(T1230-02-06) Interlaboratory Study of Multi-Attribute Method (MAM) for Antibody Therapeutics – Overview

Purpose: Monoclonal antibody has been the one of the commonest popular biotherapeutics. Therapeutic antibodies are expressed by using mammalian cells such as Chinese hamster ovary (CHO) cells. Biological process causes the antibody modifications. Extracellular stress such as purification and filing stress are also known as the source of chemical modifications. These modifications may be increased during the storage period. Some of these modifications called “post-translational modifications (PTMs)” are recognized as critical quality attributes (CQAs) for the antibody therapeutics and their impact on the patient safety and efficacy have been evaluated. In the general quality control strategy, multiple chromatographic or electrophoresis based analytical methods are applied to evaluate these PTMs indirectly. Multi-attribute method (MAM) has been proposed as the direct monitoring of the PTMs by peptide map with high resolution mass spectrometry[1, 2]. MAM is one method to quantify the levels of numerous PTMs at each modification site. When the MAM is applied to GMP testing, high robustness and precision are required. These are several research about MAM interlaboratory studies, however MAM is still immature compared to other common chromatographic or electrophoresis technologies for antibody therapeutics. The "Materials Open Platforms for Pharmaceuticals" is a center of excellence (CoE) launched in 2021 by the National Institute for Materials Science and eleven Japanese pharmaceutical companies (Figure 1). Antibody Analysis Group is composed of eight Japanese companies and aims to develop and standardize MAM procedures in Japan. We aim to spread MAM technology in Japan by establishing basic knowledge about the MAM and conducting joint research that contributes to the application of MAM technology to each company’s antibody development. In this poster presentation, we will introduce the overall picture of our activities. A detail description of each type of PTMs will be presented in separate posters.

Methods: NIST reference material 8671 (NIST mAb) and its digested materials (Waters) were used. At first, each company performed sample preparation including denaturing, reduction, alkylation and enzyme digestion and the LC/MS analysis by each own procedure. As next step, to align the sample preparation procedure and analysis conditions, digested NIST mAb was analyzed in all companies using same catalog number columns and gradient conditions.

Results: In the first trials, total about 100 kinds of PTMs (modification types and the positions) were identified. However, only four were the common PTMs that were identified by all company results. It was considered that the requirements for PTM identification caused the inconsistent results. To achieve quantitative comparison among companies, the representative target PTM list was prepared considering the identified company numbers and historical reports about NIST mAb. However, the difference of the modification ratio was also large. Elimination of the impact from sample preparation by using the digested samples and applying the same analytical conditions improved interlaboratory reproducibility (Figure 2).

Conclusion: MAM is a powerful tool for direct CQA evaluation and the potential to integrate multiple test methods for antibody quality evaluations. Our research revealed that MAM results can vary drastically according to the protocol. The standardized LC/MS method reduced variations among the companies. The detail results of the interlaboratory reproducibility for each PTM are presented in our separate posters. This is the first interlaboratory MAM research in Japan. We recognized that there is still large room for improvement especially for sample preparation steps. Our future plans are also presented.

References: 1. Rogers, R.S., et al., Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics. MAbs, 2015. 7(5): p. 881-90.
2. Rogstad, S., et al., Multi-Attribute Method for Quality Control of Therapeutic Proteins. Anal Chem, 2019. 91(22): p. 14170-14177.

Acknowledgements: All authors are employees of each affiliation.

Figure 1 Organization of MOP

Figure 2 Representative modification ratio (A) NIST mAb result according to each company’s method (B) Digested NIST mAb result according to standardized LC/MS method