Sample preparation for mass spectrometry has been the topic of several recent symposia extensively covered in the pages of GEN. Mass spectrometry has grown over the years into an essential tool for macromolecular characterization due to the development of more economical and user-friendly instrumentation as well as more effective and accurate sample processing.
New wrinkles to the technology were considered at CASSS’ recent “Practical Applications of Mass Spectrometry in the Biotechnology Industries.” These include mass spec analysis of glycoforms in partial antibody fragments, more stringent characterization of disulfide bond formation, and innovative deglycosylation strategies.
Asish Chakraborty, Ph.D., a research scientist at Waters, and his colleagues advanced the quantification of antibody glycoforms using liquid chromatographic (LC) separation combined with mass spec. “This area of investigation is critical for an understanding of the stability, biodistribution, and the target binding of antibodies,” he stated.
mAbs are an excellent model system for mass spec investigations, as they are glycosylated at the asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known, according to Dr. Chakraborty. Quantification of the glycan moieties frequently involves enzymatic release of the glycans from the proteins to which they are bound through N-linkages. Alternatively, fluorescent labeling and separation of labeled glycans using normal-phase chromatography with fluorescent detection are employed.
The presence of identical glycans in the protein, however, can make interpretation difficult. A superior interpretation of the structures may be obtained by both endopeptidase digestion of the antibody molecules, which results in the release of the Fc light chain fragment, and by reducing the sulphydryl linkages of the antibody to generate light- and heavy-chain fragments.
Dr. Chakraborty’s group combined ultraperformance liquid chromatography (UPLC) and liquid chromatography/electrospray ionization with time-of-flight mass spectrometry (LC/ESI-TOF-MS) methods to optimize various sample-preparation schemes from several batches of the commercial recombinant IgG1 mAb trastuzumab. Using UPLC analysis of 2-AB labeled released glycans as a reference method, various LC/MS-based assays for glycan quantification were compared.
The MS-based assay of the Fc/2 fragment is accurate and provides results that are comparable to the N-glycan assay, Dr. Chakraborty explained. Each batch of antibody shares nearly the same relative abundance of each glycoform, but one specific glycoform was absent in some batches. Unlike N-glycan release assay, this LC/MS-based method cannot separate isobaric glycans.
“MS should be considered a complimentary technology to the N-glycan release assay, and though it may not give a scientist all the desired information, it can be a lot quicker and easier to use than before,” Dr. Chakraborty remarked. “The beauty of the Xevo QT MS-based technology is that it is fast and easy to use, so results are yielded in an hour or two.”
Direct Online Processing
Spark Holland, a provider of sample-prep hardware and software, specializes in online services for mass spec analysis. Dries Vrielink, SPE (solid-phase extraction) services and applications manager, and Marcel Jansen, global marketing director, discussed the company’s extraction and separation technology for analytical systems, including HPLC, MS, GC, and NMR.
“There are several reasons why customers are interested in online sample preparation,” commented Vrielink. “High recoveries and better sensitivity are the main assets of this technique. Offline sample prep using such procedures as protein precipitation are not sufficiently selective and liquid-liquid extraction is difficult to automate, slow, and labor intensive. Furthermore, because of the removal of aliquots of the samples and additional handling, there can be a substantial loss of material.”
A solid-phase extraction system that allows one unattended run with online elution into HPLC and LC/MS systems was developed by the company in the 1980s and entered the market in the early 1990s. “We see big improvements in sensitivity with a notable economic advantage,” Jansen commented. “Since the runs can be left unattended, the investigator can simply load the samples, start the method, and run them overnight. So while the first sample is being analyzed, the next sample is undergoing extraction.”
Online SPE employs a technique similar to HPLC to isolate, enrich, and purify analytes from a sample matrix applied to a sorbent. All materials not adsorbed remain in the liquid phase and pass through the sorbent to waste. High pressures are applied to distribute the SPE solvents. The extracted sample is directly injected to the analytical column by a simple valve switch to avoid loss of the analytes of interest.