Biopharmaceuticals and Protein Analysis

Barry L. Karger and James P. Grinias are the winners of the 15th annual LCGC Lifetime Achievement and Emerging Leader in Chromatography Awards, respectively, for 2022. Here, we review their achievements.

We review different approaches and coupling strategies for analyzing monoclonal antibody aggregates with 2D-LC.

Intact mass analysis is becoming increasingly useful for characterizing biologics. We describe the current application of intact mass analysis, including quantitation, sequencing, and structural characterization.

Recent developments in high-resolution mass spectrometry (HRMS) are aiding biopharmaceutical development and simplifying routine monitoring, with applications in areas like multi-attribute methods, coupling established purity methods with MS, and subunit analysis.

A validated FFF-MALS method aligned with technical specification ISO/TS 21362 for the analysis of lipid-based nanoparticles (LNPs) encapsulating siRNA and mRNA is presented.

There is a need to use more descriptive and precise terms to describe some of the properties of the products used for biopharmaceutical analysis. It is probably more correct to say “low adsorption” and “corrosion-resistant” systems.

We present the main analytical techniques for performing functional characterization of biotherapeutic products. Such assessments are particularly critical for biosimilars, where analytical testing must ensure functional comparability with the innovator product.

We explore the impact of two different stationary phases and ion-pair reagents on the retention behavior of a therapeutic peptide using reversed-phase liquid chromatography. This information is of fundamental importance for the development of reliable, selective, and fast analytical methods able to separate and identify the target peptide.

The benefits of a robustness assessment for the analysis of a NIST mAb using a wide pore C4 LC column are described.

In the second part of this review of the current state of HIC, some practical considerations are explained, including method development, selection of the phase system, combined salt systems, and possibilities to combine HIC with other chromatographic modes.

A review of the current state of HIC, focusing on retention and separation mechanisms with the aim of developing more robust methods. Can HIC be considered as a non-denaturing and non-destructive technique with advantages for protein analysis?

Recent advances in stationary phases for hydrophobic interaction chromatography (HIC) permit HIC–MS analysis of intact antibodies and other proteins using direct flow to the mass spectrometer.

New analytical workflows are needed to address the advances in biopharmaceutical product composition. A description of the multi-attribute method (MAM) is given, which has been developed to monitor critical quality attributes (CQAs) simultaneously and directly.

Recombinant adeno-associated viral therapy (rAAV) products are particularly complex. Are liquid chromatography and LC–MS the right tools for their characterization?

IMS is a valuable tool for biopharmaceutical analysis. Two formats in particular have proven useful: cyclic IMS and structures for lossless manipulations (SLIM).

This article explores the potential of microchip capillary electrophoresis–mass spectrometry (CE–MS) for the characterization of intact biopharmaceuticals, and how this technique can effectively be employed to monitor essential quality attributes of interest.

This article explores the analytical challenges associated with HCP monitoring and reviews recent advances in HCP characterization, with special emphasis on high performance liquid chromatography mass spectrometry (HPLC–MS)-based methods and HCP enrichment techniques.

The diverse nature of oligonucleotide therapeutics leads to the requirement for multiple separation methods to characterize quality attributes. Highly chemically-modified to improve efficacy and resilience to nucleases, they are challenging to analyse using a single method. This article discusses multiple chromatographic separation methods, and the benefits of mass spectrometry (MS).

Cellular and gene therapies (CGTs) have contributed significantly to the improvement of clinical outcomes for patients in the recent years. This paper discusses a range of physicochemical methods that play an important role in the difficult characterization of viral vectors, to meet the unique needs of CGT manufacturing process development, process and product characterization, and the quality control testing of these materials.

In this paper, two adeno-associated virus (AAV) genome integrity analysis workflows are introduced; a standard sample preparation protocol and an accelerated procedure, both utilizing capillary gel electrophoresis with laser induced fluorescent detection (CGE–LIF) to analyze the released nucleic acids.

If you are analyzing metal-sensitive biomolecules, and a bioinert instrument is unavailable, or insufficient, passivation or mobile-phase additives may help. Here’s how to use those solutions, with tips for avoiding potential pitfalls.

The strategies described here can improve the efficiency and analyte recovery of chromatographic methods used to characterize monoclonal antibodies and antibody– drug conjugates.

Filip Cuyckens from Janssen R&D in Belgium spoke to LCGC Europe about recent innovative approaches he and his team developed to support drug metabolism and pharmacokinetic studies, and the inventive role that two-dimensional liquid chromatography (2D-LC) plays in his laboratory to boost sensitivity, solve recovery issues, and increase overall efficiency.

Single-column (batch) chromatography, involving two or more successive single-column (batch) chromatographic steps, is a standard approach for purifying biopharmaceuticals. Step one, known as the capture step, is used to remove product-related impurities, and step two, the polishing step, is used to remove product-related impurities. Here we present and illustrate the advantages of continuous chromatography for these separations: capture simulated moving bed (captureSMB) for the capture step and multicolumn countercurrent solvent gradient purification (MCSGP) for polishing.