Paula Ladwig (Presenter)
Mayo Clinic
Authorship: Paula M. Ladwig; David R. Barnidge, Ph.D.; Paul J. Jannetto, Ph.D., David L. Murray, Ph.D.; Maria A. V. Willrich, Ph.D.
Mayo Clinic; Rochester, MN
| Short Abstract As monoclonal antibody therapeutics become humanized, tryptic peptide approaches for quantitation of biologics in serum become more challenging. An alternative is to monitor the unique molecular mass of the intact light chain. The ability to selectively extract only the IgG4 antibodies from serum should allow for an advantage in detection. The IgG4 extraction compared to the extraction of all IgG immunoglobulins allowed for a ~10-fold lower limit of quantitation for eculizumab. This approach could be beneficial for the therapeutic monitoring for IgG4 biologics that need a lower limit of detection and the specificity that other approaches are unable to provide. |
Long Abstract
Background: As monoclonal antibody therapeutics (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using LC-MS1.
Distinguishing a therapeutic mAb from a patient’s normal polyclonal immunoglobulin repertoire is the primary limiting factor when determining the lower limit of quantitation (LLOQ) in serum. Most therapeutic mAbs have an IgG heavy chain and a kappa light chain. IgG antibodies make up 75% of the serum antibodies and are categorized in four different subclasses. The subclass with the lowest concentration is IgG4 which makes up only 4% of the total IgG. The ability to selectively extract only the IgG4 antibodies from serum should theoretically allow for an advantage in detection and simplify the therapeutic monitoring of IgG4 monoclonal antibody therapies.
Methods: Eculizumab (a humanized IgG2/4 mAb against complement factor 5) was obtained from our pharmacy and the drug was spiked into pooled serum. We compared extraction of all IgG immunoglobulins using ThermoFisher Melon™ Gel IgG Purification Kit (MG) to extraction of just the IgG4s using Life Technologies CaptureSelect™ IgG4 (Hu) Affinity Matrix (IG4). For MG, 20 mcL of serum is added to 200 mcL Melon™ Gel slurry and mixed for 15 minutes. 20 mcL of supernatant is reduced with 20 mcL of 50 mM ammonium bicarbonate and 10 mcL 200 mM DTT at 55ºC for 30 minutes.
Serum was also extracted using the IG4 method after 100 mcL of resin is washed twice with 500 mcL PBS. 50 mcL of serum is loaded with 450 mcL of PBS and 50 mcL of an IgG4 monoclonal protein used as internal standard, and mixed for 1 hour. The resin is washed twice with 500 mcL water. IgG4 is eluted by adding 200 mcL 5% acetic acid. Elution is reduced with 100 mcL 100 mM DTT in 1M ammonium bicarbonate.
The reduced samples from both extractions were analyzed using an Eksigent Ekspert MicroLC connected to an ABSciex 5600 Triple TOF® mass spectrometer. A volume of 2 mcL was injected onto a Poroshell C3, 1x75mm column heated at 60 C with the LC running a 24 minute gradient at 25µL/min.
Results: A linear curve from 75 mcg/mL to 400 mcg/mL was obtained for the MG preparation. Intra-assay and inter-assay precision was analyzed at three levels 75, 150, and 300 mcg/mL and acceptable at <15% coefficient of variation. A dramatic decrease in the amount of protein injected onto the mass spectrometer was observed with the IG4 preparation when compared to the MG preparation. The IG4 preparation had a linear curve from 1 to 50 mcg/mL. Intra-assay precision at 3 mcg/mL is 16.4% and 6% at 15 mcg/mL. The use of the IG4 preparation made it possible to obtain a ~10-fold lower limit of quantitation, when compared to MG without compromising precision.
Conclusion: We demonstrated the ability to lower the limit of quantitation at least 10 fold for eculizumab using the Life Technologies CaptureSelect™ IgG4 (Hu) Affinity Matrix. This approach could be beneficial for the therapeutic monitoring for other IgG4 monoclonal antibody therapies that need a lower limit of detection and the specificity that other approaches are unable to provide.
References & Acknowledgements:
1Barnidge 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.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Mayo Clinic DLMP |
| Board Member | yes | Clinical Laboratories Standards Institute |
| Stock | no | |
| Expenses | yes | Waters |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |