Anurag S. Rathore

This article discusses the challenges and effective solutions for high performance liquid chromatography (HPLC)-based analytical characterization of virus-like particles (VLPs).

Biopharmaceutical analysis is a rapidly evolving field that requires the development of new technologies and methods to keep pace with the increasing complexity of biologics. One of the most promising areas of research is the use of single-cell omics and microfluidic chips for the analysis of biopharmaceuticals. Single-cell omics has revolutionized our understanding of cellular heterogeneity, while microfluidic chips have enabled high-throughput analysis of single cells that provide an understanding of the complex biological network that complements the genomics and transcriptomics studies. This article will explore some of the emerging trends and technologies in biopharmaceutical analysis, with a particular focus on single-cell omics and microfluidic chips. We will also discuss the developments in ambient ionization mass spectrometry such as sub nanoampere ionization and the potential of low current ionization in studying cell-to-cell heterogeneity and its role in metabolomics.

The authors discuss key findings from recent biosimilarity assessments they conducted on biosimilars of granulocyte-colony-stimulating factor (G-CSF), insulin glargine, rituximab, and trastuzumab.

We present formulation and delivery of gene therapy products, focusing on adeno-associated virus (AAV) formulations. We also discuss the analytical technologies most used to characterize these products.

When used appropriately in RPLC, ion pairing agents can increase the separation and retention of charged analytes. But should they be explored in other modes of HPLC as well?

Characterizing lipid nanoparticles (LNPs) is a developing delivery modality in biotherapeutic analysis. We address some of the current challenges and opportunities in this field, including an examination of the most common tools used to characterize LNPs.

Some traditional MAM workflows are being substituted with iMAM workflows that rely on analyzing the intact mass of mAbs in their native form, resulting in significant improvements in high-throughput sample analysis.

A drug stability program is a fundamental part of ensuring product quality, safety, and efficacy. Here, we summarize essential guidelines, differences between large- and small-molecule stability, and the analytical methods used.

What is the right number of replicates in biopharmaceutical analysis? Does the answer depend on the type of analysis?

In response to regulatory concerns, host cell protein (HCP) analysis is now often conducted using LC–MS/MS. Unlike ELISA, LC–MS/MS can positively identify and quantify specific HCPs and characterize the total amount of HCPs present.

Recent assessments of four biosimilars illustrate how a combination of orthogonal, high-resolution tools is used to demonstrate analytical and functional biosimilarity.

Charge detection mass spectrometry (CDMS) is a useful tool to characterize larger, more complex biopharmaceuticals like bispecific antibodies and ADCs.

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.

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.

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.

Multidimensional separations, in which two or more separation methods are coupled, are a valuable analytical tool for higher peak capacity and improved selectivity for the analysis of complex samples like biotherapeutics.

Advanced separation and mass spectrometry methods enable comprehensive profiling of the inherent glycan heterogeneities of protein therapeutics. In particular, reversed-phase HPLC–based multiattribute methods (MAMs) provide a wealth of information, and other techniques, such as HILIC and CE-MS, also continue to evolve.

Coupling CE with MS presents some challenges. Here, we discuss the advantages of CE–MS over LC–MS, and the parameters that are important to obtain a stable CE–MS profile.

The analytical techniques used for characterizing biotherapeutics have evolved. We review the utility of the traditional tools and discuss the new, orthogonal techniques that are increasingly being used.

To ensure the reliable and accurate characterization of biotherapeutics, an arsenal of orthogonal analytical techniques is needed.

A look at techniques for charge-variant analysis of monoclonal antibodies and the question of whether pH gradients are really better than salt gradients

Several key applications of biolayer interferometry in pharmaceutical development have emerged recently. Here, we evaluate its use for easuring product titer from fermentation, and compare the strengths and weaknesses of the technique to those of HPLC.

In this article, we discuss the use of CE-MS (sheath flow interface) for analysis of intact proteins as well as of protein digests. We discuss the unique aspects that the user needs to be aware of while testing biotherapeutics versus small molecule drugs. We also highlight that the optimization of CE and MS parameters together result in the creation of a more robust and reproducible protein analysis approach. Finally, we list some of the most common errors that are likely to occur during CE-MS analysis and suggest ways to overcome them.

Glycosylation of monoclonal antibody (mAb) therapeutics is widely recognized by the regulators and the industry as a critical quality attribute (CQA). Hence, it is necessary that glycosylation is measured and adequately controlled during production. This instalment reviews the various process parameters and raw material attributes that affect glycosylation, as well as the different analytical tools that are used for characterization, with greater emphasis on the chromatographic methods of analysis. Key recent advancements that have occurred in the past five years are also discussed briefly. While significant progress has been made in the monitoring of glycosylation, its real time control has yet to be demonstrated.

Glycosylation of monoclonal antibody (mAb) therapeutics is widely recognized by the regulators and the industry as a critical quality attribute (CQA). Hence, it is necessary that glycosylation is measured and adequately controlled during production. This installment reviews the various process parameters and raw material attributes that affect glycosylation, as well as the different analytical tools that are used for characterization, with greater emphasis on the chromatographic methods of analysis. Key recent advancements that have occurred in the past five years are also discussed briefly. While significant progress has been made in the monitoring of glycosylation, its real time control has yet to be demonstrated.

A systematic approach for formation of sigmoidal gradient for analysis of biopharmaceutical proteins has been reviewed and discussed together with two case studies. The first application involves HPLC separation of the various product related variants of a microbial expressed biotherapeutic, granulocyte colony stimulating factor (GCSF). The second case study involves separation of the charge heterogeneity related variants for a monoclonal antibody (mAb) biotherapeutic product. In all cases, it is observed that the use of sigmoidal gradient successfully reduces the analysis time significantly (from 70 minutes to 15 minutes for GCSF and from 40 minutes to 4 minutes for the mAb) while retaining the selectivity and the resolution.

Here in part II of our series on assessing protein aggregation, we provide an overview of best practices for achieving this goal, including the importance of using a multimethod approach.Here in part II of our series on assessing protein aggregation, we provide an overview of best practices for achieving this goal, including the importance of using a multimethod approach.

Various analytical approaches and instrumentation for low?molecular-weight (MW), noncovalent, aggregates of proteins are discussed.

Most analytical approaches used for the determination of low-molecular-weight noncovalent aggregates in protein pharmaceuticals suffer from important limitations. This first part of this two-part column series discusses those limitations.