May 1, 2009 (Vol. 29, No. 9)

Economic Crisis and Demand for Near-Process Analytics Drive Methods Development

The success of UHPLC (ultrahigh-performance liquid chromatography) in its various forms has been a pleasant surprise. All major vendors have released, in one form or another, high-end, high-pressure systems employing columns with stationary-phase particle sizes below 2.5 microns. Not everyone agrees on the merits of ultrasmall particle technology, but the technology is definitely here to stay and is entering the mainstream.

“We have customers who are asking to transfer isocratic as well as gradient methods to UHPLC format,” observes Alessandro Baldi, Ph.D., product manager for chromatography at PerkinElmer. He calls this “an interesting development” because it means that UHPLC is no longer reserved for power-users, and is “capturing much more of the LC market than we had originally anticipated.”

Dr. Baldi refers to the three major groupings of LC column technology as tiers that include the specific instrument, column, workflow, application, and operator skills. “Classical HPLC” refers to the pressure domain up to about 6,000 psi, with particle sizes toward the upper end of the 3–5 micron range. “Near-UHPLC” consists of systems operating in the 6,000–10,000 psi range, with particle sizes between 2 and 2.5 microns. “True UHPLC” operates from about 10,000 to 15,000 psi, with particle sizes below two microns.

Porting methods from conventional HPLC to near-UHPLC takes work but is relatively straightforward. But because of radically different pressures, instrument geometries, flows, solvents, injected volumes, and buffers, switching to true UHPLC is much more difficult.

Near-UHPLC represents a compromise that nevertheless provides three- to fourfold throughput improvements. In March,  Dr. Baldi presented a paper at the “Pittcon” conference in Chicago describing a near-UHPLC system with fourfold higher throughput and 50 times greater sensitivity than conventional HPLC, and which used a fraction of the organic solvent.

PerkinElmer sells instruments that fit all three tiers, according to Dr. Baldi. In March the company introduced the Flexar™ LC platform, which is controlled by the new Chromera® Chromatography Data System. Flexar uses a sub-2 micron particle column and consumes about one-fifteenth as much solvent as conventional HPLC, Dr. Baldi adds.

PerkinElmer’s Flexar FX-15 UHPLC system uses a sub-2 micron particle column and reportedly consumes about one-fifteenth as much solvent as conventional HPLC.

Impact of Global Meltdown

The economy, and its impact on laboratories, has been one of the big instrument  stories of the past year. Due to layoffs and cost-cutting, purchasers value automation and throughput more than ever. The shortage of acetonitrile, a byproduct in the manufacture of acrylonitrile (a high-volume industrial monomer), has caused analysts to re-think solvent-intensive HPLC methods. Many companies are substituting solvents, for example methanol, but that requires re-validating methods, which in regulated industries can be expensive and time-consuming.

“Users are looking to cut back on solvent consumption through use of smaller-diameter columns with lower flow rates and shorter analysis times, not to mention alternative methods, like ion chromatography, that do not use acetonitrile,” says Phil DeLand, market development manager at Dionex.

Dionex claims its “reagent-free” RFIC™ ion chromatograph reduces equilibration time, calibration, method verification, and troubleshooting compared with standard ion chromatographs. Reagent-free refers to RFIC’s ability to manufacture eluent on demand through electrolysis and controlled formation of potassium hydroxide or methanesulfonic acid. 

Dionex also offers a rapid, high-resolution HPLC, the UltiMate® line, which includes modules for performing rapid separation LC (RSLC) and nanoflow LC/MS.

Dionex’ Rapid Separation LC (RSLC) solution combines hardware, software, and chemistries and is available in binary, quaternary, and x2 dual system variants.

Helmut Schulenberg-Schell, marketing manager for liquid chromatography at Agilent Technologies, agrees that the global economy will have a deep impact on instrument markets with speed, efficiency, and higher-quality data being the prime drivers. HPLC systems, he argues, will need to be a lot more flexible to address issues of diverse workflows and operator competencies. “Whether the issue is sub-2 micron technology or high flow, purchasers feel locked in. We want to change that.”

The Infinity LC system, Agilent’s first major HPLC introduction since the debut of the 1200 series three years ago, provides the flexibility to utilize columns and chemistries from any vendor, according to Schulenberg-Schell. The Infinity binary pump module uses “active damping,” which incorporates firmware to reduce ripples in pump output and associated background noise. Noise is further reduced by Jet Weaver gradient mixing technology, which delivers solvents in picoliter steps for precise mixing.

Another feature of the Infinity is a new UV diode array detector with (reportedly) twice the sensitivity of other available detectors—significantly higher than what is required of an FDA-validated assay, according to company spokesman Stuart Matlow. Optofluidic waveguides provide ultralow detection limits and high signal-to-noise ratio, while use of noncoated fused silica eliminates special-care requirements.

Most vendors tout pressure ranges for HPLC systems but, according to  Schulenberg-Schell, a more relevant parameter is power range, the product of flow rate times pressure. Agilent claims the highest power range available for its Infinity binary pump, up to those typical in UHPLC. Agilent has also reduced the delay volume to about 10 microliters, two orders of magnitude smaller than for typical HPLC systems. “The practical implication of this is separations in 30 seconds instead of 20–30 minutes,” says  Schulenberg-Schell.

Perhaps the biggest selling point for Infinity is its backward compatibility with pressures, columns, and established methods. “Whenever users are ready to upgrade a method, that capability will exist for them in the Infinity,” notes Schulenberg-Schell.

Agilent Technologies reports that its 1290 Infinity LC system provides full backward compatibility with existing columns and methods while providing the capability for true UHPLC operation.

Is Sub-2 Micron Worth It?

For Simon Robinson, HPLC product manager at Shimadzu Scientific Instruments, the most significant trend in HPLC is fast instrumentation that delivers rapid results at higher resolution. “Fast LC has stimulated a lot of instrument sales, and is causing users to look closely at what they’re doing, how their columns work, and which stationary or mobile phases are best for their application.”

Shimadzu’s Prominence HPLC product line includes the Prominence UFLC, an “extremely fast” system operating at standard pressures, the higher-pressure Prominence XR, and the Prominence Nano, a nanoflow system for proteomics.

Robinson delineates cutting edge HPLC instrumentation as those in the 2–3 micron particle size range and sub-2 micron. The former, he says, is optimal in terms of physics, specifically with respect to the ratio of height-equivalent theoretical plates and backpressure. Shimadzu has long played down sub-2 micron systems as too complex for the average user.

“Higher backpressures present more consequences you have to deal with, not to mention higher initial investment and maintenance.” Separating fact from fantasy on particle size, he says, is simple. “The hard part is getting people to believe it. What they learn is that running at 10,000 psi and up is an absolute nightmare on your sanity and wallet. The worst part is when the customer finds out optimizing at a lower pressure can frequently produce better results.”

Tom Jupille, an LC trainer and consultant with LC Resources, is another advocate of optimizing larger particle size systems to avoid the complexities and costs of ultrahigh pressure operation.

Firm in its convictions, Shimadzu promotes LC systems optimized for columns employing 2.2 micron particles which, says Robinson, may be run under normal-pressure conditions.

Bioprocess quality efforts have traditionally been after-the-fact activities rather than pro-active. The notion of Quality by Design, versus testing-in quality, was the impetus behind FDA’s process analytic technology (PAT) initiative. PAT projects have been occurring, particularly at large bioprocessors, but not with the frequency, fanfare, or urgency that might be expected.

For example Shimadzu has an ongoing collaboration, with a major pharmaceutical company, to interface one of its HPLC systems to a bioreactor through an auto-sampler. But this project is “still in the prototype stage,” according to Robinson.

What’s holding back HPLC-based PAT, says DeLand, is “lack of confidence” by an increasingly conservative market. “These reactions and fermentations are worth a lot of money, and a lot is invested in process development,” he says. “But thanks to initiatives from regulators, companies are beginning to open up and embrace the idea of PAT.”

Nevertheless, the tools for PAT exist. Last December, Dionex introduced a new system, the Integral™ process analyzer platform, that works with the company’s ion chromatography and rapid separation LC systems. Integral draws a sample and passes it on to the LC for analysis. The system is suitable for use in process development, pilot, and production systems.

Standing PAT

According to George Barringer, Ph.D., CSO of Groton Biosystems, HPLC/PAT has been slow to catch on because it requires changes “in process and focus on the part of users and suppliers.” More complex analyses usually go to an external analytical lab, far from the processing area.

“Moreover practitioners lack the facilities for HPLC, and using it online will require a mindset change.” The other hurdle, Dr. Barringer says, has been instrument suppliers, who focus on analytical laboratories to the detriment of process groups.

Yet HPLC is ideally suited for feedback control, particularly for quantifying the appearance of the main product or consumption of amino acids, carbohydrates, and other nutrients. Groton has demonstrated this with its flagship products, in the ARS line of automated auto-samplers and feedback control systems that connect up to eight reactors to four analytical instruments.

The company has collaborated with Agilent (on HPLC systems), DASGIP (bioreactors), and YSI (bioprocess analyzers). Thus far Genentech, Pfizer, Novozymes, and Biogen IDEC have evaluated real-time analysis that combine Groton autosamplers and various analytical instruments.

Groton Biosystems says that its Automated Reactor Sampling System facilitates online HPLC analysis, cell viability, and nutrient monitoring.

Process optimization is the principal driver behind greater use of techniques like LC and LC/MS for peptide mapping and intact protein analysis, says Jeff Mazzeo, Ph.D., biopharmaceutical business director at Waters. This idea also ties in with Quality by Design, which seeks to control critical post-translational modifications through process parameters.

“We’re seeing that users want to use mass spec more and more for protein characterization, even during the later stages of process development,” Dr. Mazzeo adds. These customers generate LC/MS datasets easily, but spend an enormous amount of time analyzing, annotating, and reporting that data. Waters has continuously upgraded its LC and LC/MS software for automating information-creation from raw data. Most notable is the latest iteration of BiopharmaLynx software.

The company has similarly been focusing on systems for faster, higher-resolution, higher-sensitivity peptide maps and extended its branded UPLC product line to intact protein separations, including the introduction of the Acquity® UPLC BEH Glycan, a new UPLC column for glycan analysis.

Regulators have recently been emphasizing host-cell protein (HCP) analysis. Traditionally bioprocessors obtain a number quantifying HCPs generally through immunoprecipitation with polyclonal antibodies. The problem with this approach is a lack of discrimination between benign and immunogenic HCPs. Waters has applied its proteomics technologies to this problem, which allow picking out and quantifying specific HCPs.

One such technique is single-dimension LC/MS. Waters recently introduced a two-dimensional technique based on its nanoAcquity nanoscale UPLC system. Normally, 2-D LC/MS on low-abundance proteins uses ion exchange in the first dimension and reverse phase (RP) in the second.

Waters’ method employs high-pH RP followed by low-pH RP, which provides orthogonal separations, which are sought after in 2-D methods. The benefit is the higher resolving power of RP vs. ion exchange.

One does not normally consider nanoscale methods where sample is not limited, but here the advantage is to exploit instrument sensitivity to overcome the huge concentration dynamic range differences between the therapeutic protein of interest and trace HCPs. Dr. Mazzeo claims a sensitivity of 20–30 ppm. “We’ve seen tremendous interest for this system from customers,” he says.

The new Waters® Acquity® UPLC BEH Glycan Column is used for the identification and quantification of the complete glycan profile of biopharmaceutical proteins.

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