In-situ Reverse Phased HPLC Analysis of Intact Antibody-Drug Conjugates.

The field of oncology has recently seen an exponential growth in antibody-drug conjugates (ADCs) as a biopharmaceutical class with seven ADCs being launched onto the market in the last ten years. Despite the increase in the industrial research and development of these compounds, their structural complexity and heterogeneity continue to present various challenges regarding their analysis including reaction monitoring. Robust and simple reaction monitoring analysis are in demand in the view of at-line in-process monitoring, and can instill control, confidence and reliability in the ADC manufacturing process. Aiming at providing chromatographic methods for conjugation monitoring, we evaluated herein the potential of utilizing reverse phase HPLC analysis, without sample pretreatment, for characterization of traditional cysteine-based ADCs. This analysis can be used for estimation of drug antibody ratio (DAR), which has shown the same trends and results as other well-established HPLC techniques. This methodology was also applied to three ADCs derived from three different antibodies. Additionally, we analyzed unpurified ADC samples existing in a complex reaction matrix and separated ADC species and payload compounds. This investigation was conducted using three different ADCs based on different payloads. The results described herein indicate the potential application of this RP-HPLC methodology in reaction monitoring studies.

Analytical Comparison of Antibody-drug Conjugates Based on Good Manufacturing Practice Strategies.

The production of antibody-drug conjugates (ADCs) has been in great demand in the field of cancer therapeutics. Although cysteine-based conjugation is the most common and well known process for producing ADCs, multiple analytical methods are required for accurate drug-antibody ratio (DAR) determination due to the heterogeneity of the ADCs. Here we report various analytical methods for DAR analysis of traditional cysteine-based ADCs; additionally, apply a good manufacturing practice (GMP) strategy to produce a four hundred milligram ADC batch for use in good laboratory practice (GLP) studies. The work described herein not only evaluates several analytical performances but also provides guidance for future phase appropriate ADC production while establishing a unique analytical strategy.

Synthesis and Biological Evaluation of Antibody Drug Conjugates Based on an Antibody Expression System: Conamax.

Antibody production for ADCs (or in general) is commonly performed by CHO-based platforms and limited by volumetric productivity, expensive downstream purification, and extended optimization timelines. The Conamax platform is a novel microbial-based protein production and secretion system. A suite of synthetic biology tools have enabled high volumetric productivity (>1 g/L/d) and glycoengineering to produce simple and consistent human-like post-translational modifications. Conamax can be engineered to secrete genuine, functional monoclonal antibodies that have been successfully used to make antibody drug conjugates (ADCs) via cysteine-linked conjugation. Specifically, we evaluated ADCs derived from both a Conamax-produced anti-HER2 antibody and comparable commercially sourced Chinese hamster ovary (CHO)-produced material in an NCI-N87 gastric cancer xenograft model. Conjugation efficiency and resulting analytical data indicated comparable ADC quality and attributes. No statistical difference was observed between Conamax- and CHO-derived test articles thereby indicating similar efficacy and function. These results further demonstrate the potential of Conamax as a useful platform for the discovery and production of therapeutic antibodies and ADCs.

Good Manufacturing Practice Strategy for Antibody-Drug Conjugate Synthesis Using Site-Specific Chemical Conjugation: First-Generation AJICAP.

The development of antibody-drug conjugates (ADCs) is in great demand in the oncology field. With the goal of maximizing the therapeutic index, the conjugation technology to produce ADCs has been shifted to a site-specific manner; however, it is still challenging to establish robust and scalable synthetic processes. We have developed a chemical conjugation platform termed AJICAP for site-specific ADC synthesis using IgG Fc-affinity peptides. Here, we report the preparation of site-specific ADCs based on first-generation AJICAP technology for use in good laboratory practice studies. Analysis of the final ADC product was conducted using validated systems and good manufacturing practice. This work may not only prompt further biological studies of AJICAP-ADC but also establish a strategy to provide well-documented manufacturing data to enable new drug application filings (e.g., investigational new drug applications) for site-specific ADCs.