An alternative to UHPLC columns are monolithic columns, so-called because they are made from a single piece of porous material.
Because of the manufacturing process, Merck KGaA’s monolithic silica columns are surrounded by polymeric polyether ether ketone (PEEK), and cannot withstand the same kind of back pressures as traditional stainless steel particle packed columns can. But that doesn’t seem to be a problem since they possess a high permeability and are designed to yield fast, high efficiency separations using “conventional low pressure and cheap HPLC systems,” says Karin Cabrera, Ph.D., head of R&D for analytical chromatography at Merck Millipore.
In monolithic columns, it is the size of the through-pores (macropores) that determines permeability, while the mesopore size determines the surface area of the material and thus the separation efficiency. “We can independently design the macropore size and the mesopore size,” extolls Dr. Cabrera.
This is not possible with the classical particle packed column, where the interstitial volume corresponds to the macropore size, she continues. Smaller particles yield a smaller interstitial volume and require a correspondingly higher back pressure to operate. Yet separation efficiency is dependent on the particle size as well.
As there are no frits to clog, “dirty” samples can be injected directly on to monolith columns without the need to filter them. In addition, because the column is a single rigid piece, it has been shown to last much longer than packed columns, which are subject to shifting and cracking over time.
The new Chromolith HR columns, with a 1.1 µm macropore size, can operate at 3.5 mL/min at a back pressure of only 200 bar, with a measured efficiency surpassing that of a 3.0 µm packed particle column. “We started with 2 µm [macropore] and now with the second generation we have 1.1 µm; now of course we can go down further—to 0.9 µm, 0.8 µm , 0.7 µm, 0.5 µm, etc.,” Dr. Cabrera says, predicting that in the next five years “we will reach efficiencies that cannot be obtained any more with smaller particles.”
Pass the Salt
Ion chromatography (IC) columns use an organic sheathing as well, and thus cannot use ultra-high pressure systems—but for different reasons. “There are no metal parts in IC instrumentation,” points out Yury Agroskin, director of R&D at Thermo Scientific. “We cannot use stainless steel pumps because we use a very broad range of pH—from 0 to 14. Stainless steel material can’t withstand this continuous corrosive environment.”
Yet the same rules still apply—the system’s resolving power is inversely proportional to the particle size. Thermo has introduced the new Thermo Scientific Dionex ICS-5000 Reagent-Free™ HPIC™ system with Eluent Generation, a high-pressure capillary IC system.
It uses columns with 4.0 µm particle size and runs at up to 5,000 PSI. While these numbers seem tame (UHPLC systems can push 20,000 PSI through columns packed with 1.7 µm particles), traditionally IC standard pressure is below 3,000 PSI, with 8–12 µm particles.
The high-pressure system is combined with a 0.4 mm ID capillary column to offer an “always-ready” chromatographic system that can run continuously—literally months and months—without human intervention,” Agroskin noted. “The flow rate is extremely small—we’re talking in the range of 10 µL/min.”
This, he points out, simplifies things for the customers, who don’t need to worry about starting up, equilibrating, and calibrating the system each time they need to use it. It also minimizes the consumption of environmentally unfriendly substances.
Mind the Carbs
Characterizing proteins is an important task of chromatography. And while modern HPLC- and UHPLC-MS techniques have brought us a long way toward solving many of the challenges of working with glycan structures, the heterogeneous nature of glycoproteins continues to cause a bottleneck in the biopharmaceutical industry.
Critical quality attributes of post-translationally added structures may affect the safety and efficacy of the proteins they are found on. Bioactivity, distribution, metabolism, and clearance, for example, may all be dependent on the makeup and placement of sugar trees decorating a protein-based drug.
It’s important to the people in the industry to “make sure they have a consistent process and they get a consistent glycan distribution, not only in terms of those glycan structures that are added to the protein, but the site of that addition as well as the relative amounts or distribution of these,” notes Steven Cohen, Ph.D., RDE life sciences director at Waters. This is especially true for biosimilars, which by definition are produced in a different cell line.
There are three levels at which these structures are typically analyzed, each yielding its own set of information, and all combining to give a fuller characterization of a protein: the intact molecule, which yields no information about where the glycan is attached; glycopeptides; and the glycan itself, released by enzymatic or chemical means, “which allow you to get in-depth information about both the glycan composition and well as distribution among various glycan forms,” Dr. Cohen explains.
He sees the underappreciated in-between stage of glycopeptide analysis—that can show to which peptide the particular glycans are attached—as having greatly benefitted of late by improved separation technologies.
“Although you can get complete analyses in 30 minutes with a UHPLC-type column, those columns allow you to get very good screening information in under 10 minutes. That’s very useful for high-throughput analysis.”