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Delivering New Biologics to the Marketplace

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Dr. Thomas Kofoed, CEO, Alphalyse

Characterization and quantification of host cell proteins (HCPs) in biopharmaceutical development and manufacturing is a critical step to ensuring product safety. While this can be achieved using ELISA, mass spectrometry using the SCIEX TripleTOF® 6600 System is more specific and enables the identification and quantitation of each of the individual proteins present.

Danish company Alphalyse is a contract research organization (CRO) specializing in protein chemistry, mass spectrometry, and bioinformatics. The company was founded in 2002 as a subsidiary of ACE Biosciences, with strong ties to the Protein Research Group at the University of Southern Denmark, which is well known for MS analysis of proteins. Following a management buy-out three years later, co-founders Chief Executive Officer Thomas Kofoed and Chief Operating Officer Ejvind Mørtz decided to focus on protein analysis, and today the company provides protein characterization, identification, and quantification services to a global customer base.

Thomas explained, “An increasing number of drugs coming onto the market are proteins rather than small molecules, for example, antibodies and vaccines. Protein analysis is a crucial part of the drug development process.”

He continued, “As our customer base has grown, we have noticed that requests for characterization and quantification of host cell proteins, an important stage in the development of novel biologics, has increased. Host cells, such as yeast or Chinese hamster ovary cells, are used to produce protein drugs, which are then purified. However, even with rigorous purification processes, small amounts of host cell proteins will always be present in the final product.

Traditionally, these have been analyzed by ELISA which, although currently the best method for total host cell protein quantification, is not specific and cannot supply any information about which particular contaminant proteins are in the drug. Neither does it tell you how much of each protein is present. MS is perfect for this type of analysis, detecting even very low amounts of these proteins, which is important as even ppm quantities can adversely affect the drug product or provoke immunogenicity.”

“Two years ago, we were awarded a Danish Government grant to develop an MS-based method for host cell protein analysis. After evaluating a number of instruments to determine which system was most suited to this application, we chose the SCIEX TripleTOF 6600 System with SWATH® Analysis. Typically, MS instruments operate in ‘data dependent’ mode, scanning for selected masses and performing MS/MS analysis on the most intense peaks appearing at particular time points. If the same sample is run multiple times, most of the proteins identified will be the same. However, there will be some variation in the fragmented peptides and host cell proteins identified because the analysis relies on the instrument responding to what it sees, and this cannot be done reproducibly. The process must be repeated many, many times to obtain good statistical data. In contrast, SWATH analysis is ‘data independent,’ with MS/MS analysis performed across small mass windows.

The beauty of SWATH is that it collects data for everything, which means that if a sample is run several times the data retrieved will always be the same. This ability to generate reproducible data is vital for drug development studies and was the reason we chose this system. It allows us to reliably identify and quantify each host cell protein in drug batches, and provide a service that customers can trust.”

“We’ve had the instrument for about six months now, and are creating a library of MS/MS data to identify the proteins and peptides we want to analyze. The next step is SWATH analysis, generating large amounts of data and quantifying the proteins present in the samples. We use a variety of software packages to interpret the data, including ProteinPilot™MarkerView™, and BioPharmaView™ and are collaborating with SCIEX to develop the software even further. Recently, we received a second grant for validation and qualification of the assay, and will shortly begin establishing the reproducibility, accuracy, and precision of the analysis.”

“Time to market is important to our customers, and the traditional approach of developing ELISAs is both expensive and time-consuming, particularly if it is in addition to MS analysis. Over time, we hope that MS will replace ELISA, allowing us to quantify host cell proteins in individual batches and between batches, and demonstrate clearance of these proteins as the purification processes proceed. The additional sensitivity of the TripleTOF 6600 is another advantage, as the quantity of host cell proteins is minute in comparison to the amount of drug product. SWATH analysis is just perfect for that kind of sample. It’s a huge benefit,” Thomas concluded.Find out more about the SCIEX TripleTOF 6600 System >

This article is from the 2017 edition of the SCIEX VISION journal. You can download the full version of the journal which includes over ten customer research stories. Download now > 

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