Quantification of Infliximab in Human Serum by LC-MS/MS Using A Full-Length Stable Isotope Labeled Internal Standard

Kevin Ray, Pegah R Jalili

MilliporeSigma, St. Louis

Infliximab, a chimeric monoclonal antibody, is used to treat rheumatoid arthritis, psoriatic arthritis, Crohn’s disease, and other autoimmune diseases. Clinical responses are different among patients due to inadequate amount of drug circulating in the blood. Therefore, there is a growing demand for reliable LC-MS/MS assays to support quantification of serum Infliximab in clinical applications. The accurate quantitation of Infliximab is enabled by early introduction of an internal standard that behaves identically to the native target protein throughout the analytical workflow. We have developed and characterized a full-length stable isotope labeled Infliximab internal standard and demonstrate its use to achieve sensitive, accurate, and reproducible quantification of serum Infliximab in an LC-MRM assay.

Methods

SIL-Infliximab was expressed in CHO cells which were grown in serum-free medium enriched with 13C6 15N4 Arg and 13C6 15N2 Lys. The SIL-Infliximab was analyzed at the intact protein level and after trypsin digestion. Intact mass analysis (SEC-MS) was used to confirm the amino acid composition of the protein and level of glycosylation. The sequence and isotope incorporation were determined at the peptide level after trypsin digestion.

For quantification, samples were prepared by spiking 25 μg/mL of SIL-Infliximab as an internal standard into human serum containing 0.5 - 100 μg/mL of Infliximab target antibody. Samples were precipitated by adding saturated ammonium sulfate, reconstituted with 50 mM ammonium bicarbonate, and digested using trypsin. Tryptic peptides were separated on a Supelco BIOshell A160 Peptide C18, 2.7 μM fused core particle column; 10 cm x 500 μM. Detection was performed in MRM mode on Sciex QTRAP 5500 system. Transitions of four unique Infliximab peptides, GLEWVAEIR, SINSATHYAESVK, YASESMSGIPSR, and DILLTQSPAILSVSPGER, were monitored.

LC-MS/MS Method for Infliximab

Figure 1. Extracted ion chromatogram (XIC) of four Infliximab-specific peptides.

Table 1. Infliximab-Specific Peptides Liberated from SIL-Infliximab Tryptic Digest.

Peptide Sequence	Location	Literature Reference
DILLTQSPAILSVSPGER	Light Chain	1
YASESMSGIPSR	Light Chain	2
SINSATHYAESVK	Heavy Chain	1
GLEWVAEIR	Heavy Chain	2

SEC-MS Analysis

Figure 2. UV trace and deconvoluted mass spectra resulting from SECMS analysis of the reduced SIL-Infliximab standard. Theoretical molecular weights assume 100% isotopic incorporation and unreduced intrachain disulfide bonds.

Isotopic Incorporation of SIL-Infliximab

Figure 3. Incorporation of 13C6 15N4 labeled arginine in two unique surrogate peptides liberated from SIL-Infliximab was > 99%.

Binding Kinetics of SIL-Infliximab vs Remicade™

Figure 4. Binding curves for SIL-Infliximab and authentic Remicade antibody. TNF-α binding of SIL-Infliximab was equivalent to the therapeutic antibody.

Quantitation of Infliximab in Human Serum

Calibration Curves for Two HC Infliximab Specific Peptides

GLEWVAEIR (HC)

Row	Component Name	Actua Con	Num. Val	Mean	Standard Dey	Percent CV	Accuracy	Value f (1	Value #2	Value #3
1	GLEWAEIR.+2y4.light	0.50	3 of 3	5.237e-1	6.490e-2	12.39	104.73	5.884e-	5.239e-1	4.586e-1
2	LEWAEIR.+2y4.light	1.00	3 of 3	9.770e-1	5.519e-2	5.65	97.70	9.479e-1	1.041e0	9.425e-1
3	GLEWAEIR.+2y4.light	5.00	3 of 3	4.660e0	2.116e-1	4,54	93.20	4.544e0	4.532e0	4.905e0
4	GLEWAEIR.-2y4.light	10.00	3 of 3	9.998e0	1.488e-1	1,49	99.98	1.005e1	1.011e1	9.829e0
5	GLEWAEIR.+2y4.light	25.00	3 of 3	2.706e1	2.955e-1	1.09	108.25	2.739el	2.699e1	2.681e1
6	GLEWAEIR.+2y4.light	50.00	3 of 3	4.786e	9.127e-1	1.91	95.72	4.735el	4.732e1	4.891e1
7	GLEWAEIR.+2y4.light	100.00	3 ci 3	1.004e2	1.220e0	121	100.42	9901el	1.012e2	1.010e2

Calibration Curves for Two LC Infliximab Specific Peptides

YASESMSGIPSR (LC)

Figure 5. Calibration curves of four Infliximab specific peptides obtained by spiking 25 μg/mL of SIL-Infliximab as an IS into human serum containing 0.5 - 100 μg/mL target antibody. The statistical values are shown for one heavy chain and one light chain peptides. Calibration curves show reproducible and linear results from across the tested range without enrichment. A lower limit of quantitation of 0.5 μg/mL was obtained with less than 15% CV and ±15% accuracy for triplicate calibration standards.

Row	Componen Name	Actual Co	Num. Val	Mean	Standard Dev	Percent CV	Accuracy	Value#	Value #2	Value #3
1	YASESMSGIPSR.+2y10.lig	0.50	3 of 3	5.314e-1	5.735e-2	10.79	106.29	5.259e-1	5.913e-1	4.770e-1
2	YASESMSGIPSR.+ +2y10.lig	1.00	3 of 3	9.094e-1	2.625e-2	2.89	90.94	9.145e-1	9.328e-1	8.810e-1
3	YASESMSGIPSR.+2y10.lig	5.00	3 of 3	4.787e0	1.990e-1	4.16	95.75	4.979e0	4.581e0	4.802e0
4	YASESMSGIPSR.+2y10.lig	10.00	3 of 3	1.007e1	4.265e-1	4.23	100.71	1.012e1	1.047e1	9.620e0
5	YASESMSGIPSR.+2y10.lig	25.00	3 of 3	2.764e	3.606e-1	1.30	110.57	2.772et	2.725e1	2.796e
6	YASESMSGIPSR.+2y10.lig	50.00	3 of 3	4.819e1	1.476e0	3.06	96.38	4.686e	4.978e1	4.792e1
7	YASESMSGIPSR.+2y10.lig	10000	3 of 3	9.937e1	1.541e0	1.55	99.37	9.847et	1.011e2	9.849e1

Summary

• Stable isotope labeled full-length SIL-Infliximab antibody has been produced with high purity and isotopic incorporation >99%.
  
• We demonstrate that the use of a full length SIL-Infliximab internal standard enables sensitive, accurate, and reproducible quantification Infliximab in human serum*.

References

1. Peng X, Liu B, Li Y, Wang H, Chen X, Guo H, Guo Q, Xu J, Wang H, Zhang D, et al. 2015. Development and Validation of LC?MS/MS Method for the Quantitation of Infliximab in Human Serum. Chromatographia. 78(7-8):521-531. https://doi.org/10.1007/s10337-015-2866-2

2. Willrich MAV, Murray DL, Barnidge DR, Ladwig PM, Snyder MR. 2015. Quantitation of infliximab using clonotypic peptides and selective reaction monitoring by LC?MS/MS. International Immunopharmacology. 28(1):513-520. https://doi.org/10.1016/j.intimp.2015.07.007