Regina Römling discusses UHPLC analysis of therapeutic antibodies
Aqueous size exclusion chromatography (SEC) has become a mainstay for the analysis of protein aggregates in the biopharmaceutical industry. As the technique has increasingly been used for quality control of biologics since the early days of recombinant protein development for pharmaceutical purposes, various method improvements have evolved. Coupling SEC with advanced detection, such as mass spectrometry, light scattering or surface plasmon resonance, makes it a versatile tool for numerous applications beyond aggregate analysis.
Timelines in analytical laboratories are increasingly tough. On the other hand, there is a strong drive to explore and understand biologics more in detail. With new biopharmaceutical formats in the pipeline – bispecific antibodies and antibody-drug conjugates, for example – rapid and thorough characterisation will be even more important. In this context, the task for separation science is to increase resolution and plate numbers in shorter analysis time. Applying the latest ultra-high performance liquid chromatography (UHPLC) column technology improves the quality of separation and thereby the reliability of results.
ANTIBODY AGGREGATE ANALYSIS
The separation of monomers from their aggregates is one of the standard assays during development and manufacturing of therapeutic antibodies. SEC is the method of choice for these analyses and for decades, TSKgel G3000SWXL has been the standard for aggregate detection. UHPLC, a standard technology when analysing small-molecule drugs, is now getting increasingly popular for large molecules, such as biopharmaceuticals. A new UHPLC column – TSKgel UP-SW3000 – featuring the same pore size and surface chemistry as G3000SWXL but smaller particle size, is an ideal UHPLC column when transferring established HP-SEC methods to UHP-SEC.
Fig. 1 demonstrates the advantages of the TSKgel UP-SW3000 column (4.6mm x 30cm, 2 µm, 250 Å, flow rate 0.35mL/min) used with a UHPLC system for mAb analysis versus the use of a TSKgel G3000SWXL column (7.8mm x 30cm, 5µm, 250Å, flow rate 0.5mL/min) with a conventional HPLC system. TSKgel UP-SW3000 offers higher resolution of both the high molecular weight species and the fragments. In addition, the analysis was completed in half the run time.
UHP-SEC AND LIGHT SCATTERING
Coupling SEC with multi angle light scattering (MALS) detection enables absolute quantification of a molecule’s molar mass. SEC-MALS has become a valuable tool for verifying purity of monoclonal antibodies (mAbs). Transferring SEC-methods to UHPLC requires special attention to the instrumentation to keep the system’s dead volume small. Although all modern UHPLC systems provide extremely low dead volumes and small UV detector cells, this is more complicated when it comes to MALS detection. The Wyatt µDAWN, a three-angular MALS detector, is ideally suited for UHP-SEC and was used with TSKgel UP-SW3000 to analyse the aggregates of a therapeutic antibody (Fig. 2). A comparison of the MALS chromatogram with the UV and refractive index signals (data not shown) revealed that the small peak width achieved by the UHPLC column was retained for all three detectors.
UHP-SEC-MS FOR BISPECIFIC ANTIBODIES
More potent antibody formats, such as bispecific antibodies (bsAbs), are on the rise. Bispecific antibodies recognise two different epitopes, which increases the potency of these molecules compared to mAbs and expands the range of possible applications. SEC coupled with mass spectrometry (MS) is increasingly being used to identify the accurate molecular mass. SEC-MS, however, requires the use of volatile mobile phases and the use of columns that do not exhibit particle shedding interfering with the MS signal.
A bispecific T cell engager (BiTE) consisting of two single-chain variable fragments (scFvs) recombinantly linked by a five-amino-acid chain was analysed by SEC-MS (Fig. 3). The deconvoluted mass spectrum of the BiTE shows a main peak at m/z 54,143; adjacent peaks correspond to different salt adducts.
SUMMARY
Today, UHPLC is increasingly used for the analysis of biomolecules. The transfer from established HPLC methods to UHPLC is not as easy as for small molecules. For SEC, the TSKgel UP-SW3000 UHPLC column offers an easy and straightforward method transfer. 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 characterisation of new biopharmaceutical formats.
Regina Roemling is with Tosoh Bioscience
