Looking ahead, how can platform technologies for analytical methods be improved? Can methods for detecting contaminating proteins,
host-cell proteins, and protein level be standardized? What new technologies or methods could help?
Analytical methods, by definition, should theoretically be amenable to platform standardization. As mentioned, the primary
purpose is to detect process- and product-related impurities. The challenge for process-related impurities is that each upstream
platform produces different impurities, such as type and amount of HCPs. Unfortunately 'generic' commercial kits are often
poor substitutes for process-specific detection methods, but do serve a purpose when used consistently in a platform.
For product-related impurities, the challenge is similar to that for downstream processing, and depends on specific molecular
variants. In addition, the analytical technologies employed are not yet standardized. Charge variants, for instance, can be
detected by at least four different methods, none of which effectively discriminate amongst several types of variants (e.g.,
sialic acid content, deamidation).
If the platform methods are developed in parallel with the process, and used and controlled consistently, then they can be
useful within the portfolio they are employed. Process-related impurities are better understood and controlled, and minor
modifications can be made to address product-related impurities. However, the relative utility of the platform is lessened
when applied more broadly across product portfolios.
Ideal analytical methods would both separate and identify unique molecular species. A high-throughput, quality-control friendly
functional equivalent to an LC–MS method would be desirable.
Most straightforward analytical methods such as A280, capillary electrophoresis, polyacrylamide gel electrophoresis, isoelectric
focusing, and size-exclusion high-performance liquid chromatography, are flexible and lend themselves for upstream and downstream
analyses. More complex methodologies, particularly for unique post-translational modifications and potency, are not as easily
standardized, particularly those requiring high-end analytical endpoints such as mass spectrometry (MS), nuclear magnetic
resonance (NMR), surface plasmon resonance for binding kinetics, and cell-based bioassays.
Once projects progress to the clinical-trial stage, it is advisable to take a closer look at standardized methods and optimize
them for the molecule at hand.
The first requirement for being able to facilitate the use of an analytical platform is that the master cell line, expression
vector, and upstream and downstream process steps are standardized as much as possible. The better characterized and standardized
the process, a combination of anti-sera reactive against known impurities and HCPs can be created from premade anticontaminant
libraries to provide sufficient coverage and sensitivity. Protein-A detection methods are relatively easy to platform while
HCP methods tend to be the most challenging.
Newer surface-plasmon resonance instrumentation is providing for significant improvement in the throughput and robustness
required for ligand binding and binding kinetics assays. With particular molecule classes (e.g., mAbs), standardization of
common reagents and capture approaches can improve and simplify the method development of specific binding activity method
For cell-based bioassays (potency), the use of common cell-based systems, either off the shelf or specifically designed, and
activity read-outs for classes of activities (e.g. cytokine production, cell migration, etc.), can significantly reduce the
amount of de novo method development. Also, the standardization of read-outs such as chemiluminescence or enzyme-generated colorimetric measurements
in a microtiter plate format can further improve throughput.