June 1, 2018 (Vol. 38, No. 11)

High-Performance Liquid Chromatography (HPLC) Never Plateaus

Large, complex, and subtly heterogeneous, biopharmaceutical compounds present countless separation challenges. Fortunately, these challenges are neither so great nor so many as to daunt the biopharmaceutical industry’s separation specialists, the analysts who make use of high-performance liquid chromatography (HPLC).

Analysts eager to climb peak HPLC may want to study the scientific program drawn up by the organizers of HPLC 2018, the 47th International Symposium on High-Performance Liquid Phase Separations and Related Techniques. Scheduled to take place from July 29 to August 2, 2018, in Washington, DC, HPLC 2018 will show that professional heights may be reached by many paths.

Selected previews of HPLC 2018 are presented in this article. They cover the resolution of protein variants by reversed-phase chromatography, the separation of carbohydrates at high pH by anion exchange chromatography, and the automation of offline sampling to streamline quality control.

Resolving Protein Variants

Innovative reversed-phase column technology has been developed by Waters to separate monoclonal antibodies (mAbs), mAb fragments, and antibody-drug conjugates (ADCs). According to a recent study (Bobály et al., 2018, J. Chrom. A), the technology outperforms silica-based and silica-hybrid C4 bonded materials, delivering superior protein recovery, and exceeds conventional C4 or C18 bonded phases, providing enhanced selectivity for mAb subunit peaks and ADCs. Also, with the new column, users can run the mobile phase with lower concentrations of trifluoroacetic acid (0.02–0.05%) without causing a significant decrease in protein recovery or peak capacity.

Waters’ BioResolve RP mAb Polyphenyl Column provides a high surface coverage polyphenyl bonding on superficially porous silica-based solid core particles. The patent-pending bonded phase is created through a multistep reaction that generates short, rigidly constrained phenyl moieties.

“We designed this new column technology to facilitate higher resolution with more [mass spectrometry]-compatible mobile phases, higher throughput, and less carryover,” says Matthew A. Lauber, Ph.D., consulting scientist, Waters. Another key goal was to be able to deliver these advantages for separations performed at lower temperatures. The comparative study by Bobály et al. showed that acceptable recoveries for most mAbs could be achieved at temperatures below 80 °C.

Dr. Lauber will be giving a keynote presentation entitled “A Novel Phenyl-Based RPLC Stationary Phase for High-Throughput, High-Resolution Characterization of Protein Therapeutics” at the HPLC 2018 conference.

Waters developed the polyphenyl column in response to customers’ need for a more efficient, higher resolution mAb separation method. “Although mAb therapies have become mainstream, the analytical tools are still lacking,” notes Dr. Lauber. “We’ve seen that resolution is too low in reversed-phase separations of proteins and especially in MS-compatible reversed-phase separations. These are tests needed to learn the molecular weight and confirm the sequence of a mAb.

“People need to run these separations to look at product-related impurities and to profile what variants are being made. Sometimes these may be critical quality attributes tied to something important about the product, and they need to be developed and monitored thereafter to ensure product quality, safety, and efficacy.”

Waters’ BioResolve RP mAb Polyphenyl Column is designed to improve the resolution, fidelity, and mass spectrometry (MS) compatibility of monoclonal antibody (mAb) subunit profiling. With this new column technology, separations can be performed with lower temperatures and lower concentrations of ion-pairing acid to reduce on-column degradation and increase signal-to-noise ratios, improving the quality of MS detection.

Separating Carbohydrates at High pH

When Christopher Pohl, vice president, chromatography chemistry, Thermo Fisher Scientific, set out to build on an ion exchange chromatography technique he developed in the early 1980s, he intended to find a better way to separate carbohydrates at high pH. Because he was looking forward, Mr. Pohl did not expect his research to lead him to literature from the 1940s. In that early literature, the researchers described how they had used large particle size ion exchange resins to separate carbohydrates. They also reported long separation times: 5–15 hours.

In these early experiments, researchers ran afoul of the tendency of carbohydrates to become unstable at high pH. After the researchers added base to the mobile phase, they observed that carbohydrates degraded over the course of the separations. The researchers’ conclusion, recalled Mr. Pohl, was simple: “You shouldn’t try to separate reducing sugars at high pH.”

While still unaware of this literature, Mr. Pohl and colleagues tried a similar approach. They were also unaware of how advantaged they were to be using a chromatographic material that required analyses of only 3–15 minutes. These analyses are short enough, Mr. Pohl and colleagues discovered, to avoid significant degradation of the carbohydrates during the separation.

The motivation behind Mr. Pohl’s research was the desire to use electrochemical detection of carbohydrates instead of the more conventional use of refractive index detection with HPLC. This necessitated performing the separations at high pH, in the 13–13.5 range.

Mr. Pohl was working with borax complexes, which were usually separated on anion exchange media at a pH between 8 and 9. Adding base to raise the pH after completion of the separation can be problematic if there is too much variability in the mixing of the mobile phase and the base. Instead, base was added to the borax complexes before the separation, and surprisingly, the results of the chromatography were better than when run at pH 9. When Mr. Pohl went back and studied the process, he found that after base was added, the borax complexes were no longer present.

Mr. Pohl’s group has since developed various products based on the basic principles outlined in these findings. “One of the keys we discovered early on is that we need to design the stationary phase so that the ion exchange sites are very concentrated,” he explained. “Then the pH will be high enough to efficiently ionize the carbohydrates.”

The group’s current research builds on all this knowledge to create a new version of the hyperbranched chromatographic stationary phase Mr. Pohl previously developed. The hyperbranched material would enable anion exchange separation of carbohydrates at high pH with greater selectivity.

As this work progresses, “the trick is to design the stationary phase so that the reaction sites are close together but still have this branched architecture,” advises Mr. Pohl. At HPLC 2018, in a keynote presentation entitled “High pH Anion Exchange Separation of Carbohydrates: Past Present and Future,” Mr. Pohl will describe his research and how it relates to a new stationary phase that is being developed by Thermo Fisher Scientific.

Automating Online Sampling

Traditionally, assessment of product quality has relied on offline sampling. For example, in GlaxoSmithKline’s continuing manufacturing processes, offline sampling has been combined with reversed-phase liquid chromatography to allow the monitoring of drug substance quantities and impurity levels. By conducting offline sampling repeatedly and frequently, GlaxoSmithKline can take snapshots at short intervals, gathering information to improve its understanding of continuing processes and confirm product quality.

With offline sampling, however, sample extraction may occur too infrequently. Worse, so much time may be required for sample preparation and analysis that quality assessments may become available only after processing ends.

To avoid these problems, GlaxoSmithKline transitioned from offline sampling to an online UPLC analytical method. The company’s experience doing so will be described by Elyse Towns, Ph.D., process analytical technology scientist at GlaxoSmithKline, in an HPLC 2018 presentation entitled “Monitoring Drug Substance in Continuing Manufacturing Processes at GSK with Online UPLC.”

According to Dr. Towns, the main disadvantage of offline sampling is the long turnover for results (several hours typically) and the resources required to run the analysis. The desire to monitor the manufacturing process more frequently and gain more process information, she explains, led to the introduction of an automated sampling system.

“We have implemented an online UPLC system that is located by the continuous rig in the production area,” says Dr. Towns. “The automated system removes a small amount of material automatically from the line, dilutes the material, and injects it to the LC for analysis, providing a measure of the product quality every 20 minutes throughout the continuous chemistry process. This allows us to get results without analyst intervention at high frequency to constantly verify that the process is running as expected.”

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