Researchers from the New Jersey Institute of Technology (NJIT) have unveiled a brand-new lab technique that they claim marks a “paradigm shift” in how pharmaceutical laboratories test and create novel protein-based medications, like the therapeutic monoclonal antibodies being developed to treat a range of illnesses, from cancer to infectious diseases.
According to researchers, their electrochemistry-based approach, which was published in the journal Analytical Chemistry, may make it possible to test the quality and safety of future biotherapeutics in a fraction of the time needed by conventional methods, which typically call for the time-consuming and expensive production of specific biomaterials used for sample testing.
A $379,397 grant from the National Institutes of Health helped fund the study, which was carried out in collaboration with scientists from Ohio University, Johnson & Johnson, and Merck.
According to Hao Chen, corresponding author of the paper and professor at NJIT’s Department of Chemistry and Environmental Sciences, “this method we’ve developed at NJIT has the potential to have a major impact in quantitative proteomics and represents a paradigm shift in the pharmaceutical industry in terms of monitoring biopharmaceutical product and process impurities for quality control.”
“With this study, we have now proven a method for quickly and precisely quantifying impurities in drug products and manufacturing processes. In the future, we anticipate it to be very beneficial in facilitating the development of therapeutic protein and vaccines for the treatment and prevention of various diseases.”
Such testing, also known as protein quantitation, traditionally entails the labor-intensive preparation of synthetic isotope-labeled peptides that are used as internal standards to measure total protein concentrations in a sample. This method aids researchers in actively monitoring the efficacy and safety of therapeutic protein components throughout the drug development process.
The coulometric mass spectrometry (CMS) method for absolute quantitation of proteins without the use of standards was created by Chen’s lab to get around this restriction. Instead, the technique uses an electrochemical flow cell and liquid chromatography-mass spectrometry to quickly quantify and identify changes in target proteins or peptides based on electrochemical signatures.
“CMS quantitation experiments could be performed immediately, as opposed to the traditional methods that required waiting weeks to obtain standards or reagents. In order to ensure their efficient clearance through process optimization and control, it would facilitate tracking drug impurities discovered during the process “added Chen.
According to the paper’s first author, Yongling Ai, a Ph.D. student at NJIT, “Such an apparatus allows us to separate peptides after protein digestion with liquid chromatography, monitor peptide oxidation in the electrochemical flow cell to produce an electric current, and measure the oxidation yield with mass spectrometry.” For absolute quantitation of peptides and proteins, the combination of electric current signals and the oxidation yield is sufficient.
The team’s CMS method was used to successfully quantify several proteins (lactoglobulin B, lacalbumin, and carbonic anhydrase) in a mixture in one run without the use of any standards during their study.
Notably, the team also demonstrated how the technique can identify protein deamidation, a common degradation process in therapeutic protein brought on by physical or chemical stresses during production and storage.
Several protein degradation products, including a crucial intermediate of protein degradation — the formation of succinimide — were successfully quantified by the team, which, according to the study’s authors, has never been done with absolute quantification before due to a lack of standards.
The difficulties in their de novo synthesis, according to Chen, are to blame for the lack of standards. “Accurate quantification of the deamidation products and intermediates could provide better understanding of therapeutic protein degradation and possibly lead to a new approach to study disease pathologies and aging processes.”
The new method will now be used by Chen’s lab to quantitatively analyze thousands of proteins at once. Additionally, they intend to increase the sensitivity of their CMS analysis to enable the quantification of extremely low protein concentrations in complex biological samples. This work could aid in research projects ranging from precision medicine to clinical diagnostics and drug discovery, which both require the identification and measurement of samples at the molecular level.
It is difficult to overstate the significance of absolute protein quantitation, according to Chen, because proteins carry out a wide range of biological functions in organisms. “CMS should expedite processes for disease diagnosis, drug discovery, and development, and it now opens a new door for biologists and biochemists to explore quantities of proteins in the human body that may serve as disease biomarkers and drug targets.”
The research, which was funded by a National Institutes of Health grant (1R15GM137311-01), also served as the basis for a fresh project idea that was recently funded by the National Science Foundation (CHE-2203284). Harsha P. Gunawardena from Johnson & Johnson, Xuanwen Shawn Li from Merck, Howard Dewald from Ohio University, and Yong-Ick Kim from NJIT are among the authors of the paper.
