Analytical Comparability to Evaluate Impact of Manufacturing Changes of ARX788, an Anti-HER2 ADC in Late-stage Clinical Development
Wayne Yu, Mysore P. Ramprasad, Manoj Pal, Chris Chen, Shashi Paruchuri, Lillian Skidmore, Nick Knudsen, Molly Allen, Ying Buechler
Abstract
ARX788 is an anti-HER2 antibody drug conjugate (ADC) developed using Ambrx proprietary Engineered Precision Biologics technology. The manufacturing process of ARX788 has been optimized during the course of early to late-phase clinical development. A comprehensive evaluation of side-by-side comparability between pre- and post-change process for ARX788 drug substance and drug product from a quality perspective was conducted based on ICH Q5E guidelines consisting of batch release assays, physicochemical and biophysical characterization, biological characterization, and forced degradation studies. All results have substantiated a high degree of similarity between the pre- and post-change ARX788 drug substance batches and drug product lots, demonstrating that the process manufacturing changes did not impact product quality.
Introduction
Antibody-drug conjugates (ADCs) are a class of therapeutics that combines an antigen-specific antibody backbone with a potent cytotoxic payload, resulting in an enhanced therapeutic index. The US FDA has currently approved 14 ADCs spanning several targets for hematological and solid tumors. The development of anti-human epidermal growth factor receptor 2 (HER2) agents has been one of the most meaningful advancements in the management of metastatic breast cancer, significantly improving survival outcomes [1]. Two anti-HER2 ADCs, Kadcyla® and Enhertu®, have been approved by the FDA in HER2-positive breast cancer. ARX788 is a next-generation, site-specific anti-HER2 ADC currently in global clinical trials for the treatment of HER2-positive metastatic breast cancer and gastric cancer [2–6]. It is comprised of a humanized HER2-targeting monoclonal antibody (mAb), conjugated to a cytotoxic tubulin inhibitor payload linker, Amberstatin (AS269), via a nonnatural amino acid incorporated into the two heavy chains of the antibody (as shown in Fig 1). Site-specific conjugation occurs when the hydroxylamine in AS269 reacts with the ketone group of the nonnatural amino acid, para-acetylphenylalanine (pAF), forming a highly stable oxime bond.
Materials and methods
Her2 binding ELISA
An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARX788 binding to target receptor HER2. Serially diluted ARX788 DS or DP samples were incubated in HER2-coated ELISA plates, and binding was detected with the addition of anti-human kappa-HRP antibody and incubation with 3,3’,5,5’-Tetramethylbenzidine substrate. Sulfuric acid was added to stop the colorimetric reaction, and plates were read at absorbance wavelength 450 nm. The relative potency of pre-change or post-change ARX788 samples was calculated by comparison of its half-maximal effective concentration (EC50) value obtained from a 4-parameter sigmoidal fit of the dose response curve to the EC50 value of an ARX788 reference standard run on the same assay plate.
Results
Side-by-side batch analysis
The batch release data of the pre-change and post-change DS and DP batches were compared side by side as shown in Table 2 and Table 3, respectively. For DS batches, all the purity assays including SEC-HPLC, CEX-HPLC, Capillary Electrophoresis-Sodium Dodecyl Sulfate Non-Reduced and Reduced (CE-SDS NR and R), and HIC-HPLC showed similar results. Likewise, the potency assays including HER2 Binding ELISA and HCC1954 Cytotoxicity Assay also showed similar results except for the first DS post-change batch ELISA at 125% and third post-change batch cytotoxicity at 74%, which is likely due to assay variability since the corresponding DP lot’s ELISA and cytotoxicity results are 94% and 103% (see Table 3), respectively. The protein concentration was increased from 10 mg/mL to 20 mg/mL in the post-change lots to reduce lyophilization cycle time during DP manufacturing. Trehalose concentration was also increased by 1.5% to help improve the stability of the drug product. These two changes accounted for the increase in osmolality from ~157 to 210 mOsmol/kg. Likewise, the DP lot release analysis yielded similar results in most assays except for the expected differences in protein concentration and osmolality (3). In summary, both pre- and post-change DS and DP batches passed the acceptance criteria for batch release and showed similar results.
Discussion
HIC-HPLC
The drug to antibody ratio (DAR), along with the unconjugated mAb, 1-drug, 2-drug, and impurities in the thermally stressed (up to 4 weeks at 40°C) drug product lots were determined by HIC-HPLC. The data are shown in Table 10. The results show that after 4 weeks of thermal stress, Pre-change and reconstituted Post-change 1 Lyo drug product lots show a small increase in relative abundance of DAR1 species and a minute decrease in the relative abundance of DAR2 species indicating gradual loss of the AS269 drug. These results correlate well with free drug assay results where a minute increase in free drug was observed under identical forced degradation conditions, namely 4 weeks at 40°C (see below). The nominal DAR for all lots of thermally stressed DP however remains the same (1.9), as the changes were very small.
Conclusion
In summary, side-by-side comparison of pre-change and post-change Drug Substance and Drug Product was conducted by evaluating different chemical, biochemical and biological functions and also included a comparison of degradation profiles under thermal stress conditions. All results have demonstrated a high degree of similarity between the pre- and post- manufacturing change ARX788 materials. Therefore, it is anticipated that the changes made to optimize the ARX788 manufacturing process will not adversely impact product quality, safety, pharmacokinetics, potency, and efficacy.
Acknowledgments
The Authors thank Ji Young Kim for her supervision of the ADCC effector function analysis, and Karen Cha for her expert guidance on global regulatory submissions.
Citation: Yu W, Ramprasad MP, Pal M, Chen C, Paruchuri S, Skidmore L, et al. (2023) Analytical comparability to evaluate impact of manufacturing changes of ARX788, an Anti-HER2 ADC in late-stage clinical development. PLoS ONE 18(7): e0284198. https://doi.org/10.1371/journal.pone.0284198
Editor: Oksana Lockridge, University of Nebraska Medical Center, UNITED STATES
Received: March 24, 2023; Accepted: June 27, 2023; Published: July 10, 2023
Copyright: © 2023 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0284198#abstract0
