Cell and gene therapies represent a radical departure from protein therapeutics. With more than 350 such treatments currently under investigation worldwide, developers and regulators must rethink critical quality attributes and how to assess them.
A typical viral vector production train using HEK293T cells involves cell expansion and transfection, vector purification, formulation/fill/finish, and drug product manufacture. QA/QC occurs throughout, but methods and strategies that were appropriate for protein manufacturing must be tweaked for non-protein products. In addition to contaminants arising from “ingredients” (media, feeds, and supplements), there are vector-related impurities such as empty capsids, and host cell-related contaminants similar to those found in protein-based processes, which include host cell genes and host cell proteins (HCPs).
Every cell-based biomanufacturing platform, regardless of the end-product (protein, gene, virus, or cells themselves), must incorporate unit operations that clear HCP contaminants during purification, and to characterize those that carry through. HCP mitigation is most challenging for non-antibody therapeutic protein processes in which structural characteristics, particularly affinity (e.g., during protein A capture), are not easily exploited for HCP clearance. However, even in such cases HCPs will carry forward through most downstream processing unless specific steps are implemented to remove them. Consequently, HCP profiles are a critical attribute for all cell-based biomanufacturing processes and products.
The main characterization tools for HCPs are enzyme-linked immunosorbent assays (ELISAs) and liquid chromatography-mass spectrometry (LC-MS), each with their advantages and drawbacks.
ELISA, the most straightforward method of HCP identification and quantitation, provides a rapid, semi-quantitative yes-no answer regarding the presence of HCPs. ELISA is well-suited to quality testing, and reagent kits are available for significant mammalian production cell lines to identify potentially problematic HCP impurities throughout the product’s lifecycle. ELISAs are less expensive to acquire than LC-MS and require only technician-level training to run. The major drawback of ELISAs is that investigators must know beforehand what they are looking for.
As with all quality assays, an HCP ELISA must be validated and qualified. Since ELISA-based HCP analysis results are variable, and reproducibility is poor for low-abundance HCPs, developers often employ orthogonal HCP analysis methods to provide more reliable HCP coverage and greater reliability of results. For example, HCPs may be further analyzed using a suitable capture or concentration step followed by 2D Western blot or Antibody Affinity Extraction™, from Cygnus, followed by 2D polyacrylamide gel electrophoresis, two-dimensional difference gel electrophoresis, capillary electrophoresis, LC-MS, or LC-MS/MS.
In addition to confirming the identities and concentrations of HCPs, orthogonal methods also provide other useful information such as capsid protein ratio and/or structure, and transgene length.
ELISA kits are most often used during discovery and early development, up to manufacturing for clinical phases 1 and 2. Once the therapy is promoted to phase 3, sponsors must decide whether to validate the commercial assay they have been using, or to develop a new protocol specifically for the HCPs of greatest interest to their process. Assay vendor companies can usually assist in either objective.
LC-MS, the current gold standard for identifying and characterizing proteins, has many advantages over ELISA. Where antibody-based assays require specific reagents for specific protein analytes, LC-MS employs much more extensive databases to identify proteins. Additionally, MS can operate in “discovery” mode to characterize and quantify unknown species, including previously unencountered HCPs and potential variants. LC-MS is also more reliably quantitative than ELISA, so it provides both identity and concentrations of species of concern. The method’s speed and automation, moreover, provide answers on a timescale consistent with continuous process improvement.
Like ELISA HCP panels, LC-MS methods can take many months of development and validation time. But here the advantage goes to ELISA-based methods which, since the vendor has done most of the development work and validation, may be used right out of the box.
HCP mitigation and characterization is part of every bioprocess involving cells. For viral vectors the challenges include utilizing cell lines with favorable host protein expression profiles, designing effective purification trains, and at all stages working with orthogonal analytical methods that confer confidence in results.
To learn more about HPC analysis for gene therapy and vaccine development, visit https://www.cygnustechnologies.com/gene-therapy-and-vaccine-development.