Biological drugs, also known as biologics, are medications derived from living organisms. Unlike traditional small molecule drugs, which are chemically synthesized, biologics are produced using biotechnology methods, such as recombinant DNA technology or protein engineering. They are used to treat various medical conditions, including autoimmune diseases, cancer, and rare genetic disorders. Biologics are often administered through injection or infusion, and they have become an increasingly important part of modern medicine.

In general, therapeutic proteins exhibit high molecular complexity, making them sensitive to various environmental factors, such as temperature, light, and pH. Even minor changes can cause the biologic to become unstable, leading to decreased efficacy or harmful side effects. Therefore, proper analytical characterization and usage of state-of-the-art techniques allow for mitigating potential risks.

Mass spectrometry (MS) has become an essential tool for characterizing proteins in modern biotechnology and pharmaceutical research. This technology is based on the principles of ionization and mass-to-charge (m/z) separation. It allows researchers to identify and quantify the individual components of a protein sample with a high degree of accuracy and precision. Undoubtedly, MS is a valuable tool for confirming the primary structure of a protein and identifying any post-translational modifications that may affect its function.

Primary structure determination

Primary structure confirmation using mass spectrometry allows for identifying and confirming a protein’s amino acid sequence. It involves breaking down the protein into its constituent peptides and analyzing these peptides to determine their mass-to-charge ratio (m/z). The first step is to cleave the protein into smaller peptides using a protease enzyme, such as trypsin or others. The resulting peptides are then purified and separated using liquid chromatography. The complex peptide mixture can be separated by a nano-LC system where the separation of the analytes takes place into capillary columns to enhance sensitivity. Next, the peptides are ionized using an electrospray ionization source and introduced into the mass spectrometer. The difficulty of obtaining complete coverage of the protein sequence using MS alone is probably the main limitation of this technique, especially for larger proteins. Additionally, some amino acids may be challenging to detect using MS, such as those that are modified or have unusual chemical properties. Therefore utilization of MS-based analysis requires thorough experience. 

Modifications of proteins

One of the key applications of mass spectrometry in protein characterization is the identification of post-translational modifications (PTMs). PTMs are chemical modifications that occur after the translation of a protein and can significantly alter its biological activity and function. Almost all proteins, whether produced as recombinant proteins or isolated from natural sources, will carry, to some degree, modified amino acids. Modification may also occur during the sample handling process. Therefore, they need to be monitored to ensure the consistency of production batches. Modifications, like deamidation, oxidation, pyroglutamate formation, etc., may affect protein functionality. Hence, identifying protein modifications and their position is essential in protein characterization. 

Among others formation of disulfide bridges significantly influences protein structure and function. Incorrectly paired disulfide bonds result in changing protein properties. For that reason, their mapping is a significant step for confirming proper tertiary structure. Mass spectrometry can be used to identify the specific sites of PTMs, such as phosphorylation or glycosylation, and to determine their abundance. The glycosylation-related heterogeneity of proteins arises from the differences in localization and occupancy of the glycosylation sites and the diversity of the glycan structures expressed on a specific site. To address both levels, glycopeptides analysis and site occupancy evaluation should be utilized. 

Aggregation and oligomerization

Oligomers, aggregates, and fragments are biologically active protein proteoforms and common product-related impurities in biopharmaceuticals that impact efficacy, safety, and stability. Identification of low-level impurities may involve labor-intensive chromatographic fraction collection and follow-up experiments. In addition, a significant part of the total protein aggregates can be caused by non-covalent molecular interactions, which are dissociable in denaturing buffers and cannot be characterized by conventional mass spectrometry. Using ammonium acetate solution for SEC allows for preserving non-covalent protein interactions and registers them by MS with high sensitivity and accuracy. 

Besides the assessment of biomolecule size variants, native MS allows the characterization of charge heterogeneity through the combination of ion exchange chromatography and native MS. Characterization of variants such as deamidation, which are traditionally unattainable by an intact mass due to their minimal molecular weight differences, can be measured unambiguously by mass and retention time.

Conformational dynamics analysis

Hydrogen–deuterium exchange mass spectrometry (HDx-MS) allows insight into the behavior of the protein in the solution and the correlation of HDx with the structure and dynamics of the molecule. The technique enables the analysis of proteins in terms of interactions with ligands/drugs, other proteins, and lipids or the study of the effect of mutations and post-translational modifications under the same experimental conditions in solution. The main application of HDx-MS is the comparative analysis of different protein conformers. HDx-MS is a complementary method to 3D static structures, allowing for a “dynamic” image of a protein that can explain many biological processes.  

In conclusion, mass spectrometry is a powerful tool for the characterization of proteins in modern biotechnology and pharmaceutical research. It allows for identifying post-translational modifications, protein-protein interactions, and quantifying protein abundance, among other applications. With ongoing advances in mass spectrometry technology and sample preparation techniques, mass spectrometry will continue to play a critical role in protein characterization and the development of new therapeutics.

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