November 2024: Antibody Reliability

As antibody-drug development accelerates, ensuring consistent binding affinity, stability, and specificity is critical to improving treatment outcomes. Recent advances in antibody sequencing, artificial intelligence, and high-throughput screening have bolstered efforts to produce reliable antibody candidates more efficiently.

A recent paper describes the development of a new paradigm for optimized experimental design in cIEF platforms aimed at an accurate robust and reliable mAbs charge-variant assessment. The success of monoclonal antibodies (mAbs) and biosimilars emphasizes the need for precise control of their purity, identity, and stability. Imaged capillary isoelectric focusing (icIEF) has emerged as a key technique in assessing charge variants. However, traditional approaches using charge variant profile assessments (CVPA) face inconsistencies due to reliance on reference standards and variable outputs across instruments. By refining calibration methods, researchers have improved the accuracy of isoelectric point (pI) measurements and developed unbiased experimental designs (UEDs) to minimize bias and enhance resolution across pH gradients. This innovation not only ensures the quality and safety of mAb-based drugs but also extends to biosimilars and other biologics, offering significant potential to improve healthcare outcomes.

Meanwhile, another study evaluated the effectiveness of surfactants in preventing mAb adsorption on medical surfaces using a novel device and protocol to detect and quantify mAb adsorption directly on medical plastic bags, ELIBAG. The research reveals that surfactant effectiveness depends on the specific antibody, surfactant type, concentration, and surface material. Findings highlight differences in adsorption behavior between model and real medical surfaces and underscore the importance of using actual medical surfaces to better understand and optimize mAb stability in formulations. This research offers insights into enhancing drug product development and ensuring material compatibility in clinical applications.

To validate antibodies themselves, new initiatives such as YCharOS aims to enhance antibody reliability by rigorously testing specificity and selectivity. Collaborative efforts involving suppliers, researchers, and organizations are pushing for standardization, the adoption of recombinant antibodies, and unique identifiers like RRIDs to improve tracking. While cultural shifts in lab practices remain challenging, the scientific community is hopeful that these efforts will lead to lasting improvements.

Unfortunately, stability of therapeutic mAbs is still a major issue, as aggregation and fragmentation of mAbs is common during various stages of product life cycle due to stress factors like thermal and air/liquid interfacial agitation. These aggregates may reduce mAb efficacy and trigger adverse immune responses, making stability a critical focus throughout the product lifecycle. A recent review provides a broad overview of mAb aggregation, including types, sizes, causes, analytical techniques, and permissible limits, as well as factors influencing aggregation, methods used to study stress-induced aggregation, and strategies to enhance stability, offering valuable insights for improving mAb formulation from manufacturing through patient administration.

In a recent paper, scientists focused on identifying previously undetected microprotein impurities in antibody drugs produced using Chinese hamster ovary (CHO) cells, a common cell line for monoclonal antibody (mAb) and Fc-fusion protein production. By employing ribosome profiling (Ribo-seq), they discovered thousands of novel short open reading frames (sORFs) likely encoding microproteins, which are often overlooked in CHO cell biology and host cell protein (HCP) impurity analysis. Using an extended protein database, they analyzed eight antibody drugs via mass spectrometry (MS), uncovering microprotein impurities that vary with cell growth and culture conditions. Their findings enhance HCP detection accuracy in antibody drugs and expand knowledge of non-canonical translation in CHO cells, providing insights to improve therapeutic protein quality and manufacturing efficiency.

Besides ribosome profiling, researchers have also used size-based electrophoresis via a CZE-MS generated spectral library to rapidly identify antibody impurities. By applying forced stress to antibodies, the research mimicked bioprocessing and storage conditions to generate degradation products. Unlike traditional chromatographic methods, this approach improved accuracy without the need for deglycosylation. Partial validation using CZE-MS/MS highlighted the potential for future enhancements through advanced mass spectrometry techniques like electron capture dissociation (ECD). This method offers a robust tool for ensuring antibody quality and safety, with broad applicability in characterizing complex biotherapeutics, particularly multi-specific antibodies for cancer treatment.