Podium Presentation in Steinbeck 1 on Thursday at 14:00 (Chair: Ruben Y. Luo / Meng Wang)
MSACL 2023 Abstract
Self-Classified Topic Area(s): Proteomics > Assays Leveraging MS > Tox / TDM / Endocrine
Time to Fly; TOF Mass Spec for Quantitation of Therapeutic Monoclonal Antibodies
Paula M. Ladwig, M.S., MLS(ASCP); Alex Barbeln, Mindy Kohlhagen, Maria A. V. Willrich, Ph.D.; David L. Murray, M.D., Ph.D.
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester MN
|Paula Ladwig, MS, MT (ASCP) (Presenter)
Presenter Bio: Paula M. Ladwig, M.S., MT (ASCP), is a Principal Developer with the Clinical Mass Spectrometry Development Laboratory, Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, MN. She has over 15 years of experience in the development and validation of new mass spectrometry tests. Her interests include the implementation of therapeutic drug monitoring of monoclonal antibody therapeutics by mass spectrometry.
Therapeutic monoclonal antibodies have drastically improved the treatment of autoimmune disorders, cancers and other rare conditions. As recent as 5 years ago, clinical laboratories were struggling to develop quantitative methods for this class of proteins, based on large molecule drugs, as traditional methods for small molecules were not directly applicable.
Application of research proteomics mass spectrometry methods have increased choices for this class of therapeutics. The first mass spec methods were tryptic peptide and detection of unique peptides in the drug. The peptide methods have been very successful for chimeric therapeutic antibodies, such as infliximab for example (previously published). As therapeutics became more humanized, the peptide method loses some of its specificity due to overlapping peptides within the human proteome. Intact methods were then developed for these more humanized therapeutics such as eculizumab and vedolizumab for example (previously published). These methods use an enrichment method followed by Orbitrap detection.
Recent commercial advances in both hardware and software has allowed for TOF mass spectrometers to enter the arena as both a screening and quantitation tool for therapeutic monoclonal antibodies. The beauty of the TOF platform is the ability to screen or quantitate multiple analytes from the same injection without the loss in sensitivity found when utilizing an Orbitrap; sensitivity and resolution having an inverse relationship. This allows for more versatility in types of testing along with advantages for multiplexing panels.
To provide proof of concept TOF mass spectrometry methods for the quantitation of therapeutic monoclonal antibodies
Samples were enriched utilizing Life Technologies CaptureSelect™ Affinity Matrix. In a 96-well PVDF plate, 100 mcL of resin was added and washed twice with 200 mcL 1xPBS utilizing positive pressure to move waste to a reservoir. 30 mcL of sample, along with 30 mcL of a stable isotope labeled or surrogate internal standard, were added and incubated shaking for 1 hour. The enrichment was washed 3 times with 150 mcL water. 200 mcL of 5% acetic acid was added and plate was incubated shaking for 15 minutes. Enriched proteins were eluted by positive pressure into a 2mL 96-deep well collection plate. Enrichment was reduced with 100 mcL 100 mM DTT in 1M ammonium bicarbonate; at 55°C for at least 30 minutes.
The enriched and reduced samples were analyzed using an Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF™ 7600 mass spectrometer. A volume of 10 mcL was injected onto a Poroshell 300SB C3, 2.1x75 mm, 5-micron column heated at 60 °C. Mobile phase A was water with 1% formic acid and mobile phase B was 10% isopropanol 90% acetonitrile with 0.1% formic acid. The therapeutics eluted during an 8 min gradient from 25%B to 33%B. The diverter valve was used to direct 6.5 minutes of the gradient into the mass spec; otherwise, the LC was diverted to waste. The mass spec using positive ESI was run using intact protein workflow; CUR 30, CAD 7 GAS1 35, GAS2 30, and temperature 500 °C. TOF MS data from collected from 1000 to 2500 m/z; DP 175 and CE 10.
Sciex OS was used for instrument control, data acquisition, along with qualitative and quantitative data viewing and processing of data. Sciex OS Analytics was used for batch quantitation. The +11, +12, and +13 charge states (±0.2 m/z) for the light chain mass of the therapeutic and internal standard were used for data extraction; combined to give the extracted ion chromatograph which is integrated and utilized for quantitation. A quadratic curve was drawn from the standards and unknowns back calculated from the curve.
Extracted trays (N=7) for the current clinical eculizumab quantitation LDT test method on our ThermoFisher Scientific QExactive Plus platform were re-injected on the Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF™ 7600 mass spectrometer and QC (N=12) and patient results (N=56) where compared. Inter day precision for the QE and TOF (respectively) compared; QCLow 19% and 16%, QCMed 11% and 10% and QCHigh 7% and 5%. Patient comparison gave a Passing-Bablok fit y=1.04x+(-4.476) for those within the AMR.
We also performed N=5 proof of concept runs for a test method for pembrolizumab and nivolumab from 5 to 500mcg/mL. 7 levels of standards were spiked with both pembrolizumab and nivolumab. 4 levels of QC were used to determine the accuracy and precision of the method. Precision was within 20% and accuracy within ±10% of expected.
We demonstrate the ability to utilize the Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF™ 7600 mass spectrometer for quantitation of therapeutic monoclonal antibodies.
|CLSI Expert Panel for Evaluations Protocol Chair
|Mayo Clinic (expense support)
|Planning to mention or discuss specific products or technology of the company(ies) listed above: