The biotech industry strives to produce high-quality drugs with targeted structure-activity-relationships. The key to delivering these therapeutics is not only in understanding the active pharmaceutical ingredient (API) itself, but also understanding process-related residual components that are inevitably present in the final product. Identifying and quantifying these components is not just a regulatory requirement – it feeds back to process control mechanisms resulting in improved drug quality.
One area where significant developments have recently taken place is in the analysis of Host Cell Proteins (HCPs). These proteins are derived from the host cell expression system and are targeted for removal through the purification process. HCPs should be assessed from early product development in order to show that the downstream purification processes are effectively reducing and removing HCPs.
Traditionally ELISA, often in kit form, has been the method of choice for HCP analysis but there is an increased regulatory expectation that ELISAs will be complemented by MS-based HCP investigations. This orthogonal approach has now been supported by a new United States Pharmacopoeia (USP) General Chapter 1132.1 (Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry) which is open for public comment until 31st July 2023. This USP chapter provides an excellent and detailed overview and covers various considerations such as:
The principal aim in any HCP assessment is to identify and quantitate proteins down to very low-level abundance and then use this knowledge to determine if and where further purification is required. Mass spectrometric techniques are ideal for this as MS has a wide dynamic range, up to 5 orders of magnitude sensitivity, allowing for detection of low levels of HCPs amongst the high concentration of drug product.
As with all methods, there are factors that can limit HCP analysis by MS, including:
Hence, HCP-MS has not been put forward to replace the more common ELISA-based assessments, but to supplement them.
Dr. Christina Morris, Senior Scientist and HCP-MS expert at BioPharmaSpec says, “Bringing mass spectrometry to HCP detection can generate meaningful data and enhance the understanding of HCP profiles by providing an orthogonal analysis to extend ELISA-derived information. I often present this table to illustrate the advantages and disadvantages of these two complementary techniques.”
Quantitation of HCPs can be achieved using both labelling and label-free methods like multiple-reaction-monitoring (MRM) or sequential window acquisition of all theoretical mass spectra (SWATH-MS). The SWATH strategy has seen an increase in popularity, benefitting from simple, fast and cost-effective workflows. Through a combination of IDA (Information Dependent Acquisition) and SWATH MS acquisitions of proteolytic digests of product material, HCP identification and quantitation is performed by matching the MS/MS spectra to theoretical masses of equivalent peptide fragments in an HCP-specific sequence database.
To make sense of the large and highly complex HCP-MS datasets generated from these workflows, bioinformatics tools are used for peak-picking and filtering of potentially thousands of HCP identifications. The algorithms that sort through these spectra are critical to the HCP-MS strategy in terms of time-efficiency, but a degree of manual assessment (as is recognized in this new USP chapter) is an important cross-check to look for any HCP mis-assignments or false positives and is especially important in identifying low-abundance peptides.
With the new HCP-MS General Chapter 1132.1 proposal, the USP clearly recognizes the unique role that MS-based HCP analysis can play in the understanding of product impurity profiles. The ability to identify and quantitate HCPs provides a means to understand the product to a greater extent by generating complementary and orthogonal data to the ELISA-derived information and, through protein identification, accumulate useful information for manufacturing process development and ultimately improved product quality.
BioPharmaSpec’s approach to HCP-MS is flexible and proceeds through clearly defined stages, meaning that you receive data that is useful and applicable at your stage of product development and allows you to meet your specific objectives.
Whether you want to ensure a robust control strategy, gain better process understanding or compare HCP profiles throughout your process and in the final product, BioPharmaSpec’s experienced scientists will help you implement reliable and repeatable LC-MS based HCP methods for your development project.
