An article published by X. Gao and Y. Xu et al at Merck & Co., Inc. in the January 2021 issue of the Journal of Pharmaceutical and Biomedical Analysis demonstrated a full validation of a therapeutic monoclonal antibody (mAb) using Mitra® devices based on VAMS® technology. The investigators included both nonhuman primate and clinical samples in their study. The paper is entitled “Volumetric absorptive microsampling (VAMS®) in therapeutic protein quantification by LC-MS/MS: Investigation of anticoagulant impact on assay performance and recommendations for best practices in method development.” It describes the first use of LC-MS/MS for quantitation of mAbs from VAMS extracts taken from venous blood (collected from a tube) and capillary blood (collected using a finger-prick). Furthermore, the paper highlights the importance of evaluating the impact of anticoagulant and choosing the correct internal standard when comparing capillary vs. stabilized venous blood.
Studies on Monoclonal Antibody Therapy
Over the last three decades, monoclonal antibodies (mAbs, a class of large protein therapeutics) have been gaining popularity as an immunotherapy approach to treat some diseases. One such drug that has gained popularity is Humira, a tumor necrosis factor-blocker, which acts to reduce joint swelling and inflammation in conditions such as arthritis.
While early studies of mAbs therapies relied on liquid venous blood samples, in recent years there have been several papers in the literature validating dried capillary blood VAMS extracts for mAbs. One such paper demonstrated successful validation of seven mAbs drugs using ELISA. However, up until the article from Gao and Xu et al from Merck & Co. that is reviewed here, an LC-MS/MS method had not been previously reported using VAMS extracts for mAbs.
Quantitative analysis of large protein therapeutics for bioanalytical studies raises a number of key challenges when compared to small drug molecules. This includes developing assays which are both sensitive and specific enough for modern analytical validation requirements. For small drug candidates, LC-MS/MS is the preferred analytical platform, because it offers both high specificity and sensitivity. For large protein therapeutics, there have been a number of bioanalytical approaches developed, including ligand binding assay (LBA) such as immunoassay, and hybrid LBA-LC-MS/MS and LC-MS/MS of digested protein such as pellet digestion. The latter technique involves protein precipitation of a biological fluid like plasma and then reconstitution of the proteinaceous precipitate in a buffer where the sample can then be digested using trypsin. The resultant peptides then act as signatures for the original protein of interest. Due to their small size, these peptides can then be measured on standard LC-MS/MS equipment used for small molecule analysis.
Due to the benefits of LC-MS/MS, the Merck & Co. team decided to use the pellet digestion method for a protein therapeutic. They stated, “LC–MS has demonstrated unique advantages including high specificity, short development time, and the ability of multiplexing.”
Sample Extraction Study Methods & Findings
A preclinical validation was successfully conducted using samples from Rhesus monkeys in accordance with industry accepted bioanalytical guidelines.
A further focus was also placed on validating the method for HCT dependence where no HCT impact was observed. Furthermore, dilution integrity was evaluated and relative extraction recovery was found to be in the range of 90-104%. Long-term stability was established at 2-8°C for 98 days. Up to 24h stability was observed at both RT and 45°C in 75% humidity.
In terms of PK study design, 3 monkeys were dosed intramuscularly and 3 intravenously. Samples were collected weekly over 1 month. Serum, venous blood, and capillary blood was collected at each time point. No difference was observed between venous blood and capillary blood both collected on Mitra with VAMS (R2 = 0.9861). The whole blood VAMS sample showed ~60% of the concentration compared to serum and this was attributed to no partitioning into the hematocrit fraction (~40% blood volume).
The method was then used in a phase 1 study IV dosing healthy volunteers. Where serum samples (from IV) were collected & stored at -80°C and finger-prick samples (from Mitra devices) were collected and stored at 2-8°C.
Interestingly, internal standard (IS) responses for the capillary samples were significantly higher than the EDTA samples. The group then investigated how to identify the root cause of the problem.
They ruled out any lot-to-lot variability and they reported “No distinguishable IS response difference was observed between the two formats as well as between two lots of VAMS tips in the same 96-well format.”
Using spiking studies, the group narrowed down the root cause to the EDTA in the venous sample. They hypothesized that the EDTA was chelating to divalent metals, thus impeding the efficacy of the Trypsin. Indeed, one experiment showed that addition of calcium to the EDTA sample extract recovered the IS values.
They commented that the reason why they didn’t see the same phenomenon in the monkey extracts was possibly due to differences in the abundance of cations between the human and monkey blood, or maybe differences in the ETDA concentration of each blood origin.
They proposed that this phenomenon should be tested early on in the method development using pooled blood with or without the presence of the anticoagulant EDTA.
One final observation was that as they used full-sized stable labelled (SIL)-mAb rather than a SIL-surrogate peptide as an internal standard. This allowed them to observe the issue with EDTA. However, it is worth noting that if they had used a SIL-surrogate peptide, they would have missed this phenomenon as they would not have seen differences in digestion efficiency compared to non-EDTA blood. This suggests that using full-sized stable labelled IS for protein analysis on VAMS is best for LC-MS/MS.
Study Authors' Discussion & Conclusions
A whole blood VAMS assay was fully validated for a mAb compared to serum.
The bridging pilot study from monkeys showed “excellent correlation between measured mAb1 concentrations in serum and in VAMS, including venous VAMS and capillary VAMS samples.”
IS discrepancy from the clinical data showed EDTA was the culprit when comparing capillary to anti-coagulated venous blood.
The authors recommend that during development investigators should compare preparing pooled blood and capillary blood from volunteers to rule out issues with the clinical study.
Use a full-sized SIL-protein as IS like the investigators in this study did.
The team found that “Overall, the work presented here demonstrates that VAMS is a reliable sampling technology for the quantification of monoclonal antibodies.”
Care must be taken with IS and choice of anticoagulant.
As Mitra devices with VAMS technology are deployed into more and more diverse assays, it is crucial to always investigate any unusual observations such as the effect EDTA was having on the sample preparation. Blood is a much more complex matrix to work with compared to serum and, although assays such as this study by Merck & Co. can be successfully validated and deployed into clinical trials, factors such as digestion efficiency much be considered to prevent other unforeseen issues down the line.