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A faster path to analysis for monoclonal antibodies as therapeutic agents
Monoclonal antibodies (mAbs) are a growing class of therapeutic agents used for targeted treatment of cancer and neurodegenerative diseases, among other conditions. These biologics show great promise due to their high specificity, activity, and favorable pharmacokinetics (PK). With PK studies occupying a critical early stage in drug development analysis—evaluating parameters like bioavailability, clearance, half-life, and metabolic profile—researchers are looking for key indicators for how well a drug candidate will perform further along the development pipeline. With increasing interest in this area, scientists often find themselves in need of faster, more efficient tools and methods to streamline the work of antibody engineering and purification, as well as preparing these molecules for analysis.
Researchers at Merck & Co., Inc., in collaboration with Takara Bio scientists, recently looked into various methods used for the protein processing workflow prior to liquid chromatography tandem mass spectrometry (LC-MS/MS), a technique gaining popularity in protein characterization and quantitation as it pertains to assessing drug candidates (Robinson et al. 2020). While immunoaffinity purification (IP) and enzymatic digestion play key roles here, by traditional methods, both processes add considerable time to sample preparation for LC-MS-based analysis. Robinson, et al. tested unique membrane-based technologies as possible alternatives to these long protocols (Figure 1). Their experiments showed a substantial reduction in the time required for processing, with sensitivity more than sufficient for the downstream PK studies required for drug development. As visualized in the image below, these methods for rapid membrane processing play important roles in creating a shorter path through the necessary stages of protein preparation prior to LC-MS analysis.
A question of time
The researchers directly compared the standard protein processing methods with corresponding Capturem technology—a novel, flexible system for membrane-immobilized reagents in spin columns and plates. Each experiment used samples of both light and heavy IgG1 λ stable isotope labeled universal mAb standards (SILuLite and SILuMAb, respectively). Typical IP and digestion, as performed by automated cartridge IP and overnight in-solution trypsin digestion, respectively, were pitted against Capturem products for IP via protein A and digestion via trypsin. They reported that the standard protocols took about 20 hours in total, compared to just 3 or 4 hours for the Capturem individual spin columns or 96-well plates, respectively (Figure 2). The most impressive time-savings came from the digestion step, a protocol that is most often a notable bottleneck in these protein preparation workflows. The authors reported that they expect to see even better results once they fully automate and optimize the protocols for the 96-well plates.
Evaluating the antibody digestion profile
In order to fully test the membrane-based digestion capabilities, the authors varied how many times the samples were run through successive passes of the trypsin column. While sequence coverage was highest for a single pass (heavy chain: 83%, light chain: 96%), missed cleavages were also at their highest levels (heavy chain: 25%, light chain: 39%). Both measurements went down slightly with each successive pass, and after four passes, peptide digestion was greater than that reached by in-solution digestion. Robinson et al. noted that this indicates the possibility of tuning the membrane digestion to match a particular desired performance or to exceed that of the traditional method.
Even with missed cleavages present, identification of distinct antibodies via mass spectrometry was still adequately enabled. While it is common to anticipate the exact same peptides as one would see via in-solution digestion, such a result is unnecessary, provided key peptides are still present. Indeed, these key peptides were consistent throughout the experiments, and it is exactly that consistency which enables peptide and antibody identification and quantification. As such, the sequence coverage and cleavage results seen with Capturem Trypsin were sufficient for this process as well as the downstream PK studies.
Meeting limit of detection needs
Quantitation for both traditional and membrane-based methods was assessed with an eight-point calibration curve in rat plasma for six surrogate peptides derived from heavy and light antibody chains. The curves were processed in triplicate, with all results showing linear relationships between area ratio and concentration. The lower limit of quantitation (LLOQ) was determined for each peptide, taking into account the percent bias of the calibration standards at each concentration. For the most sensitive peptide using each method, the LLOQ for the Capturem workflow was 0.1 ng/µl compared to 0.05 ng/µl for the standard workflow. Even with the higher LLOQ, the yield from the membrane-based method can still be easily detected, and the authors noted it remains well within range for many applications.
Membrane-based technology proves its value
In particular, the authors used the 96-well membrane workflow to conduct a PK study in which rats were dosed with Herceptin, a monoclonal antibody used for breast cancer and stomach cancer treatment. From this study, 5 µl of plasma were processed with both the 96-well Capturem workflow and the standard workflow. GPS and TPE were identified as two peptides that performed well in previously described quantitation tests, and were therefore used for quantification and confirmation, respectively. In this case, the LLOQ was the same for both workflows: 0.5 ng/µl. Additionally, the confirmation values for TPE were calculated to be within 6% for both methods. Altogether, these measurements indicate that membrane-based protein preparation method comes with the benefits of a faster, more streamlined lab experience while still retaining the ability to produce meaningful PK data.
An easier journey to drug discovery
As researchers move forward in the quest to produce targeted treatments for a wide range of debilitating and life-threatening diseases, it is important that the tools they use keep pace with their advancing needs. With so much at stake, even the smallest savings in time and funds can make a huge difference when it comes to the ultimate goal of improving and saving lives the world over. The drug development pipeline is a complex, demanding series of workflows and analyses, and large molecules like monoclonal antibodies are becoming more and more popular travelers on this path. We are proud to see multiple Capturem membrane technologies used to support more high-throughput, streamlined process development, and further heartened to see that those who work hard in this important field are able to pave the way for greater progress in human health.
Robinson, M.R., et al., Improving the throughput of immunoaffinity purification and enzymatic digestion of therapeutic proteins using membrane-immobilized reagent technology. Analyst 145, 3,148–3,156 (2020).
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Immunotherapy uses the patient's immune system to control and destroy cancer cells and holds several key advantages over traditional therapies. Explore this section to learn about adoptive T-cell therapy, bispecific monoclonal antibody therapy, and T-cell receptor profiling, an important tool to guide treatment strategies.Explore
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