Biopharmaceuticals and Protein Analysis

A top-down approach to quantifying intact proteins from biological fluids using liquid chromatography–triple quadrupole–mass spectrometry (LC–MS/MS) offers the potential for more absolute quantitation than bottom-up approaches. To make this broadly viable, however, commercial sample preparation tools are needed.

A method was developed for the molecular weight characterization of heterogeneous polymer mixtures, such as heparins and glatiramer acetate, noting that single molecular structures are not adequate for creating a molecular weight calibration curve. That limitation is overcome in this work, which demonstrates method validation and application to process samples.

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.

To fully characterize a protein biopharmaceutical, it must be broken down into smaller segments (peptides). Several high performance liquid chromatography (HPLC) techniques can be used to provide a wealth of information on everything from post-translational modifications (PTMs) to the glycoprofile to information on similarity when characterizing biosimilars.

The utility of native high-resolution mass spectrometry (HRMS) in intact protein characterization is rapidly growing because of advances in both ion-exchange chromatography (IEC) as well as MS-compatible buffer systems. MS is a critical component of biotherapeutic characterization, but its combination with traditional chromatographic separations, such as size-exclusion chromatography (SEC) and IEC, has been slow because of the predominant use of high salt mobile phases, which are incompatible with MS. Recently reported methods using cation-exchange chromatography (CEX) with volatile buffer systems for pH gradient elution has given researchers the ability to use these chromatographic techniques with MS detection. In this article a robust, MS-compatible buffer system for high sensitivity IEC with pH gradient elution for charge variant analysis of intact monoclonal antibodies (mAbs) is described.

The versatile size-exclusion ultrahigh‑performance liquid chromatography (SE-UHPLC) platform method described here provides superior separation for bispecific monoclonal antibody formats compared to a previous method.

Hydrogen–deuterium exchange–mass spectrometry enables determination of higher order structures of biopharmaceuticals and direct comparisons between a biosimilar and its proprietary analog.

Given the complexity of producing bispecific antibodies, suitable analytical methods to detect and measure the levels of undesired variants are essential. Here, a novel MS-based method for variant detection is presented.

Long-term column use can lead to on-column methionine oxidation during LC–MS/MS peptide mapping of antibody-based biotherapeutics. Following the approach described here minimizes the risk of measuring oxidative artifacts, and helps generate high quality data to provide reliable quantitative information about product-related heterogeneities.

Applications of size-exclusion chromatography (SEC) are presented for characterization and quality control of novel biotherapeutic products, including antibody–drug conjugates, hydrophobic proteins, and coformulations.

In this study, a conjugation strategy for Fab–ferritin conjugates, used for drug delivery, was successfully optimized using LC–MS. Characterization of the resulting conjugates was performed using SEC-MALS-QELS.

The potential of multidimensional online peptide mapping analysis as a strategy for improving a postlabeling workflow for protein–protein interactions is demonstrated using both hydroxy radical footprinting–mass spectrometry (HRF–MS) and LC–MS/MS.

A simple and robust size-exclusion chromatography (SEC) method has been developed for characterizing a multimeric PEG–protein conjugate. A wide range of size variants of the conjugate, ranging from 50 kDa to >1000 kDa, can be resolved and quantitated.

This article investigates host cell protein analysis using micro-pillar array columns combined with mass spectrometry.

Glycosylation is the most common monoclonal antibody (mAb) posttranslational modification. What is essential to know about it?

The Column spoke to Richard Shannon from AstraZeneca about his work characterizing monoclonal antibodies (mAbs), why ion-exchange chromatography (IEC) is his technique of choice for analyzing mAbs, and offers his advice for anyone wanting to use the technique.

Glycan isomer expressions have not been well studied, due to inefficient separation and structural identification techniques. Fortunately, with the development of novel separation techniques and liquid chromatography–mass spectrometry (LC–MS) based glycan isomer identification strategies, new efforts have been made to investigate the glycan isomers in various diseases. Here, we review the recent advances of several isomeric separation techniques for both N- and O-linked glycans.

Monoclonal antibodies (mAbs) are being developed at an explosive rate and have attracted great interest from both smaller biotech firms and big pharmaceutical companies. Developing mAbs and next-generation antibody–drug conjugates (ADCs) is highly demanding in many ways. From an analytical perspective, handling mAbs and ADCs presents many new challenges. This article describes how size-exclusion chromatography (SEC) combined with high-resolution mass spectrometry (HRMS) can be applied to the detailed characterization of mAbs and ADCs.

With SEC, special care is required to achieve in practice the chromatographic efficiency that is expected from theory.

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.

Characterization of mAbs and related products requires the identification of chromatographic peaks with MS. However, the conventional salt- and pH-gradient elution techniques used in IEX are inherently incompatible with MS. Ammonium acetate- and ammonium carbonate-based mobile phase systems have been recently applied in IEX-MS, but the influence of the eluent composition on peak shape and retention has not been discussed nor studied systematically until now. The aim of the present study was to understand the impact of ionic strength, buffer capacity, and pH-response on the retention behaviour and peak shape of mAb species.

This is the first in a series of articles exploring current topics in separation science that will be addressed at the HPLC 2019 conference in Milan, Italy, from 16–20 June. In this preview, Davy Guillarme discusses multidimensional liquid chromatography approaches for the characterization of protein biopharmaceuticals.

This is the first in a series of articles exploring current topics in separation science that will be addressed at the HPLC 2019 conference in Milan, Italy, from 16–20 June.

CZE–ESI‑TOF‑MS for the characterization of the mAb infliximab and its variants is presented. Infliximab was analyzed using a middle-up approach involving either reduction or digestion with the enzyme IdeS. A multilayer capillary coating of PB-DS‑PB in combination with a background electrolyte of 40% acetic acid provided efficient separation of the obtained antibody fragments, that is, LC and HC, as well as F(ab’)2 and Fc/2 parts. C-terminal lysine variants were also resolved. Recorded mass spectra of HC and Fc/2 fragments permitted assignment of 13 glycoforms and provided a quantitative profile, with G0F the most abundant glycoform (~50%). CZE–ESI-TOF-MS represents an efficient means for the straightforward analysis of a monoclonal antibody and its proteoforms.

A discussion of hydrophobic interaction chromatography (HIC) theory and its application to the analysis of proteins and biomolecules is presented.