Advancing a product, from its conception in research laboratories to commercialization, requires a series of analytical activities that need to evolve as the product goes through various stages and expands to various markets globally. This presentation intersects and balances Science, Quality and Regulation, and aim to help navigate complexity of the international environment.

Since the introduction of the first recombinant DNA-derived insulin and the approval of first therapeutic monoclonal antibody muromonab-CD3 in the 1980s, biotherapeutics market has shown a healthy growth. Given significant challenges in the treatment of many life-threatening diseases, biotherapeutics are becoming increasingly complex. Novel modalities such as multi-specific antibodies, fusion proteins, and gene therapy have gained significant momentum as innovative therapies. Integrated analytical strategy is required to advance attribute sciences for such complex molecules. This presentation will discuss recent developments in protein analytics for characterizing key attributes, including phase-appropriate analytical strategy, orthogonal methodology. and case studies.

N-linked glycosylation is a critical influencer of pharmacological function, safety and efficacy.  As minor changes to production processes can influence both glycan structure and composition, glycans must be monitored. However, characterization of N-linked glycosylation is analytically challenging due to innate glycan microheterogeneity. Herein we report the use of the MOBIE^TM high-resolution ion mobility system from MOBILion, to improve released N-linked glycan analysis and demonstrate applicability for profiling complex glycan isomeric mixtures.

We developed a homogeneous particle-based immunoassay using upconverting nanoparticles (UNCPs) for multiplexing potency of two different monoclonal antibodies (mAb). In this study, the recombinant human protein for each target mAb was conjugated to a corresponding UCNP. Two different UCNPs-immunocomplexes transferred energy for creating detection fluorescent signals separately with signal excitation at 980 nm. Harvested fluorescent signals at two different wavelengths were used for creating dose-response curves for the target mAb. The proof-of-concept LRET-based multiplexing immunoassay is a potent tool for improving efficiency in terms of the resource and assay performance in quality control laboratories.

This presentation describes the development of an automation workflow for qPCR titer assay using traditional liquid handler and contactless liquid dispensing technology. Combination of these two technologies allowed the cost reduction of the assay by 5-fold via miniaturization, improving assay performance and overall improvement in the operation efficiency by 4-fold in comparison to the manual assay. Case examples will be provided with results from validation runs of the assay.

Use of multi-attribute methods (MAM) for analytical testing of biotherapeutics is increasing due to their potential to improve efficiency and provide detailed information on quality attributes. Based on stakeholder input, USP established an expert panel to develop a chapter on best practices for MAM. This presentation will provide an overview of the proposed chapter contents and an update on other efforts to support biopharmaceutical quality and consistency using MAM.

A goal in developing a biological assay is to advance a robust and reproducible assay for use in repetitive testing. Many factors influence the approaches taken to create such an assay. To attain optimal assay conditions, it requires assessing a combination of parameters. Multiple parameter assays (MPAs), which can curtail assay development time, are achieved by miniaturizing volumes, utilizing denser formatted 384-well plates and managing conditions in a concurrent manner rather than consecutive. Automated methods capable of running distinctive MPAs can be developed as “tools." A library of these tools can then be used for future assay development projects.

Peptide mapping is an indispensable tool for monitoring post-translational modifications (PTM) of biological drugs. The traditional manual methods for peptide mapping are tedious and time-consuming. We have developed peptide mapping methods for monoclonal antibodies and novel bispecific DART molecules using an automated Liquid Handler system. The robustness, reliability, and reproducibility of these methods will be presented. These results will contribute to implementing the multi-attribute monitoring (MAM) workflows.

Nuclear magnetic resonance is a technique recognized for being information rich and more sensitive towards changes when compared to other biophysical techniques used to address higher order structure of biologics1,2. The introduction of benchtop flow NMR, integrated into PAT software, makes NMR important for (bio)process monitoring and control3. We show examples of magnetic resonance for biologics/biosimilars analysis, from NMR in R&D4 to benchtop EPR, FT-NMR and TD-NMR in bioPAT and QC.

The current race to develop better biologics faster has led biopharmaceutical companies into optimizing all drug discovery and development processes. As part of this effort, machine learning algorithms are being developed to identify correlations between amino acid sequences and physicochemical properties. The talk will focus on the platform Merck MSD is currently developing to serve as the interface between our scientists and machine learning algorithms for in silico developability assessments.

Process Analytical Technology (PAT) instruments include analyzers capable of measuring physical and chemical process parameters and key attributes with the goal of optimizing process controls. PAT probes and sensors are intended for understanding bioprocesses with the goal to control quality and consistency at all stages of product manufacturing to achieve quality-by-design (QbD) and real-time release. The advantages, challenges, and future development will be discussed.

Glycosylation is a critical quality attribute of biopharmaceuticals. Production from bioprocess results in a heterogenous glycan profile which necessitates rapid, sensitive, and high-throughput analysis. Liquid chromatography (HPLC or UPLC) has become a standard method of separation and analysis but often the workflow for glycan derivatization is time-consuming. We present an innovative, streamlined, 96-well-plate-based platform for derivatization that is rapid and sensitive. Applications to specific glycoproteins and advantages over existing methods will be shown.

Product quality attributes of protein therapeutics that impact safety or efficacy must be monitored to ensure product quality. Post-translational modifications, sequence variants, and glycosylation can be monitored by peptide mapping using mass spectrometry. We have developed a high-throughput peptide mapping strategy that characterizes multiple attributes to support process development, process characterization, and commercial product release/stability testing in QC.

Increased use of quality-by-design strategies creates the need to quantify post-translational modifications in the primary structure of a biological therapeutic protein. We have developed a compact mass spectrometer-based platform for monitoring both specific and multiple attributes within a monoclonal antibody. By using “QC-friendly” MS-technologies, the method can analyze >250 samples per week and be easily used by a non-MS expert, demonstrating its potential in both GMP and development environments.

We constructed a mechanistic model based on the published understanding of the underlying biology and existing data to elucidate the mechanisms and bottlenecks of rAAV synthesis in HEK293 suspension-adapted cells. Through model analysis, we designed a multi-stage transfection method that successfully increased the ratio of full to total capsids in the viral harvest without compromising the viral titer.

Gene therapy products have demonstrated great potential for treating devastating diseases and are being extensively evaluated in clinical trials for many disease indications. The structural and biological properties of these products are complex and yet to be fully understood. In-depth characterization methods were developed to assess recombinant Adeno-Associated Virus vector quality attributes. A case study of the risk assessment approach to define appropriate critical quality attributes will be presented.

This session will focus on ways to accelerate analytical development to support faster development timelines. However, activities often performed during Phase 3, as assay validation, elucidation of structure and characterization of impurities, can sometimes generate surprises. This presentation is intended to provide recovery and bridging strategies when deficiencies are discovered late in the development lifecycle.

Monoclonal antibodies and novel bispecific DART molecules are being developed for a variety of indications including immuno-oncology. Sensitive, accurate, and high-throughput analytical techniques enable faster development timelines for therapeutic molecules. An overview of different approaches, methodologies, and supporting software for high-throughput process analytics and related challenges will be presented as case studies.

Clearance of residual host DNA is an important part of the biopharmaceutical process as host DNA can pose a potential risk to the patient. Using the KingFisher Presto integrated into a Hamilton liquid handling system, we have automated the entire residual DNA assay from sample preparation through qPCR plate preparation, significantly reducing FTE labor and allowing for a walk-up system for quicker turnaround and high-throughput for residual DNA results.