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. 

Figure 1. Conceptual workflow for protein processing leading to LC-MS-based analysis of proteins. In two key areas, IP and digestion, rapid membrane processing enables a significant cut in overall time required. Reproduced from Robinson et al. 2020 with permission from The Royal Society of Chemistry.

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.

Figure 2. Total approximate time for IP, reduction, alkylation, digestion, and cleanup for standard and spin-column workflows. For the standard workflow, solid areas show the 4-hour minimum of a typical nonaccelerated workflow, and the striped area shows the additional time required for overnight digestion (16 hours in total). Capturem IP reduced IP time from 70 minutes to 15 minutes for individual spin columns or 55 minutes for 96-well plates (partially automated on a liquid handler). Trypsin digestion went from 16 hours with the standard method down to just a few minutes for individual spin columns and about 20 minutes for the 96-well plates. Both systems utilized the same reduction, alkylation, and cleanup steps. Reproduced from Robinson et al. 2020 with permission from The Royal Society of Chemistry.

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.

Figure 3. PK study analysis of Herceptin doses in rat. Five samples each were processed either by membrane-based (dark green) or standard methods (light green). Concentration results for Herceptin were based on GPSVFPLAPSSK (GPS; Panel A), and TPEVTCVVVDVSHEDPEVK (TPE; Panel B) was used for confirmation. The linear log-linear trapezoidal method was used to calculate AUC. Initial concentration (C0) was determined by linear extrapolation using the first three time points (0.25, 0.5, and 1 hr). The last three time points (24, 72, and 168 hr) were used as regression points for PK calculations. Reproduced from Robinson et al. 2020 with permission from The Royal Society of Chemistry.

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.

References

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).