Mary Lame (Presenter)
Waters Corporation
Bio: Mary received her B.S. in Chemistry from the Western Connecticut State University (Danbury, CT) and her M.S. in Chemistry from Central Connecticut State University (New Britain, CT). She began her bioanalytical career in 1997 with a global Pharmaceutical company where she supported research and discovery efforts by performing DMPK studies and conducting various bioanalytical assays until 2011. Mary joined Waters in 2011 as a Senior Applications Chemist, providing pharmaceutical applications development and support for the Waters Applied Technology Group. Mary’s primary role is to support discovery bioanalysis, and develop bioanalytical methods for drug molecules, both large and small. She is responsible for sample preparation, mass spectrometry, and LC method development, and also provides customer and in-house training on these topics. Her most recent focus has been on peptide and prot
Authorship: Mary Lame*, Hua Yang*, Paula Orens*, Sherri Naughton*, and Erin Chambers*+
*Waters Corporation, +King's College London
| Short Abstract High variability in protein quantification analytical data and a general lack of expertise strongly support the requirement for a standardized, kit-based approach. In this work, commercially available kits were used to quantify monoclonal antibody-based drugs in human and rat plasma. Single digit accuracy, precision, and repeatability data was achieved in experiments performed by different analysts, on different days, and with 5 unique lots of kits. Quantification limits from 10 to 500 ng/mL were achieved for all antibodies tested. Linearity of calibrators was always greater than 0.99 while accuracy and precision of quality control samples was better than 5%. |
Long Abstract
Background: In any bioanalytical or clinical research assay, one of the greatest sources of variability arises from the sample preparation. This is especially true for protein quantification workflows which often contain many segments-each with multiple steps capable of introducing variability. This can be of particular concern when assays are transferred from site to site or from lab-to-lab within the same research or testing organization. Furthermore, given the multitude of possible options within a typical workflow, method development time and the level of expertise needed are significant. These types of workflows typically require a scientist both skilled and experienced in the various processes involved. The difficulty in implementing these assays is further aggravated by the complexity of the troubleshooting required when analytical goals are not met. At the same time, an assay is expected to meet acceptable accuracy and precision guidelines and provide reliable, reproducible results to make critical research decisions. Thus, there is a strong need for simpler, more standardized workflows. These would ideally employ generic, kitted methods that provide a “recipe” and the reagents necessary to streamline the workflow, reduce variability and allow for implementation by less experienced scientists.
Methods: Commercially available protein quantification kits containing pre-measured, lot traceable reagents and a single simple protocol were employed to digest and quantify multiple monoclonal antibody-based drugs in human and animal plasma. All samples were processed according to the kit manufacturer’s methods. LC conditions, in brief, benefit from the increased peak capacity of sub-2µm particles packed into a 2.1 X 150 mm, wide-pore C18 column eluted with simple formic acid in water and acetonitrile mobile phases. Mass spectrometry quantification of the multiply charged species of signature peptides is performed in ESI+ using a triple quadrupole instrument.
Results: Reproducibility within an assay (intra-kit) and between assays (inter-kit) was evaluated with two different (2) analysts, on different days, using a total of five (5) kits, and six (6) technical replicates per kit. Two types of kits were tested: those designed for direct digestion of whole plasma, and those designed for digestion of affinity-purified plasma. Using the generic digestion protocol provided in the kit, several signature peptides for each protein (a combination of both generic and unique), resulting from the digestion of the monoclonal antibody drugs bevacizumab (Avastin), adalimumab (Humira), trastuzumab (Herceptin) and infliximab (Remicade) in plasma, were evaluated and analyzed by LC/MS. The coefficient of variation (CV) was used to assess reproducibility, as high CV values are indicative of poor reproducibility and precision. Raw area counts for multiple tryptic peptides from the aforementioned proteins were used to make the assessment.
For the two types of kits, both intra-kit and inter-kit % CV’s on average were ≤ 10. Additionally, using two (2) analysts, on two (2) separate days, the calculated mAb concentrations of the multiple tryptic peptides, when compared, were within 10% of each other, with % CV’s across the six (6) technical replicates ≤ 15.
Furthermore, depending on the antibody and whether or not affinity purification was performed, quantification limits from 10 to 500 ng/mL were achieved for all antibodies tested. In addition, linearity (R2) of the calibrators was always greater than 0.99 and accuracy and precision of quality control samples was better than 5%, and close to 3% on average.
Conclusion: This work demonstrates the high reproducibility, accuracy and precision achievable using this kit-based approach to protein quantification. These data suggest that a high degree of standardization can be achieved across analysts, and sites implementing this approach.
For Research Use Only, Not for Use in Diagnostic Procedures.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Waters Corporation |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | Waters Corporation |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |