The path to improved safety of gene-based products

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Kerstin Pohl, Senior Global Marketing Manager, Gene Therapy & Nucleic Acid at SCIEX, looks at the application of liquid chromatography-mass spectrometry for analysing host cell proteins.

Gene-based therapeutics and vaccines hold immense potential for more efficient and personalised treatment and prevention of complex diseases. However, the increased complexity and diversity involved in the development and manufacturing of gene therapies and vaccines can cause challenges when it comes to determining the quality of the final product, particularly around the detection of impurities. Host cell proteins (HCPs) – proteins derived from the host cells used for viral production—are one class of process-related impurities.

If they are not detected and removed during purification, process-related impurities can alter drug quality or evoke undesired immunogenic reactions in the patient. In fact, a recent study, which focused on SARS-CoV-2 vaccines based on viral vectors, found a severe adverse effect called vaccine-induced thrombotic thrombocytopenia (VITT), which might be associated with higher levels of process-related impurities and HCPs1. In these cases, injection of the viral vector vaccines induced the production of antibodies recognising platelet factor 4 (PF4) found on patients’ platelets1. Subsequently, platelets were over-activated, leading to thrombosis1. The comparison of different viral vector vaccine products for Covid-19 suggested that higher amounts of process- related impurities – such as adenoviral proteins and active enzymes from the production cell line – led to a higher prevalence of anti-PF4 antibodies and capillary leakage in patients receiving a particular type of vaccine. These impurities were associated with an increased risk of thrombosis at various locations throughout the body1. Although the frequency of VITT was one in every 100,000 people, the mortality rate was high at 22%2. Given the vast number of people receiving boosters and follow-up doses, the potential threat posed by undetected HCPs cannot be neglected.

Impact of unnoticed HCPs

Process-related impurities — specifically HCPs — can be damaging in multiple ways. First, products containing undesired proteases can impair drug stability, either by degrading the drug itself or by degrading stabilising excipients of the drug formulation, which ultimately diminishes the drug’s durability. Another dangerous consequence can occur when the therapeutic or vaccine introduces proteins that can activate an uncontrolled immune response. While triggering an immune response is the goal for any given vaccine, as a preventative application, it needs to be well understood and controlled to avoid cytokine storms, which can lead to multi-organ failure. In the case of therapeutics, the aim is usually to avoid patients’ immune responses.

Impurities can integrate into the final product at various points during production. While intense purification is being performed, interactions between process-related impurities and the drug substance or delivery vehicle (such as viral vectors) or similar physical properties can lead to their co-purification with the desired product. The adverse effects vary depending on the amount and the type of HCPs in the final drug product, which is why a thorough quality control process is essential to determine the purity and safety of the product and to ensure comparability between batches of production. This high level of quality control is also demanded and regulated by regulatory agencies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), which consider HCPs a critical quality attribute (CQA). Although the acceptable range for HCP levels is approximately 1-100 parts per million (ng per mg of product)3, it can vary between individual cases.

ELISA for conventional HCP analysis

The standard procedure for HCP analysis relies heavily on enzyme-linked immunosorbent assays (ELISAs), which can reveal the HCP quantity in the drug product. With sensitivity levels of parts per million, an ELISA can detect the presence of low-level HCPs robustly with high throughput. As efficient as ELISAs are in HCP quantification, they have several limitations. These assays require large quantities of high-affinity anti-HCP antibodies and still fail to cover the complete HCP profile. In particular, ELISAs tend to neglect small-sized and weakly immunogenic proteins.

In addition, their cumbersome developmental process limits their use for fast-tracked new modalities, such as DNA-based vaccines. Most importantly, most ELISAs only provide the overall HCP amount, meaning that one cannot detect changes in the HCP composition.

Novel gene-based drugs can be severely impacted when HCPs are not distinguished and quantified individually. Dr Thomas Kofoed, Co-Founder of Alphalyse, summarises the increased complexity of impurity detection in gene therapies: “New modalities use various expression systems for the drug production, such as Chinese hamster ovary cells (CHOs), E. coli, and Pichia cells. These host cells alone contain over a thousand HCPs. In addition, there are other sources of protein contamination, such as from cell culture medium supplements — for example, bovine serum albumin.” In other words, commercial ELISA kits, which are traditionally geared toward HCP detection in CHO-derived therapeutics, may not work with new modalities with varying process-related impurity profiles. This requires the development of process-specific ELISAs, which is very time- consuming and undesirable when striving to meet global demands quickly and efficiently.

Kofoed emphasises another major challenge with ELISAs: “If the biologic has been produced using a human host cell line, these HCPs are not always recognised by the ELISA antibodies typically raised in rabbits or goats. So, commercial ELISAs can miss a substantial proportion of the HCPs in a drug product.”

Advantages of LC-MS over ELISA in HCP detection

Liquid chromatography-mass spectrometry (LC-MS) has emerged as an orthogonal method to ELISA to bridge the gaps in impurity analysis.

It can be used to detect individual HCPs, even those in low abundance relative to the main product, and unlike ELISA, LC-MS is not biased toward the immunogenicity. Its ability to discern between individual HCPs helps manufacturers make more informed decisions to optimise the purification of the product most efficiently. Furthermore, LC-MS can help manufacturers understand fluctuations of individual HCPs throughout the manufacturing process, which might go unnoticed by ELISAs. An LC-MS method for HCP detection can be adapted to new products and new modalities derived from different cell lines in a matter of days to weeks, while the development timeline for ELISAs can be months to a year. Taken together, the advantages of LC-MS pave the way for the deliberate optimisation of gene-based vaccine and drug production processes.

Accurate mass spectrometry for initial HCP identification with high confidence

Traditionally, accurate mass spectrometry was used for large-scale proteomics screening to discover potential biomarkers as a starting point for drug development. Similar approaches are used for identifying individual HCPs. The strength of an accurate mass approach comes from its ability to accurately identify HCPs within complex mixtures. Taking into account different processing stages, biopharmaceuticals and next- generation drugs can consist of up to a few thousand proteins that span large dynamic ranges, meaning they contain proteins at extremely low and very high concentrations. Peptides derived from these proteins can overlap in their mass-to-charge ratios. Furthermore, HCP peptides and peptides of the drug substance or viral carrier that share similar physical properties can co-elute during LC separation. To identify HCPs confidently and reliably, accurate mass spectrometry can be used with MS/MS fragmentation.

SWATH DIA for comprehensive impurity detection

While LC-MS is generally less biased than ELISA, MS-based HCP profiling can be biased toward abundancies. More specifically, many MS methods rely on data-dependent acquisition (DDA), which identifies a certain number of analytes per LC elution time point in the sample. This approach is biased toward the more abundant analytes and might result in missing critical impurities that were co-eluting with higher abundant analytes. In addition, DDA methods are stochastic in nature, which means the detection of HCPs can vary between replicate analyses.

To help address this, SCIEX has developed a data independent acquisition (DIA) method called SWATH DIA4. It’s an unbiased approach for recording the MS and MS/MS of every detectable analyte in the sample at every given time point. Data can then be deconvoluted with the help of protein databases to identify the proteins in the sample. With this approach, scientists can perform HCP detection without prior knowledge of what to detect while limiting the risk of missing important analytes. In addition, the SWATH DIA approach allows for relative quantification on the MS/MS level using the same data.

Triple quadrupole LC-MS for high throughput

In some instances, the highest levels of sensitivity, robustness and throughput are required for monitoring HCPs, especially for late-stage products of downstream processing. In these cases, the HCPs previously identified with accurate mass spectrometry can be tracked most efficiently by transferring to a triple quadrupole instrument. Triple quadrupole LC-MS offers the optimum combination of sensitivity and throughput for HCP monitoring with an excellent dynamic range for absolute quantification. Through multiplexing with multiple reaction monitoring (MRM), simultaneous quantification of large numbers of HCPs in a single injection is readily achievable. Lower limits of quantification (LLOQs) as low as 0.02 parts per million are feasible with state-of-the-art instrumentation.

LC-MS with triple quadrupole technology informs the manufacturer about which HCPs are present and in what relative quantities, and it is compatible with automatic processing while requiring only minimal LC-MS expertise. The sensitivity, robustness and versatility of the technology makes it ideal for complex next-generation drugs for which reliable ELISAs do not exist.

LC-MS to replace ELISAs? Future prospects

With the state-of-art technologies mentioned previously, LC-MS has the potential to not only complement ELISA, but also replace it as a robust, sensitive and reproducible HCP analysis method. May 2021 marked a milestone in that regard, as the US FDA approved an investigational new drug (IND) application for a pharmaceutical company that ran HCP analysis for their virus-like particle-based vaccine booster using only mass spectrometry data from Alphalyse. This approval could pave the way for LC-MS to transition more frequently into a good manufacturing process (GMP) environment, where it can be used as a release assay for regulatory documentation. Eventually, LC-MS can accelerate the development of emerging gene-based vaccines and therapeutics and speed their delivery to market with its flexibility and short assay development timelines compared to ELISAs.

LC-MS can prove valuable not only in quality control, but also in early development phases. The more insight developers can gain into process-related impurities, the more optimisation they can perform on their target product profile (TPP) to minimise HCP co-purification. This increases efficiency in process development, diminishes the risk of detecting harmful HCPs later in the manufacturing phase, and potentially increases yields.

To make the most of LC-MS technologies in the near future, the pharmaceutical industry must internalise the mindset of “starting with the end in mind.”

DDW Volume 24 – Issue 2, Spring 2023

References

  1. Michalik, Stephan, et al. “Comparative analysis of ChAdOx1 nCoV-19 and Ad26. COV2. S SARS-CoV-2 vector vaccines.” haematologica 107.4 (2022): 947.
  2. Pavord, Sue, et al. “Clinical features of vaccine-induced immune thrombocytopenia and thrombosis.” New England Journal of Medicine 385.18 (2021): 1680-1689.
  3. Bracewell, Daniel G., Richard Francis, and C. Mark Smales. “The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk‐based management for their control.” Biotechnology and bioengineering 112.9 (2015): 1727-1737.
  4. Collins, Ben C., et al. “Multi- laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry.” Nature communications 8.1 (2017): 1-12.

Kerstin PohlAbout the author:

Kerstin Pohl is the Sr Global Marketing Manager at SCIEX, responsible for the communication of differentiated analytical solutions for gene therapy and nucleic acids, and the support of cutting edge technology going to market. She joined SCIEX in 2015, and had various roles focusing on biopharma, protein and oligonucleotide characterisation with accurate mass spectrometry.

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