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An introduction to this special issue by our guest editor
Over the past few years, complex and novel format biotherapeutics, such as fusion products, bispecific antibodies, protein–polymer conjugates, and coformulations have been increasingly populating the pipeline of many biotech companies. At the same time, the growing demand to reduce the time it takes to develop and bring new medicines to patients has shed light on the limitations of current analytical tools and standard technical development considerations with next-generation biologics. In this increasingly competitive landscape, analytical methods that can provide improvements in speed, resolving power, and overall separation efficiency are highly desirable and critical to ensure that sufficient product characterization is performed at each stage of development.
This special issue was assembled to showcase recent advances in the development of analytical workflows and characterization strategies that are currently used for product quality control (QC) testing, monitoring of undesired variants, and identification of critical quality attributes (CQAs). This issue also highlights key considerations to be taken into account throughout the development of novel and unique formats and is aimed to provide valuable insight into how to overcome challenges that might be associated with next-generation biotherapeutics. Some of the articles are the result of key collaborations between industry and academic leaders, highlighting the necessity of synergistic efforts for the development and implementation of novel analytical workflows and strategies.
Jennifer Rea and her coauthors provide an overview of the increased complexity of characterization and quality control by size- exclusion chromatography (SEC) of complex biotherapeutics compared to standard monoclonal antibody formats. SEC is typically used to separate and quantify product variants and to monitor in particular the level of aggregates, which often represent a safety concern. Examples of SEC analysis of bispecific, antibody–drug conjugate (ADC), and coformulated products are described, as well as unique considerations for achieving desired peak separation.
Further insight into technical considerations for a product-specific SEC method for QC is provided by Lu Dai and coauthors. To increase the half-life of a therapeutic protein, a complex multi-arm polyethylene glycol (PEG) scaffold was coupled to the protein as a means to increase the size and therefore the half-life of the product in the eye. In this case, given the major impact of size on the desired half-life, the challenge was to develop a suitable SEC method to be used for QC for the separation and quantification of the product size variants, covering a much wider range (from 50 to >1000 kDa) than for standard monoclonal antibodies (mAbs).
In a demonstration of a growing trend in biotherapeutics to design molecules with a high degree of valency, Whitney Shatz and coauthors describe an alternative approach to polymer-based scaffolds. Multivalent, self-assembling scaffolds such as ferritin can be effectively used for such purpose in drug delivery. This study, like the others presented in this special issue, provides valuable insight into analytical methods that are needed to support both optimization of the manufacturing process and QC of the final product. In this work, SEC in-line with a multi-angle light scattering detector (MALS) and quasi-elastic light scattering detector (QELS) were crucial to assess the molecular size under native conditions and inform the optimization of the conjugation process.
Ming Lei and Tao Chen offer an overview of considerations and recent developments for the quantification of product-related variants of bispecific antibodies, another approach to achieve simultaneous binding to two targets with high specificity. This article highlights the crucial need of having an analytical assay that can detect the presence of the undesired homodimer variant, both during the development of the manufacturing process to facilitate the removal and during final product QC. Given that the undesired homodimer has very similar physico-chemical properties to the desired heterodimer, the limits of standard analytical methods (such as SEC), as described by Rea et al., become apparent in this situation. An alternate approach is therefore provided by Lei and Chen, where the bispecific product is reduced to its subunits using hinge-specific enzymes, prior to chromatographic separation. The authors also presented the method qualification strategy and results of an ion-exchange chromatography (IEX)-based homodimer quantitation method in anticipation of a charge patch on a bispecific antibody.
Another critical analytical method for the characterization of biotherapeutics is peptide mapping by liquid chromatography–tandem mass spectrometry (LC-MS/MS). This tool is routinely used for the identification and quantification of post-translational modifications (PTMs) such as oxidation, deamidation, and isomerization. Given that some PTMs can be CQAs, it is crucial to provide reproducible and accurate quantitative information when using this method. Michael Mølhøj and his coauthors share their findings on how oxidative artifacts can affect the quantitation of inherent product heterogeneities and therefore generate misleading information. They offer a valuable approach to mitigate the effect of on-column oxidation when performing peptide mapping.
Julien Camperi and Arthur Schick offer an overview of how multidimensional on-line peptide mapping can streamline the post-labeling workflow of hydroxyl radical footprinting–mass spectrometry (HRF-MS) analysis. This work highlights how automated on-line peptide mapping can significantly reduce sample handling and operator time compared to standard off-line procedures.
Overall, this special issue highlights challenges and opportunities for the characterization of complex and unique format molecules entering the pipeline and describes examples of analytical workflows and control strategies when standard QC platform methods and approaches are not applicable. The analytical workflows and strategies are evolving along with biotherapeutic pipelines to enable faster development of promising and novel medicines to fulfill unmet medical needs.