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Size Exclusion Chromatography and UHPLC

Chromatography: Size Matters

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The quality of SEC analysis is always dependent on a low extra column dead volume of the LC system used. Hence, transferring SEC-methods to UHPLC also requires special attention to the instrumentation besides the selection of the proper UHPLC column. While all modern UHPLC systems provide extremely low dead volumes and small detector cell volumes for UV detection, this is more complicated when it comes to MALS detection.

Fig.3: Analysis of a therapeutic antibody with UHP-SEC-MALS (three different injection volumes, Wyatt µDawn Detector). Data provided by courtesy of D. Roessner, Wyatt Technology Europe
Fig.3: Analysis of a therapeutic antibody with UHP-SEC-MALS (three different injection volumes, Wyatt µDawn Detector). Data provided by courtesy of D. Roessner, Wyatt Technology Europe
(Source: Tosoh Bioscience)

The Wyatt µ-Dawn, a three-angular MALS detector, is ideally suited for UHP-SEC and was used with the UP-SW3000 column to analyze the aggregates of a therapeutic antibody (Fig. 3). A comparison of the MALS chromatogram with the UV and refractive index signals (data not shown) revealed that the small peak achieved by the UHPLC column was retained for all three detectors.

UHP-SEC-MS for Antibodies

Fig.4: Formation of Bispecific T Cell Engager
Fig.4: Formation of Bispecific T Cell Engager
(Source: Tosoh Bioscience)

More potent antibody formats, such as bispecific antibodies (bsAbs), are on the rise. Bispecific antibodies recognize two different epitopes. This dual specificity increases the potency of these molecules compared to mAbs and expands the range of possible applications. More than 50 bsAb products are currently undergoing clinical evaluation. Characterization of bsAbs is essential to ensuring product safety and efficacy.

Size exclusion chromatography (SEC) coupled with mass spectrometry (MS) is increasingly being used to identify the accurate molecular mass of biomolecules. SEC-MS, however, requires the use of volatile mobile phases and the use of columns that do not exhibit particle shedding which will interfere with the MS signal.

A bispecific T cell engager consisting of two single-chain variable fragments (scFvs) recombinantly linked by a nonimmunogenic five-amino-acid chain (Fig. 4) was analyzed by SEC-MS using a TSKgel UP-SW3000, 2 μm column. Figure 5 shows the total ion chromatogram, mass spectrum and deconvoluted mass spectrum of the Bite. A main peak can be seen at m/z 54,143; adjacent peaks correspond to different salt adducts.

Fig.5: SEC/MS analysis of a Bite (performed by the Wistar Proteomics and Metabolomics Facility, Philadelphia, PA) a) total ion chromatogram, (b) mass spectrum and (c) deconvoluted mass spectrum
Fig.5: SEC/MS analysis of a Bite (performed by the Wistar Proteomics and Metabolomics Facility, Philadelphia, PA) a) total ion chromatogram, (b) mass spectrum and (c) deconvoluted mass spectrum
(Source: Tosoh Bioscience)

These results demonstrate accurate molar mass determination utilizing a ammonium acetate/ammonium bicarbonate mobile phase with SEC-MS compatibility. Blank injections before and between each analysis proved the absence of shedding or carryover (data not shown).

Summary

Today, UHPLC is increasingly used for the analysis of biomolecules. Considering the complexity of large proteins such as antibodies, the transfer from established HPLC methods to UHPLC is not as easy as for small molecules.

For size exclusion chromatography, the new UP-SW3000 UHPLC column from Tosoh Bioscience offers an easy and straightforward method transfer to UHPLC. At a smaller particle size it features the same pore size and surface modification as the well-stablished G3000SWXL HPLC column. Its high durability and good batch-to-batch reproducibility are the main reasons for its rising popularity in the biopharmaceutical industry.

The easy implementation in SEC-MS and SEC-MALS methods is an additional benefit when it comes to the characterization of new biopharmaceutical formats such as bispecific antibodies, antibody-drug conjugates or virus like particles.

* R. Römling, Tosoh Bioscience Separations Business Unit Europe, 64347 Griesheim/Germany

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