New Jersey Institute of Technology (NJIT) researchers have unveiled a new laboratory technique they say represents a “paradigm shift” in how pharmaceutical laboratories test and produce new protein-based drugs, such as those being developed to treat a variety of diseases of therapeutic monoclonal antibodies, from cancer to infectious diseases.
The researchers say their electrochemical-based approach, described in the journal analytical chemistrycan allow the safety and quality testing of emerging biotherapeutics to be completed in a fraction of the time required by traditional methods, which typically require long and costly production times for certain biomaterials for sample testing.
The study was conducted in collaboration with researchers at Merck, Johnson & Johnson, and Ohio University, and was supported by a grant of $379,397 from the National Institutes of Health.
“The method we developed at NJIT has the potential to have a major impact on quantitative proteomics, and it represents a paradigm shift in the pharmaceutical industry in monitoring biopharmaceutical products and process impurities for quality control,” said Hao, corresponding author of the paper. Chen said the author is a professor in the Department of Chemistry and Environmental Sciences at the New Jersey Institute of Technology.
“With this research, we have now demonstrated a method to quantify drug product and process impurities faster and more accurately than ever before. … We hope it will be very useful to advance the development of therapeutic and preventive therapeutic proteins and vaccines. There will be different diseases in the future.”
Traditionally, such tests or protein quantification have involved the time-consuming preparation of synthetic isotope-labeled peptides that are used as internal standards to measure total protein concentration in a sample – helping researchers actively monitor the efficacy and safety of therapeutic protein components throughout a drug sexual development process.
To overcome this limitation, Chen’s lab developed a coulometric mass spectrometry (CMS) method for absolute quantification of proteins without the use of standards. The method instead applies liquid chromatography-mass spectrometry and electrochemical flow cells to rapidly quantify and detect changes in target proteins or peptides based on electrochemical signatures.
“Traditional methods do not need to wait weeks for standards or reagents, and CMS quantification experiments can be performed immediately. Therefore, this will help to track drug impurities found in the process and ensure their effective removal through process optimization and control,” said Chen .
“This device allows us to separate peptides with liquid chromatography after protein digestion, monitor peptide oxidation in an electrochemical flow cell to generate electrical current, and measure oxidation yields with mass spectrometry,” explains the paper’s first author and NJIT Ph.D. . Student Ai Yongling. “The combination of the current signal and the oxidative yield provides sufficient information for absolute quantification of peptides and proteins.”
In their study, the team demonstrated their CMS method by achieving absolute quantification of multiple proteins (β-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) in a mixture in a single run, without the use of any standards.
Notably, the team also demonstrated the method’s ability to detect protein deamidation — a common degradation event for therapeutic proteins caused by physical or chemical stress throughout manufacturing and storage.
According to the study authors, the team successfully quantified several protein degradation products, including the formation of a key intermediate in protein degradation — the formation of succinimide — which had never been quantified before in absolute terms due to a lack of standards.
“The lack of standards is due to the challenges of their de novo synthesis,” Chen said. “Being able to accurately quantify deamidation products and intermediates could lead to a better understanding of therapeutic protein degradation and potentially create a new way to study disease pathology and aging processes.”
Now, Chen’s lab plans to apply their new method to the large-scale quantification of thousands of proteins at once. They also plan to increase the sensitivity of the CMS assay to allow the quantification of very low levels of proteins in complex biological samples, which will benefit research ranging from clinical diagnostics and drug discovery to precision medicine where samples are identified and quantified at the molecular level work is necessary.
“As proteins perform a wide range of functions in living organisms, the importance of absolute protein quantification cannot be overemphasized,” Chen said. “CMS should speed up the process of disease diagnosis, drug discovery and development, and it now opens a new door for biologists and biochemists to explore biology in humans that may play important roles as disease biomarkers and drug targets The amount of protein that functions or acts.”
The research was supported by a National Institutes of Health grant (1R15GM137311-01) and also formed the basis for a new project proposal recently awarded by the National Science Foundation (CHE-2203284). Other contributors to the paper include Harsha P. Gunawardena of Johnson & Johnson, Xuanwen Li of Merck, Professor Howard Dewald of Ohio University, and Yong-Ick Kim of NJIT.