Biologics—antibodies, antigens, peptides, and other proteins—are frequently used as vaccines, but also for treatment of conditions such as cancer or diabetes. Biologics are typically produced in expression systems like yeast, E.coli, or mammalian cells. Even with elaborate purification protocols, traces of host cell protein (HCP) remain in the final product that may cause side effects, elicit an immune response, or affect drug stability and efficacy.
Regulatory authorities therefore request HCP documentation as part of the Biologics License Application (BLA) quality control process. The current gold standard for HCP detection is enzyme-linked, immunosorbent assays (ELISAs). Because off-the-shelf assays often do not provide complete coverage, additional specific antibodies must be raised and validated, taking significant time and effort.
A Growing Need to Identify HCPs
While optimized ELISAs precisely quantify the total amount of HCP, they do not reveal the identity of the contaminating proteins, making it difficult to assess the impact of a particular HCP on the patient’s health. Dr. Thomas Kofoed, CEO and co-founder of Alphalyse, a company providing services to the pharmaceutical industry based in Odense, Denmark, became aware of this issue about five years ago, when more and more of his customers started asking for analyses that included not only quantification, but also characterization of HCPs.
“We believed that mass spectrometry-based proteomics would be an excellent tool to address this challenge.” Thomas says. “In a consortium formed by several universities and private partners, supported by a Danish innovation fund, we tested the whole variety of instruments available and also contacted SCIEX to see if we could run some samples on their latest liquid chromatography (LC) / mass spectrometry (MS) instrument, the LC-MS TripleTOF® 6600 System.”
Solving the Issue of Sensitivity vs. Robustness
“Robustness is a prerequisite for any regulated quality control process, but on top of that, our customers always ask for as much in-depth information as possible.” Thomas points out. However, high-performance, sensitive LC instruments tend to be unstable and maintenance-intensive, whereas simpler, more robust instruments often lack sensitivity.
In the SCIEX TripleTOF 6600 System, a powerful MS/MS unit is coupled to a microflow high-performance liquid chromatography unit to create a robust setup with excellent sensitivity. “The main reason why we chose the SCIEX instrument was because it solved the sensitivity/robustness conundrum,” Thomas says. “At LC run times of just one hour, we reproducibly achieve both peptide qualification and absolute quantification over a linear dynamic range of five orders of magnitude, allowing us to analyse both highly purified and ‘dirty’ samples.”
Harvesting All Data Sets with SWATH® Acquisition
In LC-MS/MS workflows, sample peptides are first separated by liquid chromatography (LC). Elution fractions are then introduced into the MS instrument, where peptides are separated based on their mass-to-charge ratio (MS1 analysis). In the subsequent MS/MS (MS2, or tandem MS) analysis step, peptides from the MS1 step are fragmented to generate signature ions that serve to identify and quantify the original molecule.
“Traditionally, protein characterization, including HCP analysis, has been done in Data Dependent Acquisition mode (DDA)” Dr. Ferran Sánchez, market development manager at SCIEX, explains. “In DDA, the MS instrument software selects 10-15 peptide ions representing the most abundant peaks in MS1 to be sent to MS2. However, HCP analysis is complex because it requires detection of low abundant peptides against a high background of drug substance in excess of five orders higher. In DDA mode, information on some HCPs can get lost, and even identical replicates often deliver different results.”
To address these issues, SCIEX developed and patented SWATH® Acquisition, a Data Independent Acquisition mode (DIA). “In DIA, the instrument does not select individual peptides, i.e. it is non-discriminating, but it divides the entire peptide mass range into small windows that mirror the LC elution pattern. These windows are processed one after the other, ensuring that for each analyte, full MS and MS/MS spectra are determined with high reproducibility,” Ferran points out.
Thomas agrees: “The beauty of SWATH® Acquisition is that you get a complete dataset for all peptides in your sample without having to know what to look for. Even though data files are larger, the format is so standardised that it is easy for us to create reports for our customers.”
Will MS replace ELISAs?
“MS-based data provide very detailed information on each HCP.” Thomas says. “Once our customers know the identity of contaminating proteins, they can search databases to learn about HCP properties and decide whether it is necessary to further optimize the purification process. During our service projects at Alphalyse, we monitor and report every HCP along the entire production process.”
Still, MS is considered an orthogonal technique that will not replace ELISAs in the near future. “Especially the data analysis can be challenging to standardise,” Thomas admits. “The expert eye is still hard to replace by software algorithms. To us, MS is a really valuable additional tool that complements ELISAs.”
Next stop: MAM
Having established HCP characterization, Alphalyse now takes their two types of SCIEX instruments—the TripleTOF 6600 system and the recently installed X500B QTOF System—to new exciting projects. Through an EU-funded Marie Curie program, 15 PhD students at 11 universities and biotech companies—including Alphalyse—will explore the application of multi-attribute methods (MAMs) to monitor process variables such as Critical Quality Attributes, and to achieve quantitative characterization of post-translational modifications in biologics.
“This PhD project is a perfect match, because with MAM, we address the same customer group, and literally apply the same samples as for HCP analysis”, Thomas explains. “We truly enjoy working in multi-centre collaborations, staying at the forefront of our customers’ needs and applications. Together with the excellent instrument and software support we get from SCIEX, this means a significant competitive advantage for us,” he concludes.
Images via Shutterstock, Alphalyse, and SCIEX
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