It goes without saying that biopharmaceutical products require a high level of detailed structural characterization. But do we ever really consider that the "product" is not just the active drug itself but also the other components that may be present?
Lurking in the solution are other compounds, which are, quite literally, the residue of the manufacturing process. These need to be recognized and controlled so that these unwanted residual chemicals remain below regulatory acceptable levels.
The very different chemical properties of molecules involved in drug manufacturing require development and application of different analytical methods to monitor them. Different degrees of hydrophobicity or hydrophilicity, as well as the presence or absence of functional groups or chromophores, all need to be considered when developing a method that is suitably sensitive to reliably detect and quantitate low-abundance compounds in a complex matrix.
The ability to discern the compound of interest within a matrix that may contain many other components, often at significantly higher levels of the drug product relative to the compound of interest, needs to be considered.
One important class of residuals that requires consideration is polymeric species. These large molecules can prove challenging due to their size and inherent heterogeneity. However, suitable analytical methods do exist and can be developed for this class of compound.
For hydrophilic polymers, liquid chromatography can be used along with mass spectrometric detection, but this will be impacted by background signals at low polymer concentrations, which will adversely impact both limits of detection and quantitation, necessitating thoughtful method design.
These limits can be improved by the use of multiple reaction monitoring (MRM) protocols. Here, specific masses and their associated fragment ions are monitored in a triple quadrupole mass spectrometer. With the quadrupoles set to pass the parent and daughter ions only, the instrument serves as a mass filter, allowing only the selected ion transitions to pass. This significantly improves the signal-to-noise ratio by reducing background to minimal levels.
This approach will not be suitable for all polymers, but for those that ionize sufficiently well and produce fragment ions, this certainly provides a means of maximizing detection.
There's another advantage. It may be possible to develop and follow multiple transitions for the polymer of interest. Thus, with various transitions to follow, the residual polymer distribution can be accessed across the polymer mass profile. This is important since different batches of polymer used in different manufacturing runs may exhibit different mass profiles. Thus, the ability to follow a wide mass series means that any bias across reagent polymer batches to higher or lower ends of the mass distribution will still be identified and quantitated, rather than potentially not observed which would result in false low detection of the residual polymer in the sample.
Not all polymeric species are amenable to mass spectrometric analysis, but this doesn't preclude their assessment. Where mass spectrometry isn't suitable, other detection techniques can step in, like evaporative light scattering (ELS). This technique still uses chromatography as an initial separation step to isolate the component of interest from the background matrix as far as possible. But the eluent is then passed into a heated drift tube for desolvation prior to detection via a light scattering detector. This technique is also suitable for species that lack a chromophore and thus cannot be detected by UV absorption methods.
One factor in this type of residual analysis needs special consideration: purification. Polymeric species, being relatively large and heterogeneous, prove difficult to purify consistently. Purification methodologies may be challenged to work efficiently without risk of loss across the entirety of the mass range of the polymer.
For this reason, techniques should be developed as far as possible to work without requiring a pre-purification step. This proviso therefore requires the development of analytical procedures that can successfully operate in the presence of what may be high levels of sample and/or buffer/matrix components. Again, techniques such as MS-based MRM methods, with their high level of mass selection, are well suited when analyzing this class of residuals.
Analysis of residual polymers is certainly something that can be undertaken with careful method development. It can frequently, although not necessarily always, be investigated by mass spectrometric means.
Careful initial investigations are required to assess the mass spectrometric profile where possible to assess generated fragment ions of the polymer across the width of its chromatographic elution. This leads to the development of MRM transition profiles that can be monitored to ensure that the full mass profile is investigated, and no bias is introduced.
This results in a resilient method for detection and quantitation of the polymer that's usable even if variation exists between polymer batches used in the production process.
In all cases, to provide the most accurate assessment of polymer levels, the sample should be analyzed alongside a representative aliquot of the polymer used in the process as a reference sample.
In this increasingly complex analytical landscape, BioPharmaSpec offers specialist expertise in developing and applying robust methods for the analysis of residual polymers and other process-related impurities. Drawing on deep experience in mass spectrometry, chromatographic techniques, and orthogonal detection strategies, the BioPharmaSpec team can design analytical approaches tailored to each polymer’s properties and process context.
