MSACL 2018 US Abstract

Topic: Proteomics

Quantitation of the Therapeutic Monoclonal Antibody Eculizumab Using High Resolution Accurate Mass Spectrometry

Ann Rivard (Presenter)
Mayo Clinic Clincal Mass Spec Dev Lab- DLMP

Bio: Ann Rivard is a Development Technologist Coordinator with the Clinical Mass Spectrometry Development Laboratory, Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, MN. She has over 9 years of experience in clinical mass spectrometry laboratory, and 3 years of experience in the development and validation of new mass spectrometry tests. Her interests include the implementation of small molecule quantitation by mass spectrometry and a new fondness for therapeutic drug monitoring of monoclonal antibody therapeutics by mass spectrometry.

Authorship: Ann Rivard; Paula M. Ladwig; Maria A. V. Willrich, Ph.D.
Mayo Clinic

Short Abstract

Eculizumab is a humanized IgG2/4¥ê monoclonal antibody therapeutic (t-mab) targeting complement component C5 of the alternative pathway. It is prescribed for rare, chronic conditions such as atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH). As an expensive t-mab, therapeutic monitoring is desired in attempt to decrease the high cost of therapy. A previously published method using the molecular mass of the intact light chain of the t-mab by microLC-ESI-Q-TOF mass spectrometry was adapted into a sensitive and specific assay, validated on a multiplexed HPLC-high resolution accurate mass spectrometer.

Long Abstract


Eculizumab (Soliris; Alexion Pharmaceuticals) is a recombinant humanized IgG2/4 kappa monoclonal antibody therapeutic (t-mAb). Eculizumab binds complement component C5, thereby blocking the terminal complement cascade, preventing inflammation and cell lysis. Therapeutic monitoring of eculizumab concentrations is desired given the high expense and chronic nature of the disease states that this therapy targets. Proteomics has been commonly applied for assay development of t-mabs. The well-established tryptic peptide digestion has been applied to chimeric t-mabs such as infliximab, which contains 30% of murine variable region sequences (1). It is increasingly difficult to develop peptide assays that monitor humanized t-mabs, with <5% animal sequences, and those fully human t-mabs. In these cases, the peptide method would lend to decreased specificity by the limited candidate peptides which do not cross-react with human sequences. Sensitivity is also sacrificed by the slim chance of those peptide sequences standing out amongst the patient"s polyclonal immunoglobulin background. A recently published alternative approach monitors the unique molecular mass of the intact light chain using miRAMM (monoclonal immunoglobulin Rapid Accurate Mass Measurement). This platform utilized microLC-ESI-Q-TOF mass spectrometry (MS) (2). A shortcoming of this approach is its difficult translation into a high volume clinical laboratory due to long run time, inability to multiplex and lack of comfort with microLC. In this report, we have translated the miRAMM method to a Q Exactive Plus Hybrid Quadrupole-Orbitrap MS with a Transcend multichannel HPLC system (ThermoFisher Scientific), allowing for multiplexing for implementation into a high volume clinical mass spectrometry laboratory (CMSL).


Eculizumab was obtained from the pharmacy and the drug was spiked into purchased pooled serum at various levels for standards and quality control. Pools were aliquoted and frozen at -20¡ÆC for daily use.

Life Technologies CaptureSelect¢β IgG4 (Hu) Affinity Matrix was used for the extraction of eculizumab from serum. Distinguishing a t-mab from a patient"s normal polyclonal immunoglobulin repertoire is a primary limiting factor when determining the lower limit of quantitation (LLOQ) in serum. The ability to selectively extract only the IgG4 antibodies from serum provided a substantial increase in sensitivity (3).

Briefly, 25 mcL serum, 25 mcL surrogate internal standard (IS; 150 mcg/mL pembrolizumab), 100 mcL washed IgG4 Affinity Matrix resin, and 250 mcL PBS were combined in a well of a 96-well 0.2 mM PVDF filter plate, and incubated rotating for 1 hour at room temperature. Positive pressure was used to wash wells 2 times with 1 mL water. Next, 150 mcL of 5% acetic acid was added to each well for elution from the resin. Positive pressure was used to elute into a 96-well collection plate. Eluate was then denatured by adding 75 mcL of 100 mM DTT in 1 M ammonium bicarbonate, and incubated rocking for 30 minutes at 55¡ÆC.

Extracts were injected (10 mcL) across 2 LC channels on a Thermo Scientific¢β Transcend TX4 HPLC system. An Agilent Poroshell 300SB C3 (2.1x75mm, 5 micron) column heated to 60¡ÆC was used for chromatographic separation running at 300 mcL/min with a total run time of 17 minutes. The gradient started at 90% mobile phase A (MPA; water + 0.1 % formic acid), 10% mobile phase B (MPB; 90% acetonitrile + 10% isopropanol + 1% formic acid). After 90 sec, the system ramped to 27% MPB over 1 minute, to 36% MPB over 6 minutes, to 50% MPB over 1 min, and to 98% MPB over 1 minute. Flow rate was increased to 500 mcL/min and column washed through 2 cycles of a 45 second ramp to 20% MPB followed by 45 second ramp to 80% MPB. Column flow rate was returned to 300 mcL/min and 10% MPB over 30 seconds and then equilibrated for 3 minutes before the next injection.

A Thermo Scientific¢β Q Exactive¢β Plus Orbitrap MS was used for detection. HESI source settings were as follows; sheath gas flow rate 50, aux gas flow rate 12, spray voltage 3.5 kV, capillary temp 300¡ÆC, s-lens RF level 65, and aux gas heater temp 300¡ÆC. A targeted-selective ion monitoring mode was run at 140k resolution (25 AGC, 125 ms maximum IT) for the inclusion list to include the +11, +12 and +13 charge states for eculizumab and the +11 charge state for the surrogate IS. The QE plus allows for a second monitoring mode to be evaluated as well. A full scan method was included to allow for information on suspected interferences of similar therapeutics; scan range 1200 to 2500 m/z, 140k resolution, 16 AGC and 500 ms maximum IT.

TraceFinder¢β Software (ThermoFisher Scientific) was utilized for sample acquisition along with data processing and quantitation. For data processing, the extracted ion chromatogram (XIC) for eculizumab included 3 isotopes from 3 charge states, while the XIC for the surrogate IS included 7 isotopes from the +11 charge state. For quantitation, an 8 point quadratic curve with 1/x2 weighting was generated for each run with analytical measuring range of 5 to 600 mcg/mL.

The method was validated for analyte and extract stability, within-run and within-laboratory imprecision, accuracy, linearity, analytical sensitivity and analytical specificity. Briefly, analyte stability was evaluated at 5 different time-points over a 28 day period at ambient temperature, refrigerated (4-8¨¬C) and frozen (-20¨¬C), in addition to 5 freeze-thaw cycles and considered acceptable if recovery of analyte was within 80-120% of original results. For extract stability, a run was re-injected 7 days after the first injection and considered acceptable if results compared within 80-120% of original run results. Precision estimates were carried out with measurement of 5 levels of eculizumab-spiked pooled serum: at LLOQ (5 mcg/mL), low (15 mcg/dL) below the analytical cutoff limit, medium (100 and 400 mcg/dL) near the analytical range, and high (550 mcg/dL) above the analytical cutoff limit. Twenty precision measurements were taken within one analytical run (within-run imprecision) in addition to more than 20 different analytical measurements over the course of more than 20 days (within-laboratory imprecision). Accuracy was accessed by spiked recoveries. Patient matrix pools were spiked at 4 levels of eculizumab concentrations; 30, 90, 150, and 300 mcg/mL. Recovery was found to be acceptable if measured was within 80-120% of calculated expected. Linearity was assessed at 5 levels (10, 25, 100, 300 and 550 mcg/mL) over 10 runs; acceptable with slope of 1.0±0.1 and R2¡à0.95. Analytical sensitivity studies included determination of the lower limit of detection (LLOD) and quantitation (LLOQ). The LLOD will be defined as the lowest concentration tested that has a peak area that is greater than or equal to the average of 20 blanks plus 3 standard deviations. The LLOQ will be determined from sensitivity and imprecision experiments; less than 20% CV over the course of 20 days. Analytical specificity was studied by testing investigated potential interferences from the presence of hemolysis, lipids, and bilirubin; as well as high total protein concentrations (> 7g/dL), elevated IgG4 samples (above laboratory established reference interval), and cross-reactivity with other t-mabs in serum. Carryover was addressed by injecting extracted blanks after the highest standard. Carryover was deemed acceptable if the extracted blanks calculated below the LLOQ. Ion suppression was verified by spiking equal levels of eculizumab into extracted matrix and compared with spikes into 5% acetic acid, the final solution in the method. Surrogate internal standard was added in the final steps of extraction. Percent difference from spiked matrix results was calculated and was expected to be within 20%.


Stability studies were performed to determine optimal specimen transport conditions along with storage time and temperature; 28 days ambient, 28 days refrigerated, 28 days frozen and 5 freeze thaw cycles. Prepared extract stability was tested out to 7 days and found to be acceptable. Intra- and inter-assay precision were accessed for 4 levels (15, 100, 400, and 550 mcg/mL) along with at the lower limit of quantitation (LLOQ); 5 mcg/mL. Intra-assay precision of 20 replicates at each level analyzed in one run were all <10%CV with 15% at the LLOQ. Inter-assay precision of the 4 levels run over > 20 runs over 2 months with >50 data points gave ¡Â10%CV with 11% at the LLOQ. Accuracy experiments comparing spiked levels of the t-mab recovered within ¡Â±20% of the original value. Carryover was less than the LLOQ. Standards and controls were utilized as unknowns to verify linearity for 10 different runs; y=1.063x+0.6011, R2=0.9801. This assay will not utilize a reference range as eculizumab is not endogenous, but a drug administered via intravenous infusion, and as such no eculizumab should be detected in a healthy population. We verified with the analysis of 20 normal value donors that not been administered eculizumab; < LLOQ was detected in this population. LLOD along with the LLOQ were determined through measurements of 0 (blank), 1, 2, and 5 mcg/mL of eculizumab over >20 days. The LLOD was found to be 2 mcg/mL while the LLOQ was set to 5 mcg/mL with a CV of 11%. Analytical specificity was verified through interference studies. Six monoclonal antibody therapeutics (rituximab, infliximab, nivolumab, vedolizumab, adalimumab, and ipilimumab) were shown not to interfere and be separated both chromatographically and by mass/charge. Grossly hemolyzed or visibly lipemic samples were found to impact quantitation and will be rejected. Samples with high protein along those with high IgG4 were found not to interfere. Ion suppression was calculated to affect results by less than 20% difference.

Conclusions & Discussion

In summary, we found that a humanized t-mab may not fit into the well-established tryptic peptide approach most often utilized for proteins and implemented in the past for a chimeric t-mab4. This method report used the core principles of miRAMM, which was originally developed using microLC and Q-TOF instrumentation, and applied them to the Q Exactive platform with the TLX Transcend. This move gave us the advantage a high resolution accurate mass instrument with normal flow and the ability to multiplex, making the test suitable for a high-volume environment.

References & Acknowledgements:

(1)Willrich MA, Murray DL, Barnidge DR, Ladwig PM, Snyder MR., Quantitation of infliximab using clonotypic peptides and selective reaction monitoring by LC-MS/MS. Int Immunopharmacol. 2015 Sep;28(1):513-20.

(2)Barnidge DR, Dasari S, Botz CM, Murray DH, Snyder MR, Katzmann JA, Dispenzieri A, Murray DL., Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy. J Proteome Res. 2014 Mar 7;13(3):1419-27.

(3)Ladwig PM, Barnidge DR, Willrich MAV., Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry. J Am Soc Mass Spectrom. 2017 May;28(5):811-817.

(4)Ladwig PM, Barnidge DR, Willrich MAV., Mass Spectrometry Approaches for Identification and Quantitation of Therapeutic Monoclonal Antibodies in the Clinical Laboratory. Clin Vaccine Immunol. 2017 May 5;24(5).


We would like to acknowledge Sydney Schmidt for her help in processing the verification runs for the validation of the eculizumab assay.

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