The number of protein based drugs coming onto the market is at an all-time high, particularly those produced with a host cell system. With host cells come their own proteins. These host cell proteins (HCPs) constitute a major part of process-related impurities and can adversely affect drug safety, so it is critical that they are identified and quantified accurately.

HPCs can go undetected when using traditional characterization and quantification methods, the most common being immunoassays, such as enzyme-linked immunosorbent assays (ELISAs). But recently, orthogonal analytical methods, particularly mass spectrometry (MS), are becoming more prevalent to ensure previously unknown HCPs are not missed.

In a recent article published in The Column’s Biopharmaceutical Analysis Issue, we explore the advantages of universal data-independent LC-MS/MS workflow for HCP analysis compared with immunoassays.Read the Full Article >

ELISA Is Good, but Is It Good Enough?
Let’s start by exploring some of the drawbacks of ELISA – a method based on polyclonal antibodies raised against the host cells used in the fermentation process. It has been the workhorse method for HCP testing because of its high throughput, sensitivity, and selectivity.  

Although assays can take up to eight months to develop, they are typically capable of detecting most HCPs, particularly those proteins that are highly abundant or highly immunogenic. However, ‘the majority’ simply isn’t good enough for ensuring biotherapeutic safety. Lower abundant proteins can still be immunogenic or not elicit an immune response in ELISA animal models, and therefore go undetected.

As a result, orthogonal detection and monitoring techniques using as LC-MS/MS are often needed to supplement ELISA methodologies to fully detect and characterize HCPs in a biologic preparation.

How Does Mass Spec Compare?
The analytical technology that is emerging as the major alternative is mass spectrometry (MS). It offers the opportunity to not only monitor and measure the host cell protein and product impurity profile, but also the ability to identify what is, and is not, present in any sample, including low abundance proteins.

Until recently it was unclear whether the solutions available could provide the sensitivity and dynamic range necessary to detect and quantify trace HCP contaminants, and at speeds to complete confident analyses in a reasonable time span. However, a technique that meets the standard, and is already in use for HCP analysis, is quadrupole time-of-flight tandem MS combined with unbiased SWATH^® Acquisition.

Introducing Mass Spec with Unbiased SWATH Acquisition
Enter SWATH Acquisition! Data independent SWATH Acquisition on a TripleTOF^® 6600 System provides an unbiased method for detecting low-level HCPs – even those that were previously unknown or did not generate an immunogenic response in animal studies. The technique is comprehensive, sensitive, fast and reproducible, enabling the profiling and identification of the HCP complement of 1,000s of proteins at a sub-ppm level in a single 1-hour run. However, rather than being used as a supplement to ELISA, SWATH Analysis is an alternative to ELISA for fully characterizing and monitoring HCPs.

SWATH Acquisition works by collecting MS and MS/MS data across the entire data range in an unbiased data-independent fashion. Because of the speed, sensitivity and dynamic range of the TripleTOF System, and the intelligent design of the SWATH workflow, the full HCP complement can be detected at sub-ppm levels in 1 hour. And contrary to other LC-MS/MS techniques employed for HCP analysis, no complicated and extensive LC fractionation is required. As described by SCIEX researchers in a recent webinar and also in this informative slide presentation, this can have profound effects on the efficiency and overall cost of a biologic through process development, with time and cost savings of 20x for a typical study.

Additionally, because SWATH Acquisition is unbiased, the approach provides substantial benefits over other MS strategies by capturing comprehensive, quantitative MS and MS/MS information for every analyte in the sample. Thus, sequence variants and low-level modifications to the biologic can also be captured in the same data set. In fact, protein contaminants from any source can be detected along with the HCPs. Because of the comprehensive nature of the SWATH data file, the digital record that is created can be used as a digital archive of the current state of a sample. Any protein contaminant concerns that may emerge in the future can be tracked over time by mining the data retrospectively.

The method is mature and has been used extensively in the microflow regime where the LC details have been optimized for easy implementation. The workflow can also support high flow and nanoflow chromatography where it may be advantageous to perform HCP analysis in the early development stage when there is not as much product available for characterization.Read more about SWATH Acquisition on a SCIEX TripleTOF 6600 System for HCP analysis in The Column’s Biopharmaceutical Analysis Issue >