John Carpenter is a Professor of Pharmaceutical Sciences in the Department of Pharmaceutical Sciences at the University of Colorado, Anschutz Medical Campus. His laboratory is involved in researching the causes and control of aggregates and particles in therapeutic protein products which occur during manufacturing, shipping, storage and delivery to patients. This research is important as it contributes to improvements in product quality and therefore to reductions in adverse reactions of patients to aggregates and particles within biopharmaceutical products, which can include immune responses and dangerous infusion reactions. Also, by investigating the roles of key biophysical properties in protein stability and degradation, rapid and rational formulation strategies can be developed to help ensure that a product’s stability is properly optimized.
John Carpenter is a Professor of Pharmaceutical Sciences in the Department of Pharmaceutical Sciences at the University of Colorado, Anschutz Medical Campus. His laboratory is involved in researching the causes and control of aggregates and particles in therapeutic protein products which occur during manufacturing, shipping, storage and delivery to patients. This research is important as it contributes to improvements in product quality and therefore to reductions in adverse reactions of patients to aggregates and particles within biopharmaceutical products, which can include immune responses and dangerous infusion reactions. Also, by investigating the roles of key biophysical properties in protein stability and degradation, rapid and rational formulation strategies can be developed to help ensure that a product’s stability is properly optimized.
The team assesses and investigates a range of state-of-the-art analytical methods for the biophysical characterization of proteins and protein formulations, and for particle characterization. Throughout this research, the team collaborates closely with academic, industry and regulatory colleagues.
John’s team works with a wide range of different proteins and sample types, and encounters many different challenges related to understanding and controlling aggregation and particle formation in therapeutic protein products. For this research, it is critical to perform a comprehensive characterization of all aggregates and particles, ranging in size from soluble oligomers to nanoparticles, to microparticles through to visible particles.
Particles in the samples analyzed by the team are commonly protein-based, but may also be generated by the shedding of foreign or contaminant materials into the protein solution from, for example, processing equipment, final drug product containers and delivery systems such as intravenous (IV) administration setups. Therefore, for a given experiment, the scientists often need to be able to characterize and quantify heterogeneous particles, as well as a wide range of aggregate and particle sizes.
As John explains, “With the biophysical characterization of proteins and formulations, there comes a range of challenges including the specific problems of analyzing highly concentrated protein solutions, or new types of protein products, such as antibody-drug conjugates. It is very important to our research that the analytical instruments upon which we depend have flexible capabilities, such as the ability to analyze reproducibly at a range of concentrations, and that they are both robust and also preferably automated, in order that we can assess large numbers of samples in a short amount of time, and in a non-subjective way.”
Currently in John’s laboratory, there is a wide range of Malvern Panalytical in use, providing a comprehensive particle characterization toolset, including: NanoSight (Nanoparticle Tracking Analysis), MicroCal VP-Capillary DSC (Differential Scanning Calorimetry), Archimedes (Resonant Mass Measurement), Morphologi G3-ID (Automated Static Image Analysis twinned with Raman Spectroscopy), and Viscotek SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering).
Using Malvern Panalytical’ systems has enabled John’s team to make significant advances in characterizing particles, especially those in the subvisible range, in protein drug delivery systems. John explains, “In the past, the ability to size and quantify micron-sized particles greatly increased our ability to understand their root causes and also their potential control. But even with these data there were still many unknowns, because proteins can form assemblies ranging in size from dimers to large visible particles, and we were only able to analyze a subset of these.”
He continues, “Similarly, foreign particles shed into protein solutions from processing equipment, drug containers and delivery systems range in size from submicron to visible. When we started characterizing and quantifying nanoparticles with the NanoSight instrument, we immediately obtained remarkable new insights into the particle content of therapeutic protein products. For example, we found that intravenous saline can contain tens of millions of nanoparticles per mL, and we could actually see protein molecules associating with these nanoparticles, and so understood that they could act as nucleation sites. We also learned that current in-line filters used for intravenous administration (e.g., 0.2 µm pore size) may not be effective at removing the vast majority of nanoparticles, which fall below this size range. These tiny particles may then be delivered to patients and may play key roles in immune responses and adverse infusion reactions. Ultimately, we hope that our work will lead to improved filters, reductions in delivery of nanoparticles to patients, and ultimately to improved product safety.”
John’s team faced a second set of complications with the analysis of particles in therapeutic protein products which have been prepared in prefilled syringes, a storage and delivery mechanism rapidly growing in popularity because of its ease of administration. Silicone oil is typically used to lubricate the barrels of these syringes, causing microdroplets of oil to shed into the formulation. These oil droplets are often very difficult to distinguish from protein particles and aggregates. However, by using the Archimedes instrument, John’s team saw that the populations of silicone oil droplets and protein particles could be measured independently, enabling the quantification of each type of particle. These data can provide valuable and unique insights into product quality and some of the key factors affecting protein particle formation.
In another study, the team found that Multi-Angle Light Scattering (MALS) detection during size exclusion chromatography (SEC) was critical for determining that a protein formulation in a given syringe configuration was starting to form aggregates during storage. John explains, “With only ultraviolet (UV) detection, there was no indication that the protein monomer was being converted to aggregates during the course of the storage study. But with SEC-MALS detection, because of its much greater signal intensity for soluble aggregates, it was obvious that trace amounts of protein were progressively being converted to aggregates. With such data, one can determine early in a study if the therapeutic protein in a given drug container or formulation has propensity to aggregate.”
He continues, “Also, we have found SEC-MALS to be extremely useful in sizing aggregates and for determining that there is a range of aggregate sizes eluting in the void volume from an SEC column. Such information is invaluable for the rigorous characterization of the aggregation pathway for a given protein and for developing control strategies for aggregates.”
Finally, John’s team of scientists states that Differential Scanning Calorimetry (DSC) is an extremely valuable and widely applicable biophysical method for therapeutic protein product development. “Determining the thermal transition temperature of a protein has great utility in comparing the relative stabilities of candidate molecules for a given product, screening formulation conditions to optimize protein stability, comparing stability for a given product before and after a manufacturing change, and for comparing a biosimilar to an innovator product. With Malvern Panalytical’s automated capillary DSC system, the MicroCal VP-Capillary DSC, these types of studies can be performed with minimal hands-on work by the operator. The results obtained are by far the most reproducible of any I have seen from any analytical instrument. Incredibly, thermograms from a half dozen analyses of a given sample can be overlaid and one cannot see anything but a single line. Amazing!”
John concludes by discussing his team’s relationship with Malvern Panalytical: “We have been fortunate to have a long-term collaboration with Malvern Panalytical, in which we work together on the applications of state-of-the-art instruments and also the development of guidance for best practice for the characterization of therapeutic protein samples. We benefit from this relationship, not only because of access to the newest and best instruments, but also because of the expert guidance and advice we receive from our Malvern Panalytical collaborators. And we have a lot of fun working together!”
“Malvern Panalytical’ experts are outstanding collaborators and colleagues, and their customer service and assistance are excellent. It is a pleasure to work so closely with so many people at this leading analytical instrument company.”
