Kendall Cradic (Presenter)
Mayo Clinic
Authorship: Kendall Cradic (1), Paula Ladwig (1), Dave Barnidge (1), Michael Zorn (2), Christina Christ (2), Karl Florian Wintgens (2), Maria Willrich (1)
(1) Mayo Clinic, Rochester, MN, USA (2) Immunodiagnostik, Bensheim, Germany
| Short Abstract Quantitation of therapeutic monoclonal antibodies is important for monitoring loss of response to therapy. Mass spectrometry assays have been developed for specific antibodies, mostly utilizing tryptic digest methodologies. We developed an assay for quantitation of vedolizumab intact light chains using the Q-Exactive Orbitrap mass spectrometer. The linear range (1-150 mcg/mL) is consistent with the therapeutic range and precision at 1 mcg/mL is less than 10%. Method comparisons with a tryptic digest method (Immunodiagnostik, Bensheim, Germany) showed a slope of 1.002 with an intercept of -0.70 (r=0.991; n=84). The assay provides a simple, rapid, and accurate means to monitor vedolizumab. |
Long Abstract
Background
The use of therapeutic monoclonal antibodies (mAbs) is becoming increasingly important for treatment of a wide variety of diseases. Because of their low toxicity and high specificity for disease targets, mAbs are presenting as a frontline therapeutic option. In parallel with their prescribed use, physicians seek the ability to monitor patients for effectiveness of therapy or study causes for loss of response. This presents the opportunity for new companion diagnostics to measure mAbs. We have previously shown that mass spectrometry based assays that do not rely on mAb specific reagents can be used to quantify mAbs in patient sera (1, 2). The effort shown here is an extension of previous work performed on a lower resolution Q-TOF mass spectrometer (2). We demonstrate that a Thermo Scientific Q-Exactive Orbitrap mass spectrometer run at a high resolution setting of 140,000 can be used to detect and quantify intact mAbs by monitoring specific isotope peaks of select light chain charge states, thus providing a platform for rapid and accurate measurement for clinical use.
Vedolizumab was FDA-approved in 2014 to treat inflammatory bowel disease (IBD). It is a humanized IgG1 that binds specifically to the alpha-4-beta-7 integrin receptor located on the surface of T-lymphocytes. Since this receptor is specific to signals originating in the gut, vedolizumab can be used to reduce inflammation in the small and large intestines. Crohn’s disease and ulcerative colitis have both been effectively treated with vedolizumab.
Monitoring disease activity and therapy effectiveness in IBD requires a colonoscopy or biopsy, both invasive procedures. Measuring mAbs has become the standard of practice for monitoring loss of response to therapy for other IBD biologics, such as infliximab and adalimumab. To provide adjunct therapeutic monitoring capability to vedolizumab, we have developed a quantitative assay based on its intact light chain detected in patient serum.
Method
Residual waste serum samples from 84 patients undergoing treatment with vedolizumab were collected during various time points in therapy. Initial development of vedolizumab quantitation used a tryptic digest method (3). Technical challenges and labor requirements of that strategy motivated an attempt to quantify intact protein, by measuring the mAb light chain (4).
To 20 mcL of patient serum, we spiked nivolumab as an internal standard at 50 mcg/mL. The resulting mixture was diluted into 200 mcL of Melon Gel (ThermoFisher Scientific) slurry which binds high abundance serum proteins and leaves immunoglobulins in solution. The slurry was incubated for 30 minutes with constant mixing, followed by removal of the Melon Gel solid phase. A 200 mcL aliquot of the resulting liquid was treated with DTT (50 mM final concentration, 55 C for 30 minutes) to reduce disulfide bonds between light chains and heavy chains. This mixture was applied to an AdvanceBio RP-mAb column (Agilent Technologies) and light chains were eluted using an acetonitrile/water gradient over 7 min at 250 mcL/min.
The mass spectrometer used for detection was a Thermo Scientific Q-Exactive scanning at a resolution setting of 140K. TSIM acquisition was used to isolate windows 4 m/z wide surrounding each charge state used for quantitation. Vedolizumab was quantified using its +10 and +11 charge states. In order to optimize signal to noise, 9 individual isotopic peaks in each charge state envelope were extracted using a 3 ppm window. The peak area from the sum of the 9 extracted ion chromatograms was used for quantitation. Only the +11 charge state was used to integrate the internal standard.
Even with the high mass resolution provided by the instrument, there is still concern for interference from endogenous monoclonal proteins. To address this, we intercalated low-resolution (17K) scans into the duty cycle that cover the entire lambda and kappa light chain mass ranges. These scans provided a qualitative, high-level view of the entire mass region and can alert the user to possible M-spikes that might interfere with quantitation. This advantage was included as a precautionary measure because of the unknown prevalence of monoclonal spikes and free light chains in the IBD population, which commonly presents with hypergammaglobulinemia.
Results
The calibration curve was linear and consisted of 8 standards made by spiking vedolizumab into pooled human serum (0, 1, 5, 10, 25, 50, 100, 150 mcg/mL). This analytical range was selected to correspond to the therapeutic range in most patients. Accuracy was assessed by method comparison with a tryptic digest assay performed by Immunodiagnostik (Bensheim, Germany) using 84 residual patient specimens. The comparison showed a slope of 1.002 (95% CI: 0.955 to 1.06) and intercept of -0.70 (95% CI -2.9 to -0.03), with a correlation coefficient of 0.991 after Passing-Bablok linear regression. Our lower limit of quantitation at 1 mcg/mL showed an inter-assay coefficient of variation of 8% with an average signal-to-noise of 3 (n=19). Imprecision at 3 mcg/mL was 8% (n=19) with an average signal to noise of 7.
Discussion
The newly developed method offers a rapid and accurate assay to monitor intact vedolizumab in patients with IBD. Using high resolution accurate mass measurement it was possible to reach an LOQ of 1 mcg/mL, which covers the entire therapeutic range, a 10-fold gain when compared to a lower resolution Q-TOF instrument (3). At trough, therapeutic concentrations of vedolizumab are expected to be above 5-8 mcg/mL. The highest concentration observed after testing residual patient serum a few days after infusion of the mAb was < 150 mcg/mL. Because we are measuring intact protein, we have eliminated the troublesome and laborious tryptic digest techniques that are common with many proteomic assays. The method is technically simple and reagents are inexpensive. Typical hands-on time is less than 60 minutes for the average batch, and with multiplexed HPLC, results can easily be reported within 24 hours of sample receipt in contrast to 2-day pre-analytical steps and overnight incubation of most trypsin digest methods. By combining high-resolution scans for quantitation and low-resolution scans for observation of interfering proteins, we are alerted to interfering monoclonal proteins.
The method is simple, reliable, and sensitive enough to monitor the effective therapeutic range of vedolizumab. The novel approach described combines the measurement of reduced light chains from vedolizumab on high resolution mass spectrometry using the Q-Exactive Orbitrap detector, and can provide additional layers of specificity to quantitation of other monoclonal antibody therapeutics.
References & Acknowledgements:
1. M. A. V. Willrich, D. L. Murray, D. R. Barnidge, P. M. Ladwig, M. R. Snyder, Quantitation of infliximab using clonotypic peptides and selective reaction monitoring by LC-MS/MS. Int Immunopharmacol 28, 513-520 (2015).
2. J. R. Mills et al., Using Mass Spectrometry to Quantify Rituximab and Perform Individualized Immunoglobulin Phenotyping in ANCA-Associated Vasculitis. Anal Chem 88, 6317-6325 (2016).
3. P. M. L. W. Katrangi, D. R. Barnidge, J. R. Mills, S. Dasari, D. L. Murray, M. R. Snyder, M. A. V. Willrich, in 67th AACC Annual Scientific Meeting and Clinical Lab Expo. (American Association for Clinical Chemistry, Atlanta, GA, 2015).
4. D. R. Barnidge et al., Using Mass Spectrometry to Monitor Monoclonal Immunoglobulins in Patients with a Monoclonal Gammopathy. J Proteome Res 13, 1419-1427 (2014).
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |