Proteins, Antibodies and ADCs

The applicability of MALDI-TOF mass spectrometry to perform protein detection is well recognized in the life science field. In this field, SDS polyacrylamide gel electrophoresis and size exclusion chromatography have been historically used, however, they have drawbacks such as being time-consuming or lacking accuracy in molecular weight determination. Due to its ability to provide more accurate molecular weight information, MALDI-TOF mass spectrometry has become the primary tool for the analysis of protein primary structures. Moreover, in recent years, the analysis of proteins at the femtomole and subfemtomole levels is often required, which is increasing the demand for higher sensitivity measurements with MALDI-TOF mass spectrometry. 

In recent years, there is a significant surge in the market of biopharmaceutical products due to their popularity as alternative solution for many chronic diseases. Monoclonal antibody (mAb) is a highly complex biological macromolecule with specific therapeutic effects. It is produced from live cells in extremely complicated culture conditions. Quality control of biopharmaceuticals, especially biosimilars, is a critical step to elucidate any alteration in the primary structure as compared to the reference product (innovator). Peptide mapping and sequencing analysis of C-terminal and disulfide-bonds linked peptides are among the essential attributes for characterization of biosimilars. In this report, characterization of a bevacizumab biosimilar is described, with focusing on peptide mapping and MS/MS sequencing of C-terminal and cysteinecontaining peptides of a bevacizumab biosimilar on LCMS-9030, a Q-TOF system. 

Peptide mapping by HPLC is one of the important quality assurance tests used for verifying the primary structure of antibody drugs. Typically, following enzymatic digestion of the antibodies, separation is conducted using a traditional reversed phase column. Due to the large number of peaks that require separation, the use of small-particle columns and core shell columns for peptide analysis has spread in recent years.
In order to compare elution profiles for identity and mutation confirmation, a highly repeatable system is required. The Nexera-i integrated UHPLC is the ideal system for such an analysis. Here, the Nexera-i is used in the analysis of IgG (human immunoglobulin G) tryptic digest.

The manufacturing of antibody drugs has been gaining massive popularity due to their specificity towards target diseases, efficacy and role as potential personalised medicines. Peptide mapping plays a critical role in quality assurance of antibody drugs. It is employed for elucidating of the primary structure of antibody biosimilars. Peptide mapping of enzymatic digest is carried out using a C18 reversed phase column on a inner HPLC or UHPLC.

Recently, the term "biomedicine" is often heard in the field of pharmaceuticals. While also called biopharmaceuticals, they refer to proteinaceous drugs and antibody drugs developed and manufactured using biotechnologies including genetic recombination, cell fusion, and cell culture. In contrast, conventional medicines are referred to as "low molecular drugs" and are produced through chemical synthesis. 

Protein identification with a mass spectrometer (MS) and search engine utilizing genomic databases has now become the main stream in analysis of proteins. Although the proteins in the genomic databases are registered as precursor proteins, the expressed proteins in living cells are modified after translation and have various functions.

Mass spectrometry has become an indispensable tool for researchers looking to sequence peptides. Although effective in many cases, sequencing by In Source Decay (ISD) faces a few challenges its ability to provide reliable sequence  information including isobaric amino acids, database dependency and low molecular weight interferences. 

Monoclonal antibodies, or mAbs, have been used for over a decade in the treatment of a number of diseases but predominantly in the treatment of cancer and autoimmune diseases. Quantifying therapeutic mAbs in biological samples has been traditionally addressed by ligand binding assays (LBA's), however, there are major limitation in terms of extended method development times, reagent procurement, and matrix effects.

At present, shotgun proteomics techniques using liquid chromatography mass spectrometry are utilized mainly as high-throughput methods for identifying many different proteins in cellular cytoplasm. However, these techniques are not necessarily effective for identifying all proteins. In particular, when handling proteins separated by means of two-dimensional electrophoresis etc., the protein spots detected on the electrophoresis gel must be linked to the results of protein identification. 

This article introduces an example of measuring digests of phosphoprotein and analyzing phosphate modifications using the benchtop MALDI-TOF mass spectrometer "MALDI-8020".

This article introduces an example of measuring molecular weights of such glycoproteins using the benchtop MALDI-TOF mass spectrometer "MALDI-8020".

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals due to its wide application in medicine and biological science. The antibody’s biological activity can be attributed to its unique structural conformation.

Therapeutic proteins such as monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs) easily aggregate due to changes in temperature, pH, concentration and so on. Aggregation in the production of mAb and ADC negatively affects their efficacy and safety(1). Therefore, the amount of aggregation should be monitored. Size exclusion chromatography (SEC), which separates molecules by differences in size, is one of the most utilized analytical methods for the detection of aggregation in therapeutic proteins.

Many protein-based biopharmaceutical products, typified by antibody drugs, are synthesized in cultured cells derived from eukaryotes such as CHO (Chinese hamster ovary) cells. For this reason, there are inevitably many post-translational modifications to the biosynthesized proteins. Among these, modifications of glycans have gained attention as items for evaluating the quality of biopharmaceuticals.

Many protein-based biopharmaceutical products are synthesized in cultured cells derived from eukaryotes. For this reason, the synthesized proteins are mainly glycoproteins which comprise proteins with glycans linked to them. The glycans in these glycoproteins are broadly divided into N-linked glycans (N-glycans) and O-linked glycans (O-glycans), each having diverse and complex branching structures.

Protein glycosylation has been shown to be related to protein activity. It is therefore of great interest to study glycan structures of glycoproteins in immunology and cellular biology. On top of that, it is also essential to  analyze glycans in recombinant glycoprotein drugs to ensure consistent glycosylation profile.

Antibody drug conjugates (ADC), a type of pharmaceutical composed of an antibody bound to a drug, appeared in the 2000s with the expectation they would serve as more effective anti-cancer drugs than previous small-molecule pharmaceuticals through the combination of the antibody's high selectivity and the availability of a smallmolecule drug.