Abstract
Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.
Highlights
There is a fundamental difference between traditional drug substances and biopharmaceuticals.In contrast to traditional Active Pharmaceutical Ingredients (APIs), in which the InternationalNonproprietary Name (INN) as a rule indicates a defined structure, the InternationalNonproprietary Name (INN) of a biopharmaceutical generally does not define only one specific molecular entity, but stands for a mixture of similar compounds
In accordance with previously published data, 2D-electrophoresis showed multiple spots spreading at ~50 and ~23 kDa; numerous poorly resolved spots were observed in both chains, in HC, the most intense spots laying on the basic end [2] (Figure 1)
Two-dimensional gel electrophoresis (2-DE) experiments involving N-deglycosylation, desialination and terminal lysine cleavage demonstrated no significant changes in the pIs and Mr of trastuzumab HC
Summary
Nonproprietary Name (INN) as a rule indicates a defined structure, the INN of a biopharmaceutical generally does not define only one specific molecular entity, but stands for a mixture of similar compounds. These mixtures are mainly a result of differences in the epigenetic modification of a given protein sequence. Strict observation of the quality is of particular importance in this class of compounds It has to be kept in mind that drug efficacy may be subject to variations or even loss, due to changes in the epigenetic processing of a specific protein.
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