Protein characterization

Analytic of proteinogenic drugs

There is a growing need in society for new therapeutic approaches in the form of biopharmaceuticals such as proteinogenic agents, antibody conjugates and immunotherapies for the treatment of serious and rare diseases. For this reason, investments are being made in the rapidly developing field of biologicals with the aim of advancing the development and production of biopharmaceuticals.

The production and release of biologicals is a challenging process, mainly due to the highly complex protein structures of these molecules. These complex structures require a high level of expertise, both in production and in quality control, which forms the basis for the release of the products. For the latter, specialized primary and secondary reference standards and analytical equipment, particularly in the field of mass spectrometry, are essential in addition to expert knowledge of analytics. The technology in this area is developing rapidly and requires regular investment. GBA Group stays up to date for you and operates an extensive pool of highly specialized analytical equipment. GMP-compliant qualified instruments are available for analysis.

The GBA Group is one of the few laboratories with a GMP qualification for the Orbitrap Exploris from ThermoFisher Scientific. Measurements can be carried out with the GMP and cGMP quality standard.

Protein characterization

The analysis for the quality control of biopharmaceuticals includes as an essential element methods of protein characterization, which are regulated in the European and American pharmacopoeias (Ph.Eur. and USP/NF).

The following topics are to be addressed in detail:

Ph. Eur.:

• 2.2.43 Mass Spectrometry

• 2.2.55 Peptide Mapping

• 2.2.59 Glycan Analysis of Glycoproteins

USP/NF:

• <736> Mass Spectrometry

• <1736> Applications of Mass Spectrometry

• <1055> Biotechnology-Derived Articles – Peptide Mapping

• <1084> Glycoprotein and Glycan Analysis – General Considerations

• <212> Oligosaccharide Analysis

The characterization of proteins and glycoproteins can take place at different levels:

• Level of the intact protein and glycoprotein

• Level of the cleaved glycans

• Level of peptides and glycopeptides (peptide mapping)

• Level of monosaccharides

• Level of amino acids

Our Methods

The GBA Group offers a wide range of methods for the analysis of proteinogenic drugs and monoclonal antibodies "for human use":

Analysis at the level of the intact protein and glycoprotein

• ESI and APCI mass spectrometry: Orbitrap Exploris from ThermoFisher Scientific

• Ion Exchange HPLC / -UHPLC

• SEC-HPLC / -UHPLC e.g. determination of aggregates

• HIC-HPLC / -UHPLC e.g. for antibody-drug conjugates

• Sodium Dodecyl Sulfate – PolyAcrylamid Gel Electrophoresis (SDS-PAGE) e.g. according to Ph. Eur. 2.2.31

• native PAGE

• Capillary electrophoresis (cGE, CE) e.g. determination of charge variants

• Capillary isoelectric focusing (cIEF)

• Sialylation heterogeneity

Analysis at the glycan level

• Enzymatic cleavage using exo-glycosidase

• ESI mass spectrometry: Orbitrap Exploris from ThermoFisher Scientific

• Anion Exchange - HPLC or UHPLC - MS

• HPAEC-PAD chromatography

• Fluorophore labeling, e.g. analysis of fluorescently labeled N-glycans using Glycoworks RapiFluor-MS

• Capillary electrophoresis of fluorescently labeled glycans

Analysis at the level of peptides and glycopeptides

Peptide mapping is roughly divided into the following steps:

• Isolation and purification (isolation of proteins and glycoproteins)

• Selective cleavage of the peptide bonds

• Chromatographic separation

Identification of peptides:

• using ESI mass spectrometry: Orbitrap Exploris from ThermoFisher Scientific e.g. molecular identity determination

• with peptide sequencing by ESI-MS-MS: Orbitrap Exploris from ThermoFisher Scientific

• by means of HPLC- / UHPLC- UV/VIS

• by qualitative and quantitative determination of post-translational modifications such as oxidation methionine

Analyses at the monosaccharide level

• HPAEC-PAD chromatography

• Fluorophore labeling with RP HPLC

Analyses at the level of amino acids
see "Amino acid analysis according to Ph. Eur. 2.2.56"