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Characterising Biotherapeutics using TSK-GEL® HPLC Columns

By: Regina.Roemling, Denise Wallworth, Reporter EU Volume 41

Regina.Roemling1 and Denise Wallworth

denise.wallworth@sial.com

1 Tosoh Bioscience, Germany

Development and production of therapeutic biopharmaceuticals is a growing segment of the pharmaceutical industry. A thorough characterisation of these is a key task for the successful submission of research and production data for regulatory approval of new drugs. The introduction of the first so-called biosimilars in Europe has further increased the demand for highly efficient analysis methods. Sigma-Aldrich has now expanded its supply of the complete range of Tosoh Bioscience HPLC columns that provide the robustness and reproducibility for these applications.

The typical chromatographic modes for the separation of proteins in their native form such as size exclusion chromatography (SEC) and ion exchange chromatography (IEC) are routinely used for the characterisation of biotherapeutics. In addition, reversed phase (RPC) and hydrophilic interaction liquid chromatography (HILIC) are applied to characterise protein moieties such as peptides or oligosaccharide chains after enzymatic cleavage. TSK-GEL HPLC columns are routinely used in all of these analytical methods in the biopharmaceutical industry, and in this article some of their applications for monoclonal antibodies will be discussed.

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Determination of protein aggregates by SEC with TSK-GEL SW columns

Protein aggregation is a common issue encountered during expression, purification and formulation of protein biotherapeutics. Aggregation needs to be characterised and controlled during the development of protein pharmaceuticals such as monoclonal antibodies (MAbs); even small amounts of aggregates can alter the MAbs’ function as an effective therapeutic. High-performance SEC on silica-based columns is the industry’s workhorse for separating and quantifying soluble protein aggregates in biotherapeutics, the analysis of which using SEC is almost always required for regulatory approval.

TSK-GEL SW series columns, and TSKgel G3000XL in particular, are the industry standard for biotherapeutics. These are based on highly porous silica particles, the surface of which have been shielded from interacting with proteins by derivatisation. They contain a large pore volume per unit column volume, which results in high resolution when analysing proteins. When sample size is limited or the components of interest are present at very low concen tration, TSKgel Super SW3000 columns can be applied to achieve even higher performance. The internal diameter of these columns has been reduced from 7.8 mm to 4.6 mm and the particle size from 5 μm to 4 μm in order to provide higher sensitivity in sample-limited cases.

Figure 1 compares the analysis of MAb aggregates on both column types. Owing to the different inner diameters of the columns, the flow rates are different in order to apply the same linear flow. The figure shows that resolution of monomer and aggregate peaks is higher on the SuperSW column, as is the detection sensitivity.

Figure 1 Determination of monoclonal antibody aggregates on silica based SEC columns.

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Analysis of MAb charge variants by IEC with TSK-GEL STAT columns

Apart from isoelectric focusing, cation exchange chromatography is a method of choice to analyse charge hetero-geneity of proteins. Charge isoforms of proteins result from deamidation of asparagine or glutamine residues, or from incomplete removal of C-terminal lysine residues. The newly developed TSK-GEL STAT ion exchange columns can help to increase throughput in QC of biopharmaceuticals.

TSK-GEL STAT ion exchange columns are non-porous polymer columns with a high surface density of functional groups: quaternary ammonium for anion exchange (Q- and DNA-STAT), carboxymethyl (CM-STAT) and sulphopropyl (SP-STAT) for cation exchange. Applications include the separation of peptides and proteins, PEGylated proteins and charge isomers of monoclonal antibodies.

As an example, a TSKgel CM-STAT weak cation exchange (WCX) column was used to separate charge variants of several monoclonal antibodies. Figure 2 shows the analysis profiles for five different antibodies (A to E) on a 10 cm CM-STAT column, filled with 7 μm particles, confirming that high-resolution analysis can be obtained in around 20 minutes. In contrast, typical analysis times on conventional 25 cm long WCX columns are generally about forty minutes.

Figure 2 Separation of charge variants for 5 monoclonal antibodies on TSKgel CM-STAT

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Characterisation of protein glycosylation by HILIC with TSKgel Amide-80 columns

Another important QC parameter of MAb characterisation is the characterisation of glycosylation, since it may influence immunogenicity, pharmacokinetic and pharmacodynamic properties. Several complementary analytical techniques are routinely used to characterise, identify and quantify oligosaccharides isolated from glycoproteins. The standard method for QC analysis of glycans, for example, is the HILIC/normal phase separation of AB-labelled glycans followed by fluorescence detection.

The chemistry of amide-bonded HILIC phases such as TSKgel Amide-80 is ideally suited for the separation of charged and neutral fractions of glycan pools in one run. The retention of fluorescence-labelled polysaccharides is used for the identification of glycan structures by comparison to a labelled dextran ladder; this is used to normalise retention times in order to calculate the number of glucose units (GU values) of the separated glycans. The GU values obtained after separation of sequential exoglygosidase digests can then be used to predict the glycan structure by database query. Figure 3 shows one example of the fluorescence chromatograms of HILIC separations of 2-AB labelled N-glycans released from a recombinant ZP domain construct of murine transforming growth factor beta type 3 receptor (TGFR-3), compared to the dextran ladder.

Figure 3 HILIC chromatograms of 2-AB labelled glycans

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Summary

TSK-GEL HPLC columns are applicable to a broad range of HPLC applications used in development, production, validation and release of protein therapeutics. Some of the applications mentioned above are mandatory when submitting a new biopharmaceutical for regulatory approval. Tosoh Bioscience is continuously working on new stationary phases for the common modes of biochromatography; Sigma-Aldrich is pleased to be expanding its support for the Tosoh products throughout Europe.

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