Interview: An author’s perspective: Omnia Ismaiel on mature LC–MS/MS methodology


Omnia Ismaiel Chromatographic Sciences Department, PPD Laboratories, Richmond, VA, USA Omnia.Ismaiel@ppdi.com


In this interview, we spoke to Omnia Ismaiel (PPD) on her article, ‘Do we have mature LC–MS/MS methodology for therapeutic monoclonal antibody bioanalysis?’ published in Bioanalysis.


Find out more about her research in the original interview including; the challenges of large molecule quantification, considerations for LC–MS assays and the advantages and disadvantages for using this technique.


Q


Could you introduce yourself and provide a brief summary of your career to date?


My name is Omnia Ismaiel; I am a Research Sci- entist at Bioanalytical Lab, Pharmaceutical Prod- uct Development (PPD; VA, USA). I am also an Associate Professor of Pharmaceutical Analytical Chemistry at the Faculty of Pharmacy, Zagazig University (Zagazig, Egypt). My career started at the Faculty of Pharmacy, Zag- azig University, as an Instructor and was followed by many other advanced steps in teaching, research and supervising graduate students. I was a Postdoc- toral Fellow at Virginia Commonwealth Univer- sity (VA, USA) and also a Postdoctoral Research Associate at University of Georgia (GA, USA).


Q


Describe some of the challenges of large mol- ecule quantification in biological matrices?


There are several challenges in the quantifica- tion of large molecules in biological matrices; for example, they may be present at a much lower concentration than endogenous proteins. It is challenging to detect low levels of surrogate pep- tide sequence(s) derived from the target protein in a digest, and achieve a lower limit of quan- tification in the presence of other endogenous peptides, which often occurs at much higher concentrations. Complexity and nonselectivity of extraction protocol, instability and degrada- tion during sample processing are also considered some of the main challenges of large molecule quantification in biological matrices.


Q


Could you list/describe the key factors that need to be con- sidered in order to develop a robust LC–MS assay


for


biotherapeutics? To develop a robust


and reliable assay for biotherapeutics in biologi- cal matrices, the scientist needs to understand the complexity and differences between small molecules and protein-based compounds. There are many key factors to achieve a robust LC–MS assay such as; use of appropriate internal stan- dards, apply a proper sample cleanup procedure to improve method selectivity and also monitoring a unique surrogate peptide sequence(s) derived from the target protein.


Q


Why are internal standards important and how do you select them?


Internal standards are required for accurate and reproducible quantification. An ideal internal standard is a stable isotope internal standard (SIL- IS) that can be added at the start of sample pro- cessing. It is used to compensate for variability during immunocapture and digestion steps and also to correct for variability in chromatography and electrospray ionization. It will also correct any degradation, chemical changes or instability of the target analyte. However, a SIL-IS may not be available for each single biotherapeutic. Other options such as a general SIL-IS or peptide SIL-IS may be used.


Q


What are the advantages/disadvantages of using LC–MS/MS for biotherapeutics?


Sensitivity, specificity, wide dynamic linear range, precision, accuracy and the ability to quantify multiple analytes simultaneously are the main advantages of LC–MS/MS methods. Using LC– MS/MS for biotherapeutics overcomes many drawbacks of immunoassay methods. However, the complexity of digested proteins necessitates extensive and complex sample cleanup before – and may be after – digestion. Matrix effects and coeluting peptides may cause


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