Editor's note: Dr. Sara Carillo is a Bioanalytical Research Lead at National Institute for Bioprocessing Research and Training (NIBRT). This is the first blog post in the Biopharmaceutical Basics series.
Biopharmaceutical characterization is a critical step of biopharmaceutical development and manufacturing. This emerging and exponentially growing class of drugs is characterized by an intrinsic complexity and heterogeneity, which drives the need for a deeper knowledge of the structural features that can have huge impact on the stability, efficacy and safety of the final drug.
If you’re new to the world of biopharma analysis, here’s a helpful overview on biopharmaceutical characterization and what’s available today.
What is biopharmaceutical characterization?
Because of the complexity of biopharmaceuticals, their characterization is never performed through a single assay. Instead, more orthogonal techniques are usually required to monitor all the features that are critical to ensure their quality.
The wide landscape of techniques available for biopharmaceutical characterization could be potentially split into two categories: (1) those techniques able to monitor the overall structure and correct assembly of the protein; and (2) those looking into the fine modifications that each amino acid may bear as post-translational modification.
In the first technique, the main goal is to ensure the protein has correctly folded, and that the protein maintains stability, monitoring the presence of either smaller fragments or aggregation of the main species, with both entities indicating a degradation of the molecule of interest.
In the second technique, the heterogeneous nature of the proteins is acknowledged and investigated through the monitoring of those critical quality attributes affecting product efficacy, activity, stability and immunogenicity. The main modifications monitored are usually deamidation, oxidation, N-glycosylation, glycation and disulfide assembly. Relative abundance of these modifications can vary from 100 percent to less than 0.1 percent. Sometimes minor changes can reflect massive differences in drug performance; therefore, these modifications need to be closely monitored throughout the bioprocess.
Leading techniques for biopharmaceutical characterization are based on liquid chromatography (LC) often hyphenated with mass spectrometry (MS), as they allow simplification of the complex mixture and accurate identification of each proteoform. Nevertheless, the wider landscape of techniques used for biopharmaceutical characterization includes more and different workflows, including bioassays, capillary electrophoresis and LC-UV.
What are the different ways to characterize biopharmaceuticals and biotherapeutics?
As for all other proteins, the analytical approaches for the characterization of biopharmaceuticals can be divided into bottom-up, middle-up, middle-down, top-down and intact analysis.
Bottom-up, middle-up and middle-down analyses imply a degradation of the sample, and are usually performed through a chemical or enzymatic reaction in order to divide the molecule in smaller regions (peptides or subunits) that are easier to analyze, although the mixture complexity may increase.
Conversely, top-down and intact protein analyses analyze the biopharmaceutical in its intact form, with the only aid of MS fragmentation techniques (top-down). With this method, it’s easier to monitor those fragments or aggregates naturally present in the drug formulation, even though some of the low abundant or small modifications may be harder to monitor. The choice in approach is determined by the question we need to answer in that specific analysis or at that specific step of the manufacturing process; however, oftentimes a combination of the two levels of analysis can provide a more comprehensive determination of product features.
Why is biopharmaceutical characterization important?
Biopharmaceutical characterization is important because it allows scientists to monitor those features that ensure drug activity and safety. The characterization step allows manufacturers to improve biopharmaceutical production from the very early phases and to follow a Quality by Design approach — not only in the design of the protein but also in the production. For example, some features may indicate cellular stress during upstream processing and monitoring allows for quick correction of the culture conditions, resulting in a better yield and an overall better quality of the product. For this reason, there is a growing interest in bringing analytical techniques closer to the bioprocess pipeline or even integrating them so that real-time monitoring of the critical features can improve both process and product.
What is a real-world example?
An example of how real-time monitoring can improve the quality of biopharmaceuticals can be observed in a recent study performed by the National Institute for Bioprocessing Research and Training (NIBRT). A multi-attribute method (MAM) was employed to monitor trends of some PTMs during a bioreactor campaign and researchers could observe undesired N-glycan profiles appearing after 10 days of cell culture. The knowledge derived from this experiment helped in the optimization of the upstream process, which now ends when the desired N-glycan profile is reached. This process change helps to improve the overall quality of the final product.
What solutions are available today?
To learn more about Thermo Fisher Scientific analytical solutions for biopharmaceutical characterization and control, check out this brochure, visit the dedicated website or contact your Thermo Fisher representative.