To demonstrate the effectiveness of a simple pH gradient/ion-exchange chromatography workflow approach to the characterization of charge variant profiles of an innovator molecule (cetuximab) and a candidate biosimilar. To show the assay is simple, reproducible, easily optimized and resolves variants effectively.

To demonstrate the CX-1 pH buffer system is capable of charge variant determination for the majority of therapeutic monoclonal antibody species. To show the ease of optimization and improved reproducibility of this buffer system when compared to salt-based gradient systems used for charge variant analysis.

A UHPLC workflow for mAb aggregate analysis that operates continuously for weeks.

The biopharmaceutical industry continues to develop protein-based biotherapeutics in increasing numbers. Due to their complexity and biotechnological production, there are many attributes that need to be analyzed to guarantee their safety and efficacy. Peptide mapping is used to measure several critical quality attributes (CQA) required for the characterization of any biotherapeutic protein. The analysis is used to confirm that the correct sequence has been expressed for the protein and to check for post-translational and chemical modifications.

The Thermo Scientific Vanquish Flex UHPLC system with UV detection is applied for the charge variants analysis of NISTmAb Humanized IgG1K monoclonal antibody using strong cation exchange-based chromatographic separation on a MAbPac SCX-10 column. The use of Thermo Scientific CX-1 pH gradient buffers enabled high resolution separation of reference material monoclonal antibody charge variants using a 30 minute gradient.

The Thermo Scientific Vanquish UHPLC system with Fluorescence detection is applied for the rapid glyco-profiling of a commercial chimeric IgG1 mAb.The separation is performed on a Thermo Scientific Accucore-150-Amide HILIC column in under 2.5 minutes. The developed methodology offers an excellent analytical platform for ultrafast glycan profile comparison of different batches​ of these mAbs.

The Thermo Scientific SMART Digest Kit is applied for generation of peptides from therapeutic monoclonal antibodies in 60 minutes. Peptide mapping samples were analysed on a Thermo Scientific UltiMate U3000 RSLC system coupled to a Thermo Scientific Q Exactive HF with Biopharma Option. Separation of sample peptides was performed on a Thermo Scientific Acclaim RSLC 120, C18, 2.2 µm analytical column using a 30 minute separation gradient, yielding excellent chromatographic separation and high sequence coverage.

The Thermo Scientific MAbPac SCX-10 column is applied to the charge variant analysis of Cetuximab. Ion exchange separation was performed using a pH gradient on a Thermo Scientific Vanquish Flex UHPLC system with UV detection at 280 nm. Use of Thermo Scientific CX-1 pH gradient buffers enabled high resolution separation of monoclonal antibody charge variants over a 30 minute gradient.

The Thermo Scientific MAbPac SCX-10 column is applied for charge variant analysis of Infliximab. Ion exchange separation was performed on a Vanquish Flex UHPLC system using a pH gradient and UV detection at 280 nm. Use of Thermo Scientific CX-1 pH gradient buffers enabled high resolution separation of monoclonal antibody charge variants over a 30 minute gradient.

Ion exchange separation is applied for charge variant analysis of Bevacizumab using a pH gradient on a Thermo Scientific MAbPac SCX-10 column. The Thermo Scientific Vanquish Flex UHPLC system with UV detection at 280 nm was used with Thermo Scientific CX-1 pH gradient buffers enabled high resolution separation of monoclonal antibody charge variants over a 30 minute gradient.

The Thermo Scientific Vanquish H system with fluorescence detection is applied for the glycan profile comparison of a biosimilar candidate produced ‘in house’ using Chinese hamster ovary (CHO) cell line using HILIC-based chromatographic separation on Accucore 150-Amide-HILIC column. Developed methodology offers an excellent analytical platform for glycan profile comparison of multiples lots of the biosimilar product.

The Thermo Scientific Vanquish H system with fluorescence detection is applied for the rapid glycoprofiling of a commercial chimeric IgG1 mAb. The separation is performed on a Thermo Scientific Accucore-150-Amide HILIC column in under 2.5 minutes. Developed methodology offers an excellent analytical platform for ultrafast glycan profile comparison of various production batches.

The Thermo Scientific Vanquish H UHPC with Fluorescence detection is applied for the glycan profile comparison of trastuzumab innovator to a biosimilar candidate produced ‘in house’ using Chinese hamster ovary (CHO) cell line using HILIC-based chromatographic separation on Accucore 150-Amide-HILIC column. Developed methodology offers an excellent analytical platform for glycan profile comparison of multiples lots of both the innovator and biosimilar products

The Thermo Scientific Vanquish H UHPLC system was used for the high resolution determination of 2-AA (anthranilic acid) labelled N-glycans released from a commercial chimeric IgG1 mAb (Infliximab). The separation is performed on a 150 mm Accucore 150-Amide-HILIC column with fluorescence detection giving separation in less than 30 minutes.N-Glycans present at the asparagine 297 residue in the CH2 domain of the Fc region of IgG play a crucial role in modulating the structural stability and functional activity relationship of the antibody.

The Thermo Scientific Vanquish H UHPLC system is applied for the rapid determination of 2-AB (2-aminobenzamide) labelled N-glycans released from human serum IgG in less than 15 minutes using a 50 mm Thermo Scientific Accucore 150-Amide-HILIC column with fluorescence detection. The method is able to determine N-Glycans present at the asparagine 297 residue in the CH2 domain of the Fc region of IgG that play a crucial role in modulating the structural stability and functional activity relationship of the antibody.

Analysis of protein aggregation of five important biotherapeutic monoclonal antibodies (mAbs) by size-exclusion chromatography, showing the universal applicability of the Thermo Scientific MAbPac SEC-1 column for aggregate analysis of mAbs. The biopharmaceutical industry continues to develop mAb-based biotherapeutics have several key quality attributes that require quantification. This shows the ability to determine quickly and accurately the percentage of aggregates and fragments for five therapeutics as shown with full details in the AN21601 download.

The importance of correct UHPLC instrument setup for protein aggregate analysis by size-exclusion chromatography is shown by using the Thermo Scientific Vanquish Flex Quaternary UHPLC system. For this analysis, pre-column dispersion was reduced to show the effects of dispersion in front of the column at different flow rates. After the flow rates are reduced to 300 μL/min with the MAbPac-SEC-1 column formats of 4 mm i.d., the use of low volume tubing and fittings becomes essential in order to maintain resolution of aggregates from the main peak. Refer to attached pdf file for full experiment.