UHPLC: Evolving Better Resolution of Larger Proteins


For decades, high-performance liquid chromatography (HPLC) has been used to separate and purify molecules. Recently, ultra HPLC (UHPLC) was developed to facilitate the separation of small molecules. Thanks to better resolution and faster run times, compared with HPLC, UHPLC of small molecules has played an important role in drug development. But increasingly, with proteins being developed as biotherapeutics, researchers are focusing on UHPLC for larger molecules. Here’s an update on how scientists are adapting UHPLC methods to harness the technology’s strengths for analyzing larger biomolecules, such as proteins.

What’s so ‘ultra’ about UHPLC for proteins?

Unfortunately, proteins don’t get the same “wow” factor from UHPLC that small molecules do. Proteins tend to be much larger than small molecules, and their movements are limited by diffusion, no matter what kind of pressure or flow rates you subject them to. So what exactly is so “ultra” about UHPLC when it comes to protein analysis?

When researchers first used UHPLC for small molecules, the resulting peaks were so well separated that “they could drive a truck through them,” says Michael McGinley, manager of core products at Phenomenex. “Users could easily ‘trade in’ some of that excess resolution for reduced run times by using shorter columns and faster flow rates and gradients.” Because protein peaks are not nearly as well separated, a dramatic reduction in run time isn’t usually possible. Nevertheless, recent protein-friendly adaptations have resulted in UHPLC that can provide better resolution and somewhat shorter run times.

The prospect of better resolution is attractive to researchers who are struggling to separate big proteins—like antibodies—and generating peak profiles with minute differences between them. For example, a single amino acid change in an antibody often represents less than 0.1% difference in chemical composition. Compare this to a single amino acid change in a small peptide, which may represent a 10% to 20% change in difference in chemical composition. “When proteins start getting big, separating them starts getting dicey,” says McGinley. “The changes can quickly get hard to see.”

Biocompatible systems

Although UHPLC doesn’t let scientists see all differences between proteins all the time, it can enable them to see some differences—for example, in gross morphology, disulphide isoforms, deaminations and protein folding. Protein researchers use different chromatography modes to accomplish this, such as reversed phase (RPC), ion exchange (IEX) and size exclusion (SEC) chromatography. “Salt-containing mobile phases, used for example in IEX and SEC, can be highly corrosive to a stainless-steel system,” says Cornelia Vad, senior product manager for analytical HPLC at Agilent Technologies. The same is true of solvents with extremes of pH.

To address solvent challenges, Agilent Technologies offers the 1260 Bio-inert LC System, constructed from corrosion-resistant titanium in the solvent-delivery system and 100% metal-free materials in the sample flow path. Other suppliers offer biocompatible, corrosion-resistant systems made from biocompatible metal alloys, titanium and other biocompatible materials. These systems include Thermo Fisher Scientific’s Vanquish Flex UHPLC and Waters’  ACQUITY UPLC System.

Thermo Fisher Scientific added many new features to its Vanquish Flex UHPLC system, specifically improvements to the temperature-control functionality in the autosampler and column compartment. “To successfully separate the subtle changes [in proteins], the instrument needs to shield delicate protein samples from external factors by having biocompatibility and accurate temperature control during storage and separation,” says Evert-Jan Sneekes, strategic marketing manager for HPLC at Thermo Fisher Scientific.

Another consideration when using an UHPLC system for examining proteins is instrument dispersion. “Dispersion is an increase in the volume of the peak as it moves through the components of the flow path,” says Tom Wheat, principal scientist at Waters. “As the peak comes out of the column, it’s as narrow as it’s going to be.” Broader peaks mean less resolution, so minimizing dispersion is crucial. Wheat says that minimizing dispersion is particularly important in SEC and RPC columns, but less so in IEX columns. Stacy Squillario, product manager in bioseparations at Sigma-Aldrich, adds that instrument dispersion should be “kept at a minimum in order to maximize the chromatographic performance.”

Although GE Healthcare Life Sciences doesn’t have a dedicated UHPLC system, its range of pre-packed chromatography columns, along with the ÄKTA™ platform for medium-pressure liquid chromatography and its new Increase columns for SEC are all designed for protein separations. Small-molecule UHPLC was mainly developed using RPC columns, but working with proteins often requires the use of other techniques. “For instance, techniques like SEC, affinity chromatography and IEX are used far more often for proteins than is RPC,” says Åke Danielsson, research director for R&D, research and applied markets at GE Healthcare’s Life Sciences business.

Integrating with mass spectrometers

Many protein researchers use UHPLC to separate proteins before mass spectrometry (MS) analysis. This can work well, depending on the mode of UHPLC analysis. “Cleanup before in-line analysis [to MS] is well developed for peptide separations with RPC, but is less well developed for other protein separations,” says Danielsson. “The compatibility of MS with common buffers and salts used for non-RPC protein separations remains an issue.”

Some UHPLC systems are designed to integrate with downstream MS systems. Thermo Fisher Scientific’s UHPLC systems integrate with its Orbitrap MS instruments, and Agilent’s UHPLC systems integrate with its Q-TOF systems and Triple Quadrupole MS systems. Wheat says that Waters’ UHPLC systems can be used with many MS instruments, “depending on the scale and application.” He adds that most integration problems “come with the software,” so it’s wise to check whether the MS and UHPLC systems’ software is compatible.

Improved particle technology

Conventionally, a defining feature of UHPLC for small molecules was a column of fully porous particles less than 2 microns in diameter. These don’t work as well for larger proteins, though, as they can cause increased back pressures, column clogging and other instrument-maintenance problems. Core-shell particles (also called superficially porous, geometrically structured, fused-core or hybrid particles), which are made from a solid, spherical inner layer surrounded by a porous outer layer, are recent innovations that can provide the separation efficiencies of UHPLC when working with larger proteins—without the associated problems.

Core-shell particles work better for proteins for several reasons. The porous shell reduces the diffusion path taken by proteins in and out of particles, so peak widths tend to be narrower. “Diffusion plays a very big role with biomolecules, and core-shell materials have the advantage,” says McGinley. “You can minimize the diffusion path, and by doing this you achieve higher performance.” Core-shell particles tend to be larger, which results in lower system back pressures and longer column life. “When we’re doing protein separations, we certainly don’t run at anything resembling high pressure,” says Wheat. “With large molecules that diffuse slowly, your best separations are at lower flow rates with hybrid particles.”

Several vendors offer core-shell particles or columns for protein separations. Offerings include Agilent’s Poroshell particles, Phenomenex's Aeris™ WIDEPORE core-shell columns, Sigma-Aldrich’s BIOshell™ Protein columns, Thermo Fisher Scientific’s Accucore™ particles and Waters’ CORTECS® particles.

Applications today and in the future

Applications for UHPLC are broadening, and increasingly they include biotherapeutics. McGinley says Phenomenex’s core-shell particles often are used for separating immunoglobulins, “usually IgG therapeutics, which are the lion’s share of protein therapeutic work these days.” One of Agilent’s newest columns, says Vad, is the “AdvanceBio mAb reversed phase column for intact antibodies and their fragments, which are analyzed with reversed phase, as well.”

Another UHPLC application is glycan analysis of glycoproteins. Waters recently released new UHPLC and UHPLC-MS analytical workflows, including the GlycoWorks RapiFluor-MS N-Glycan Kit. In addition, the company’s new ACQUITY UPLC Glycoprotein BEH Amide 300Å 1.7 µm Columns have large pore sizes to separate intact glycoproteins. Agilent also offers glycan-analysis tools with its 1290 Infinity II LC system, a high-end UHPLC system “for peptide mapping and glycan analysis, where highest peak capacity is absolutely necessary,” says Vad.

The future likely holds further technological developments in core-shell particles and in applications for research and biopharma. As an intersection of chemistry and biology, UHPLC for proteins is poised to diversify into an array of interesting applications—stay tuned to see where UHPLC technology heads next.