CDMOs are becoming increasingly important for developers to reach efficient vaccine and complex biotherapeutic development for their advanced analytics capabilities throughout the development and manufacturing process. Mass spectrometry, in particular, is critical for the characterization and management of active pharmaceutical ingredients (APIs) and impurities, an essential step for downstream processing and for ensuring product quality and safety.
The global impact of COVID-19 has led to an increased role for Contract Development and Manufacturing Organizations (CDMOs) when it comes to supporting the development of vaccines and other complex biotherapeutics. This includes critical roles in downstream processing, like the characterization of APIs and managing a diverse population of impurities, which allows for effective mitigation of safety risks and ensuring product quality.
We recently sat down with Michael J. Nold, Ph.D., Senior Director of KBI’s Mass Spectrometry Core Facility in Durham, North Carolina, to discuss how advanced Mass Spectrometry plays a significant analytical role in the characterization and quantitation of APIs and impurities in complex biotherapeutics.
Why is characterization and quantitation analysis so essential to managing impurities?
Managing process- and product-related impurities in downstream processing is vital to ensure product quality and safety, and global regulatory requirements continue to demand controls and mitigation strategies around impurities. Therefore, the effective removal of these impurities is critical for developing biotherapeutics and vaccines.
What types of impurities do you encounter when developing vaccines and other biotherapeutics? Are there any common impurities that you come across?
Common impurities can be process-related, meaning that they come into play from the manufacturing process, or product-related, meaning that they come from the product itself.
Common process impurities include host cell proteins (HCPs), host cell DNA, cell culture media components, antibiotics, adventitious and endogenous viruses, chromatographic media from purification steps, buffer components, and solvents. As part of the purification process, other impurities can be introduced; these must also be removed.
Common product impurities we come across include things like aggregates, truncated and other modified forms of the desired product, precursors, and certain degradation products that can arise during manufacturing and storage. These impurities present specific challenges for the characterization and development of biotherapeutics and vaccines.
How can mass spectrometry support the development and characterization of these complex samples?
High-resolution mass spectrometry is a powerful tool used at KBI Biopharma for analytical strategies concerning complex samples. At KBI, we offer mass spectrometry services for impurity- and API-characterization in an integrated fashion leveraging our expertise at our North Carolina, Colorado, and Belgium facilities, all of which are set up with accurate mass systems employing either Orbitrap or Time-of-Flight (TOF) technology. These systems allow fast scanning speeds with high sensitivity and resolution in support of highly specific and selective methods to allow diverse and complementary analytical options.
Specifically, we have used a proteomic approach for process clearance monitoring of HCPs to confirm the robustness of clearance steps during purification. It also allows us to monitor for any observed hitchhiker effect of proteins that copurify with APIs and to provide relative quantitative insight to the specific HCPs driving the reported ppm levels from ELISAs.
How do you use targeted workflows alongside GMP compliance in your mass spectrometry facilities?
Our GMP-compliant, targeted strategies are based primarily on TOF-MRM, a high-resolution multiple reaction monitoring (MRM) method that is a multiplexed form of selective reactive monitoring, or SRM. This highly specific and sensitive mass spectrometry technique provides comparable performance when compared to classical MRM for limits of quantitation (LOQ) and detection (LOD) for targets within complex mixtures.
Our GMP compliant LCMS systems in North Carolina have Waters Acquity® UPLCs coupled with Xevo® G2-XS mass spectrometers. This platform provides automated workflows, streamlined instrument control, data processing, and reports with full audit trails – meeting data integrity compliance with 21 CFR Part 11, the U.S. Food and Drug Administration (FDA) rule governing electronic records and signatures. Our Belgium facility has a compliant Xevo G2-XS system, as well, that operates within a UNIFI software environment. In fact, the Belgium facility in December 2022 reported another successful European Medicines Agency (EMA) audit.
What approach(es) do you use for non-proteinaceous concerns?
The TOF-MRM targeted approach is a common approach, but in some cases, simply employing accurate mass-extracted ion chromatography in a non-targeted manner is sufficient for monitoring and/or semi-quantifying non-proteinaceous species.
How do you monitor for glycation?
In the artificial environment with complex chemical components present in cell culture media and feeding conditions, protein glycation is expected – but the reaction kinetics and extent to which this occurs can be challenging to predict and control.
Unlike glycosylation, glycation results from the nonenzymatic reaction between free-reducing sugars and the N-terminal primary amine or the amine group of lysine side chains. These are observed in standard peptide mapping assays.
Where do you see the most potential for future innovation supporting API and impurity characterization and quantitation?
As Thomas Edison said, “There’s a better way to do it; find it.” At our Mass Spectrometry Core, we are always looking at ways to find it through effective collaboration with clients, vendors, and our internal teams – every interaction and step along the way provides insight into impurities that need to be understood and accounted for.
Technology innovation in mass spectrometry allows us to see more and dig deeper. Improvements in performance with techniques such as ion mobility, electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) are just a few examples that can support characterization while allowing us to triage stability issues that may arise from storage.
Whether it’s sample preparation strategies and automation or chromatographic improvements or GMP-compliant data processing and workflows, there are many opportunities for innovation.
We would like to sincerely thank Michael J. Nold, Ph.D., for taking the time to talk with us. If you are interested in learning more about how KBI Biopharma’s world-class analytical services are moving biological therapeutics from transfection to clinic, please get in touch with our team.