It has been an eventful year for PharmaFluidics. Its first product, a nano-LC micro Pillar Array Chromatography column (µPACᵀᴹ), was presented to the market, commercially launched, and very well received by the user community. Together with expert centers, the company has developed a comprehensive set of application notes demonstrating the value of its ultra-high-resolution micro-chip-based HPLC columns in the fields of proteomics, lipidomics, and metabolomics. Recent results also reveal its unique capabilities in the characterization of biopharmaceuticals. µPACᵀᴹ may be well on its way to playing a prominent role in the characterization of monoclonal antibodies (mAbs) as originators, similars, or conjugates.
Perfect order for better separations
µPACᵀᴹ cartridges are unique, as they are created by lithographically etching a separation bed from a silicon chip. This ensures a perfectly ordered stationary phase that reduces dispersion to an absolute minimum and enables the achievement of a very high separation resolution at moderate operating pressures. The µPACᵀᴹ benefits are most striking in long gradient separations where the average peak width remains much smaller.
This allows the µPACᵀᴹ columns to outperform the best packed-bed columns in metabolomic, lipidomic, and proteomic profiling applications. Under optimal conditions, the achieved peak capacity and detection sensitivity can be twice that of state-of-the-art packed bed alternatives. Micro-chip columns are thus better equipped to handle the tremendous complexity and dynamic concentration range of the molecules present in small extracts of biological tissue or biofluids. This is indispensable in many research fields, such as biomarker and drug discovery, toxicology, foodomics, and plant sciences.“At the RIC, we could demonstrate that the state-of-the-art μPAC™ columns are successful for challenging applications such as lipidomic profiling of blood serum, as well as to study biotherapeutics like mAbs or ADCs [antibody-drug conjugates] in great detail. We have also found the µPAC™ column to be a very robust separation tool. Because of the lithographic etching process and the nature of the silicon, the µPAC™ has a very rigid structure. In comparison to traditional HPLC columns, it allows the analytical lab to operate without any risk of stationary-phase compression or column deterioration. In that sense, we could easily switch the same µPAC™ column used for the lipidomics experiment to the biotherapeutics study without affecting resolution or productivity.” – Dr. Koen Sandra, Scientific Director, Research Institute for Chromatography (RIC), Belgium
Tiny differences in massive molecules
Recently, the company also demonstrated the value of µPACᵀᴹ columns in biopharmaceutical applications. mAbs have emerged as an important class of therapeutics in treating life-threatening illnesses such as cancer or autoimmune diseases, and their markets have grown significantly. Today, more than 40 mAbs are marketed in the United States and Europe, of which 18 have a blockbuster status.
The detailed characterization of such mAbs can be very challenging. They are large (ca. 150 kDa) and heterogeneous molecules. Moreover, during production and in subsequent storage, small modifications are introduced, such as glycosylation, deamidation, isomerization, or oxidations of amino acids. Point mutations can arise in the cloning process or over time, altering the protein sequence. Finally, several biopharmaceutical compounds are ADC, where the antibodies are used to guide cytotoxic drugs to specific cells. This conjugation can happen at different sites, creating different isomers and further increasing complexity.
All these tiny differences can have a great impact on biological function, but they are very hard to distinguish. Nevertheless, they are essential to monitoring mAb production processes to ensure consistent quality. It is even more important to the production of biosimilars, where regulatory agencies evaluate candidate compounds for their level of similarity to the originator. Powerful analytical tools are thus essential to comparing both molecules.
µPACᵀᴹ will become an essential tool
In a series of application notes, PharmaFluidics and the RIC investigated how well µPACᵀᴹ columns can address these challenges. Using Herceptin® and a candidate biosimilar, the prevalence and location of post-translational modifications could indeed be reliably determined. In an analysis of the ADC Kadcyla®, the resolving power of µPACᵀᴹ allowed for the in-depth study of conjugation sites and achieved the separation of isomeric conjugated peptides. Finally, when comparing Remicade® to a candidate biosimilar, it was possible to identify point mutations. As regulatory authorities consider an identical primary sequence primordial, this biosimilar candidate should be ruled out for further development. The ability to do this in an early stage, or to confirm similarity in such detail, is of the utmost importance for biopharmaceutical companies and regulatory authorities alike.
When used in combination with state-of-the-art Mass Spectrometry systems, PharmaFluidics’ µPACᵀᴹ HPLC columns allow for unprecedented performance and productivity improvements in the characterization of biopharmaceuticals, mAbs and ADCs. The further development of our µPACᵀᴹ HPLC columns in this block-buster sector, where detailed insight in tiny differences on complex molecules is of critical value, opens up substantial growth opportunities for PharmaFluidics. – Johan Devenyns, PharmaFluidics CEO
