1. Introduction

Biopharmaceuticals are medicinal products obtained by biotechnological processes using molecular biology methods, which include proteins, sugars, nucleic acids, cells, tissues, used for therapeutic or diagnostic purposes in vivo^[1]. Biopharmaceuticals account for about 30% of drugs currently under development. They are substances of biological origin obtained by using biotechnology techniques such as the recombinant human technology, gene transfer and antibody production methods, genetic engineering or hybridoma technology. Any biopharmaceutical is also referred to drug obtained from microorganisms, from genetically modified organisms or from substances produced by living organisms. Biopharmaceuticals are very different from conventional chemical drugs. Synthetic drugs are, in fact, small molecules while biopharmaceuticals are macromolecules that possess complex structural requirements, specific bioactivity, and therefore a greater degree of heterogeneity than synthetic drugs^[1][2]. Biopharmaceuticals show many advantages over conventional drugs which include targeting only specific molecules, the high activity and specificity and rare side effects^[3]. However, although many advances have been made in the study of biopharmaceuticals in recent years, this topic deserves a lot of attention due to the high potential shown by this class of compounds to obtain increasingly performing products for therapeutic use in medicine. In particular, the production of biopharmaceuticals involves the need for an adequate physico-chemical characterization because, unlike small conventional drugs, even minimal alterations in the production processes can strongly modify the final product and consequently both its safety and its bioactivity. Therefore, the quality assurance of biopharmaceutical products requires a much more specific and accurate physico-chemical characterization than for small molecules. An overview of mass spectrometry and chromatography techniques, some of the most important analytical methods available for the physico-chemical characterization of biopharmaceuticals, is provided below.

2. Mass Spectrometry (MS)

Mass spectrometry (MS) is a useful and widely used methodology for the characterization of biopharmaceuticals. Advances in MS instrumentation and techniques have enhanced protein characterization capabilities and supported increased development of biopharmaceutical products.

Thanks to the MS instruments such as Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) and Electrospray Ionization Quadrupole Time-of-Flight (ESI-QTOF) MS, characterized by high mass accuracy and resolving power, is possible to determine the molecular weight of the biopharmaceutical, the first parameter to be known which exactly identifies the molecule, its subunits and confirms the primary structure.

By tandem MS (MS/MS) the primary structure, i.e. the amino acid sequence, disulfide linkages and post-translational modifications (PTMs) of biopharmaceutical, is investigated. First of all, protein is digested by suitable enzymes, the obtained peptides are then separated by HPLC and identified by tandem MS (MS/MS). The results of this analysis can reveal modifications which affect protein bioactivity and pharmacokinetic and for this reason it should be always performed during manufacturing ^[5].

The analysis of the monosaccharide content and composition are usually performed by Gas Chromatography Mass Spectrometry (GC–MS) after derivatization of the biopharmaceutical, and the oligosaccharide composition investigated by Light Chain Mass Spectrometry (LC–MS) and MALDI-TOF-MS ^[6].

Hydrogen-deuterium exchange MS (HDX-MS), chemical cross-linking MS, and ion mobility MS (IMS), give a fingerprint of higher order structure of biopharmaceuticals including secondary, tertiary, and quaternary structures, which determine the spatial arrangements of the biopharmaceutical responsible for its bioactivity^[7].

3. Chromatography

Release analysis and profiling the charge isoforms caused by enzymatic or chemical modifications of biopharmaceuticals are determined by Ion-Exchange Chromatography (IEX), a technique widely used for the detailed characterization of therapeutic proteins which can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge heterogeneity^[8]. IEX can resolve protein heterogeneities due to modifications such as C-terminal lysine truncation, N-terminal glutamate to pyroglutamate conversion, deamidation and sialylation.

Reversed phase high performance liquid chromatography (RPLC) is a common method used for physicochemical characterization and quality control of biopharmaceuticals. This technique it is mainly used for protein quantification and purity determination as well as in release tests. By direct comparison with native biotherapeutic drugs it is possible to quantify the oxidized, reduced and deamidated forms obtained^[9].

Hydrophobic interaction chromatography (HIC) is considered as a reference technique to determine the relative hydrophobicity and to separate proteins based on their hydrophobic interactions. The analysis is performed under non-denaturing conditions, and this has two advantages: 1) it allows the conservation of proteins in their native forms and 2) the separated proteins may be collected for further analysis ^[10].

Hydrophilic interaction liquid chromatography (HILIC) is a technique used for glycan or glycopeptide analysis and it is considered an alternative to normal phase chromatography. The HILIC-UV-MS results obtained by Tengattini et al.^[11] for different glycosylated proteins suggest that with the right selection of the HILIC stationary phase and optimization of MS-compatible chromatographic conditions, a very good glycoconjugate resolution can be obtained. Moreover, intact glycoform of semi-synthethic glycoprotein products can be identified without sample pre-treatment.

Size exclusion chromatography (SEC) is a common, simple, low-cost technique which requires small quantity of samples and for these reasons it is the most frequent method used for the characterization of protein aggregates. It enables the separation of the monomeric protein and other variants of lower or higher molecular weight by differential exclusion from the pores of the stationary phase. Recent advancements in SEC methodology have improved size separation range, resolution, and sensitivity for analytes of interest^[11].

4. Conclusions and future perspectives

The physico-chemical characterization of biopharmaceuticals is a very important aspect that requires the use of advanced analytical methods for the quality assurance of the final products that must meet specific safety and bioactivity requirements. This requires that the analytical methods used for daily quality control are reliable, easy to use and available at low cost. Mass spectrometry and chromatography techniques are physico-chemical characterization methodologies often used due to their ability to provide precise, confident and information-rich data. Anyway, they have to be complemented by other analytical approaches for full physicochemical characterization of biopharmaceuticals.