Let’s talk about column priming and how to address this common concern
Guest Author: Chad Eichman, Ph.D., Biopharmaceutical Industry Manager
With the emerging trend of biotherapeutics in the pharmaceutical industry, scientists have a challenging task of characterizing these large molecules. For a refresher on the basics, check out our article on the main analytical differences between small and large molecule analysis. The chromatographic separation of protein and peptides by HPLC and UHPLC is necessary in biologics characterization.
Large molecules are a delicate species and making sure they are in a “happy” environment is critical for analytical success. They come in all shapes and sizes, can be hydrophobic or hydrophilic, and their physical properties make them an intimidating class of molecules to analyze, from sample preparation to purification. Classes of compounds, including monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs), vary in their structure each time they are produced and must be monitored with high scrutiny.
Fortunately, analytical researchers have discovered a multitude of methods to accommodate for most of these protein traits by observing native, denatured, and digested states. However, some of these methods, such as aggregate analysis, remain a chromatographic challenge due to the protein properties.
One notable chromatography problem with biologics analysis is the requirement for column priming.
Typically, researchers must inject a protein surrogate, such as bovine serum albumin (BSA), multiple times to prepare the column for consistent performance. This priming procedure is necessary to create a steady state in the peak area, as well as maximizing analyte recovery. It is especially important to prime the column in size-exclusion chromatography (SEC) because protein adsorption is observed more during this analysis.
A major cause of the priming requirement is the adsorption of protein analytes on the wetted surfaces in instrument and column hardware. Companies have developed so-called “bio-inert or bio-compatible” LC systems that integrate materials with minimal protein interaction. For HPLC systems, polyether ether ketone (PEEK) is common, whereas titanium or MP35N (a nickel alloy) is integrated into UHPLC systems.
While these instrument advances are helping with adsorption issues, stainless steel is still utilized in the column hardware, which results in interaction of the protein with the metal surface. PEEK tubing is commonly used to minimize protein adsorption, but there are limitations to this technology.
First, because PEEK is an organic polymer, the tube is prone to swelling and pressure limitations. Even standard HPLC backpressures can be problematic. So packing UHPLC sub-2 μm or core-shell particles is infeasible. Further, this issue can result in variation in chromatography performance, as well as shortened column lifetime. Second, due to variability in organic polymer synthesis, column-to-column consistency is challenging due to column inner diameter variations.
So how can one create a bio-inert column without the limitations included with organic polymer tubes?
The answer lies in the utility of a bio-inert metal surface, namely titanium. This inert feature is already included in bio-inert UHPLC systems, but only Phenomenex has integrated the technology into the column hardware itself.
With the pressure limitations and column lifetime you would expect with standard metal tubing, the titanium integration alleviates protein adsorption, and the need for priming. What commonly may take 15-20 injections (and possibly more) of BSA to equilibrate the column, is now reduced to less than 5 injections to reach stability. Moreover, because the column is fully metallic, the internal diameter of the tube is consistent with each round of production creating reproducibility that cannot be achieved with PEEK tubes. Finally, titanium frits are used to make sure column lifetime is maximized and protein adsorption is minimized.
While the benefits from the titanium column hardware can be observed in reversed phase separations, the most pronounced effect is for protein aggregate analysis using SEC. Column priming is minimized, and analyte recovery is increased. In many cases only 1 or 2 injections of BSA is required to fully prime the column for SEC utility. This fact clearly improves on the limitations of stainless steel column hardware and leads to significantly less down time for researchers.