and OR lots exhibit similar degradation profiles when subjected to thermal treatments.

From Efficiency to Yield: Exploring Recent Advances in CHO Cell Line Development for Monoclonal Antibodies.

The increasing demand for biosimilar monoclonal antibodies (mAbs) has prompted the development of stable high-producing cell lines while simultaneously decreasing the time required for screening. Existing platforms have proven inefficient, resulting in inconsistencies in yields, growth characteristics, and quality features in the final mAb products. Selecting a suitable expression host, designing an effective gene expression system, developing a streamlined cell line generation approach, optimizing culture conditions, and defining scaling-up and purification strategies are all critical steps in the production of recombinant proteins, particularly monoclonal antibodies, in mammalian cells. As a result, an active area of study is dedicated to expression and optimizing recombinant protein production. This review explores recent breakthroughs and approaches targeted at accelerating cell line development to attain efficiency and consistency in the synthesis of therapeutic proteins, specifically monoclonal antibodies. The primary goal is to bridge the gap between rising demand and consistent, high-quality mAb production, thereby benefiting the healthcare and pharmaceutical industries.

Comparative Analysis of Glycoform Profiles Between Biosimilar and Originator Monoclonal Antibodies by Liquid Chromatography–Mass Spectrometry

Glycosylation is considered as a critical quality attribute for monoclonal antibodies (mAbs) and needs routine monitoring during production. This study aims to compare the glycoform profiles of biosimilar and four originator mAbs using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization-quadrupole time of flight-mass spectrometry (ESI/Q-TOF MS). The resultant mass spectrum showed that seven different glycoform pairs, including G0F–GN/G0, G0F–GN/G0F, G0F/G0F, G0F/G1F, G1F/G1F, G1F/G2F, and G2F/G2F were identified via intact mass analysis for all tested mAb samples. The correct identification of each glycoform pair was achieved by comparing the observed mass with its theoretical mass using high-resolution mass spectrometry data (with mass accuracies of less than 100 ppm). The most abundant paired glycoforms detected at the intact protein level are G0F/G0F and G0F/G1F, with relative abundance ranges of 38.45 – 43.43% and 19.32 – 22.20%, respectively. The obtained data demonstrated that biosimilar and originators have the same types of glycoform pairs, and the relative abundances of each pair were comparable among biosimilar and four originator mAb samples. Additionally, the reduced mass analysis revealed that five different glycans (G0F–GN, G0, G0F, G1F, and G2F) were attached to the heavy chain of the mAb, and the relative abundance of G0F ranged from 75.21 to 77.90%. The detected mass accuracies for reduced mass analysis were below 25 ppm. The results of the intact and reduced mass analyses showed that the biosimilar is similar to its originator in terms of glycoform percentages and molecular masses.

An evaluation of the utilisation of biosimilar monoclonal antibody drugs in Ireland and barriers to their usage.

While biologic drugs have demonstrated efficacy across a range of indications, patient access to these drugs is constrained due to their high cost. Biosimilars provide a means to increase patient access while reducing the financial burden. The primary objective was to determine the current usage of biosimilar and reference trastuzumab and rituximab in four Irish hospitals. A secondary objective involved determining barriers to biosimilar usage. This project involved a retrospective chart review to analyse the usage of reference and biosimilar versions of trastuzumab and rituximab. Additionally, a prospective cross-sectional study identified barriers to the usage of biosimilars via the distribution of a novel questionnaire to patients, pharmacists, doctors and students. The utilisation of biosimilar intravenous trastuzumab and rituximab ranged from 39 to 100%, and 0 to 89%, respectively. A total of n = 479 questionnaire responses were included. Biosimilar awareness was significantly lower among 'Doctors and Medical Students' (45.3%; 95% [CI, 33.8-57.3%]) compared to 'Pharmacists and Pharmacy Students' (97.1%; 95% [CI, 94-98.8%; comparison p < 0.001]). A significant majority of healthcare professionals agreed biosimilars should have consistent nomenclature (p < 0.001). A significant majority of patients (87.3%, 95% [CI, 81.3-92%; p < 0.001]) indicated that they would agree to commence using a biosimilar medicine. Biosimilar versions of trastuzumab and rituximab were in use to a variable extent. There remains a considerable opportunity to further increase the usage to maximise their potential benefits. A series of challenges were identified including reduced awareness among the medical profession and lack of clear nomenclature.

Automatic peak annotation and area estimation of glycan map peaks directly from chromatograms

AbstractThe present bottleneck in biosimilar bioprocess development has become evaluation of analytical results, due to recent advances in analytics, such as automated sample preparation and development of high‐throughput methods. Currently automated chromatogram integration and annotation is only efficient for simple chromatograms. In an ever more competitive field of biosimilars, this represents a serious drawback because chromatographic analytical methods that provide some of the most valuable physicochemical quality attributes of the product also require careful chromatogram integration and annotation. This work focuses on the glycan mapping analytical method as utilized in development of monoclonal antibody biosimilars, evaluating more than 2000 chromatograms spanning the life cycle of multiple biosimilar development projects. It proposes a modified workflow by implementing automatic machine learning algorithms to determine the proportion of specific relevant glycan species in a sample directly from the chromatogram. Data preparation and analysis is performed using a pipeline approach. Pipeline is a modular design of data processing where signal “travels” through various active modules in a series. Each module performs a specific function or transformation on the signal and propagates the transformed signal to the next module. The pipeline is designed in a way that modules can be independently improved and exchanged. Module functions currently implemented are chromatogram resampling by spline interpolation, baseline removal by asymmetric least squares, peak alignment using parametric time warping, and quantification of the relative proportion of a glycan species using partial least squares regression. Hyper‐parameters of the pipeline are then optimized using the Nelder–Mead method. The approach stands out for its ability to accommodate a broad landscape of samples, covering multiple different proteins in different stages of biosimilar development, analyzed using different adaptations of the glycan map analytical method. The pipeline presents an intuitive, flexible, and creatively simple method design capable of providing reliable results for a wide range of glycan species essential for biosimilar development. It enables transparent, faster, and less subjective evaluation of analytic raw data (from sample to result). Furthermore, our automated approach maintained an accuracy comparable with manual integration thus demonstrating its readiness for implementation in the conservative and highly regulated environment. The presented methodology reduces the cost and time of biosimilar development and should be applicable for any chromatogram‐based analytical method.

Temperature Upshifts in Mammalian Cell Culture: A Suitable Strategy for Biosimilar Monoclonal Antibodies?

Temperature downshifts are the gold standard when setting up control strategies for mammalian cell culture processes. These shifts are performed to prolong production phases and attain heightened levels of productivity. For the development of biosimilars, however, the bottleneck is in achieving a prespecified product quality. In a late-stage development project, we investigated the impact of temperature shifts and other process parameters with the aim of optimizing the glycosylation profile of a monoclonal antibody (mAb). We applied a design of experiments approach on a 3 L scale. The optimal glycosylation profile was achieved when performing a temperature upshift from 35.8 °C to 37 °C. Total afucosylated glycan (TAF) decreased by 1.2%, and galactosylated glycan species (GAL) increased by up to 4.5%. The optimized control strategy was then successfully taken to the manufacturing scale (1000 L). By testing two sets of set points at the manufacturing scale, we demonstrated that the statistical models predicting TAF and GAL trained with small-scale data are representative of the manufacturing scale. We hope this study encourages researchers to widen the screening ranges in process development and investigate whether temperature upshifts are also beneficial for other mAbs.

Investigation of Immunogenicity Assessment of Biosimilar Monoclonal Antibodies in the United States.

Immunogenicity is critical for biologics. However, reference biologics labeling documents do not necessarily mention immunogenicity impact, rendering the development of biosimilars more challenging. We aimed to investigate the comparative assessment of immunogenicity profiles between biosimilars and their respective reference biologics in the review reports of the biosimilar monoclonal antibody applications approved by the Center for Drug Evaluation and Research (CDER), US Food and Drug Administration (FDA) as of March 13, 2022, covering 22 applications approved between April 5, 2016, and December 17, 2021. The maximum differences in anti-drug antibody (ADA) and neutralizing antibody (NAb) incidences between biosimilars and reference products mostly fell within ± 15% (-13.6% to 12%) and ± 20% (-17.4% to 17.1%, except extreme values of -23.4% and 66.7%), respectively. In comparison with antineoplastic agents, more immunosuppressants had ADA-positive (11/11, 100.0% vs. 8/10, 80.0%)/NAb-positive (11/11, 100.0% vs. 3/10, 30.0%) subjects, and the distribution of the aforementioned incidence differences was wider. The investigated biosimilars with available data for analysis demonstrated a high degree of consistency with their reference products in terms of the impact on pharmacokinetic parameters. No increase in immunogenicity was found in available switching studies. Most (16/22, 72.7%) biosimilars were issued post-marketing requirements that were not directly related to immunogenicity concerns. The FDA considered the totality of evidence assessing clinical consequences of immunogenicity differences, if any. Additional information on titers and subgroup analysis may be warranted to elucidate the critical attributes of immunogenicity impact and to aid in forming cost-effective strategies for biosimilar development.

Comparative Safety Profiles of Oncology Biosimilars vs. Originators in Europe: An Analysis of the EudraVigilance Database.

In the last decades, the clinical management of oncology patients has been transformed by the introduction of biologics. The high costs associated with the development and production of biologics limit patient access to these therapies. The expiration of exclusive patents for biologics has led to the development and market introduction of biosimilars, offering the reduction of costs for cancer treatments. Biosimilars are highly similar to the reference products in terms of structure, biological activity, efficacy, safety, and immunogenicity. Therefore, the monitoring of biosimilars' safety in real-world clinical practice though pharmacovigilance is essential. This study aimed to analyze the post-marketing pharmacovigilance data of biosimilar monoclonal antibodies used in oncology and compare them with respective reference products. Data of a 2-year period (1 January 2021-31 December 2022) were retrieved from EudraVigilance, and descriptive and comparative analysis were performed using the Reporting Odds Ratio to evaluate the distribution of medicine-reaction pairs related to biosimilars of three antitumor biological products and their corresponding reference products: bevacizumab, rituximab, and trastuzumab. The results showed that most frequently reported ADRs for biosimilars were non-serious and consistent with the safety profiles of reference products. These findings provide reassurance regarding safety equivalence of biosimilars and support their use as valid alternatives to originator biologics.

RWD30 Adverse Event Reporting of Marketed Biosimilar and Biological Monoclonal Antibody Cancer Treatments in the United States

By September 8, 2022, 10 biological monoclonal antibody (mAb) biosimilar products for cancer treatment had been approved and marketed in the United States (U.S.). These biosimilars were approved based on bioequivalence studies conducted among healthy volunteers, but their post-marketing investigations for safety are limited. This research aims to examine adverse event (AE) reporting patterns and disproportionate reporting signals for mAb biosimilars in the U.S. compared to their originator biologics.

Adverse event reporting of marketed biosimilar and biological monoclonal antibody cancer treatments in the United States

ABSTRACT Background By 8 September 2022, 10 biological monoclonal antibody (mAb) biosimilar products for cancer treatment had been approved and marketed in the United States (US). This study examined adverse event (AE) reporting patterns and disproportionate reporting signals for mAb biosimilars in the US compared to their originator biologics. Research design and methods The US Food and Drug Adverse Event Reporting System database was used to identify AE reports for biological rituximab, bevacizumab, trastuzumab, and their marketed biosimilars. Proportions of patient age, sex and type of reporters of AEs were described for these reports. Reporting odds ratios (RORs) with 95% confidence intervals were calculated to compare reporting disproportionality in serious, death, and specific AEs between mAb biologics/biosimilars (index) and all other drugs. Breslow-Day statistic was used to determine homogeneity in RORs between each mAb biologic–biosimilar pair at p < 0.05. Results We observed no risk signals of serious or death AE reporting for all three mAb biosimilars. A signal of disproportionate reporting of death was detected between biological and biosimilar bevacizumab (p < 0.05). Conclusions Our findings support the similarity in signals of disproportionate AE reporting between mAb originator biologics and biosimilars, except for death between biological and biosimilar bevacizumab.

Adverse event reporting of marketing biosimilar and biological monoclonal antibody cancer treatments

Adverse event reporting of marketing biosimilar and biological monoclonal antibody cancer treatments

Trastuzumab Charge Variants: a Study on Physicochemical and Pharmacokinetic Properties.

Post-translational modifications in bioprocessing and storage of recombinant mAbs are the main sources of charge variants. While the profile of these kinds of variants is considered an important attribute for the therapeutic mAbs, there is controversy about their direct role in safety and efficacy. In this study, the physicochemical and pharmacokinetic (PK) properties of the separated charge variants belonging to a trastuzumab potential biosimilar, were examined. The acidic peaks, basic peaks, and main variants of trastuzumab were separated and enriched by semi-preparative weak cation exchange. A panel of analytical techniques was utilized to characterize the physicochemical properties of these variants. The binding affinity to HER2 and FcγRs and the PK parameters were evaluated for each variant. Based on the results, the charge variants of the proposed biosimilar had no significant influence on the examined efficacy and PK parameters. During the development and production of biosimilar monoclonal antibodies, evaluating the effect of their charge variants on efficacy and PK parameters is needed.

Stability Evaluation of the Biosimilar Monoclonal Antibody Using Analytical Techniques.

Determination of the drug substance (DS) and drug product (DP) stability is especially important for biosimilar monoclonal antibodies since it can affect the quality, efficacy, and safety of the drugs. The main objective of this study was to determine the stability of the biosimilar candidate (TUR01) using state-of-the-art (current) analytical techniques. Analytical techniques used in this study were isoelectric focusing on capillary electrophoresis, capillary electrophoresis-sodium dodecyl sulfate, size exclusion chromatography-ultra-high performance liquid chromatography, binding affinity, and physicochemical and microbiological tests. DS was kept in polyethylene terephthalate copolyester, glycol modified (PETG) bottles at ≤-65.0°C and 5.0 ± 3.0°C for 18 months, where the pre-filled syringe stability study was conducted at 5.0 ± 3.0°C for 24 months and 25.0 ± 2.0°C/60% ± 5 relative humidity (RH) for 6 months. The accelerated condition for DS was accepted as 5.0 ± 3.0°C, while it was 25.0 ± 2.0°C for the DP. The results indicated that TUR01 DS was stable when it was stored under long-term storage conditions at ≤-65°C and at 5 ± 3°C at least 18 months. Also, TUR01 DP was stable at 5 ± 3°C for 24 months and at 25 ± 2°C with 60.5% RH for 2 months without any significant changes. State-of-the-art analytical techniques proved to be invaluable tools for evaluate the stability of the TUR01 DS and drug product.

Efficacy and Safety of Proposed Biosimilar Natalizumab (PB006) in Patients With Relapsing-Remitting Multiple Sclerosis

Proposed biosimilar natalizumab (biosim-NTZ) PB006 is the first biosimilar monoclonal antibody therapy developed for multiple sclerosis (MS) treatment. To evaluate matching efficacy, safety, and immunogenicity between biosim-NTZ and reference natalizumab (ref-NTZ) in patients with relapsing-remitting MS (RRMS). The Antelope trial was a phase 3, parallel-group, randomized, active-controlled study, conducted between October 2019 and March 2021, with last patient follow-up visit on August 23, 2021. The study took place in 48 centers in 7 countries. Of 531 patients with RRMS aged 18 to 60 years screened, 266 were excluded before randomization in line with study criteria. Eligible participants had 1 or more documented relapse within the previous year and either 1 or more gadolinium-enhancing T1-weighted or 9 or more T2-weighted brain lesions, Kurtzke Expanded Disability Status Scale score of 0 to 5.0 (inclusive), and John Cunningham virus index of 1.5 or less at screening. One patient withdrew consent before dosing. Intravenous infusions every 4 weeks of biosim-NTZ, 300 mg, or ref-NTZ, 300 mg (1:1 randomization), from week 0 to week 44 (end-of-study visit: week 48). At week 24, the ref-NTZ group was rerandomized and 30 patients were switched to biosim-NTZ for the remainder of the study. The primary end point was the cumulative number of new active lesions on magnetic resonance imaging (new gadolinium-enhancing T1-weighted lesions and new/enlarging T2-weighted lesions without double counting) over 24 weeks. Additional end points included further magnetic resonance imaging parameters, annualized relapse rate, and Kurtzke Expanded Disability Status Scale score. Safety, tolerability, and immunogenicity assessments included adverse events, laboratory evaluations, and positivity for anti-John Cunningham virus antibodies and antinatalizumab antibodies. A total of 264 participants (mean [SD] age, 36.7 [9.38] years; 162 [61.4%] female) received treatment with biosim-NTZ (n = 131) or ref-NTZ (n = 133). At week 24, the model-based mean difference in cumulative number of new active lesions between biosim-NTZ and ref-NTZ treatment groups was 0.17 (least square means [SE]: biosim-NTZ, 0.34 [0.34]; ref-NTZ, 0.45 [0.28]; 95% CI, -0.61 to 0.94 within the prespecified margins of ±2.1). No significant differences between treatment groups were observed across secondary efficacy end points, safety, tolerability, or immunogenicity assessments. Biosim-NTZ matched ref-NTZ in efficacy, safety, and immunogenicity for patients with RRMS in the tested setting. This phase 3 trial supports proposed biosim-NTZ as a biosimilar alternative to ref-NTZ for treating RRMS. ClinicalTrials.gov Identifier: NCT04115488.

A Data Driven Approach to Support Tailored Clinical Programs for Biosimilar Monoclonal Antibodies.

Biosimilar monoclonal antibodies (mAbs) have been approved in the European Union since 2013 and have been demonstrated to reduce healthcare costs and to expand patient access. Biosimilarity is mainly established on the basis of demonstrated similarity of relevant quality attributes (QAs), determined by comprehensive physiochemical and functional analyses, and demonstration of bioequivalence. In addition, comparative efficacy/safety studies have been requested for all approved biosimilar mAbs so far, although the European Medicines Agency (EMA) Guidelines state that such confirmatory clinical trials may not be necessary in specific circumstances. In order to evaluate the degree of analytical similarity, how residual uncertainty regarding biosimilarity was resolved, and the value of clinical data, we analyzed the quality and clinical data packages for authorized adalimumab (7 products) and bevacizumab (5 products) biosimilars. The percentage of biosimilar batches meeting the similarity range for QAs, as defined by the biosimilar manufacturer based on a comprehensive characterization of the EU reference product (RP), was determined and clinical data were reviewed. Our analyses show that QAs of approved adalimumab and bevacizumab biosimilars have varying concordance with the EU-RP similarity range. In this study, we found that clinical efficacy data played a limited role in addressing quality concerns. Therefore, we encourage a regulatory review of the standards for clinical data requirements for mAb and fusion protein biosimilars. This study outlines a quality data driven approach for facilitating tailored clinical programs for biosimilars.

The Mannose in the Mirror: A Reflection on the Pharmacokinetic Impact of High Mannose Glycans of Monoclonal Antibodies in Biosimilar Development.

Biosimilar development has a well-documented foundation of product quality and extensive comparative analytics providing the bulk of the "totality of the evidence" that a proposed product is biosimilar to its reference product. This work provides a retrospective evaluation of a single critical quality attribute-high mannose glycans for monoclonal antibody biosimilars. Given the well-established conclusion that high mannose glycans can impact pharmacokinetic (PK) profile, we performed a retrospective evaluation of 21 monoclonal antibody biosimilar programs (those licensed before April 2022), their levels of glycans, and the methods used to study them. We provide herein a summary of the methods used and their relative performance. We also present a subset analysis for seven biosimilar products with levels of high mannose that differ from the corresponding reference product (and where other differences in quality attributes between the two that may influence PK profile were not observed or considered minor) and compared the PK profiles. Critically, this analysis has demonstrated that the measurement of glycan profiles is highly precise, reproducible within and across programs, and can detect differences in mannose levels, even those that do not impact PK. These results provide support that analytics rather than pharmacokinetic data may be sufficient to predict whether differences within a certain magnitude of this attribute are likely to impact PK. This work enhances the Agency's understanding of this issue allowing for better understanding of challenges faced by the biotechnology industry developing biosimilars.

Comparison of originator and biosimilar monoclonal antibodies using HRMS, Fc affinity chromatography, and 2D-HPLC.

Due to the complex manufacturing process of therapeutic monoclonal antibodies, it is hardly possible to produce an identical copy of the original product (originator). Consequently, follow-on products (biosimilars) must demonstrate their efficacy being similar to the originator in terms of structure and function. During this process, a variety of analytical methods are required for this purpose. This study focuses on three particularly relevant analytical techniques: high-resolution mass spectrometry, fragment crystallisable (Fc) affinity chromatography, and two-dimensional peptide mapping. Each analytical method proved able to identify specific differences between originator and biosimilar. High-resolution mass spectrometry was used to characterize the glycan pattern. It was shown that a trastuzumab biosimilar did not have the G0:G0F sugar modification identified in the originator. The application of affinity chromatography to rituximab showed that originator and biosimilar interacted differently with the immobilized Fc receptor. Furthermore, 2D-HPLC peptide mapping demonstrated the influence of orthogonality of separation dimensions, leading to differentiation of a rituximab originator and biosimilar.