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Feb 15, 2008 (Vol. 28, No. 4)

LC Applications Are Expanding

Range of Approaches in Life Sciences to Be Addressed at “Pittcon 2008”

  • A tremendously useful tool for characterizing biotherapeutics is LC/MS of intact mAbs. Scott Berger, Ph.D., a principal scientist in the biopharmaceutical sciences group at Waters, will tackle this topic at the annual “Pittcon” conference in New Orleans next month. A number of other speakers will also focus on LC/MS applications in the life sciences.

    Possible only since the early 1990s, MS analysis of intact proteins was enabled by the introduction of soft electrospray ionization methods. Researchers now recognize the wealth of information available through intact protein mass determinations, which provides a holistic view of the molecule and every transformation it has undergone.
    “The mass represents the molecule’s entire history,” notes Dr. Berger, “including oxidations, cleavages, and other post-translational modifications. All these modifications contribute to the heterogeneous structure populations reflected in the intact mass spectrum.”

    Dr. Berger will specifically address high-throughput methods of intact protein mass measurements suitable for guiding clone selection during early development. The objectives of this exercise are to uncover a robust cell line that produces proteins reproducibly. Ultimately, one would like to convince regulators that process-control understanding has been achieved. “The product doesn’t have to be 100% identical every time,” notes Dr. Berger, “as long as it falls within a specified range of criteria.”

    Waters’ LC/MS method, built around its Acquity UPLC® system and time-of-flight mass spectrometer, involves two gradient methods optimized for intact and reduced mAbs that permit rapid desalting and reproducible characterization of antibody structures. Data is processed using Waters’ BiopharmaLynx™ software, which compares intact protein data and automatically assigns deconvoluted masses to proteins and variants.

    Getting Small—Really Small

    Pittcon 2008” will feature close to 100 posters and lectures on microfluidics and nanotechnology, mostly from U.S. universities. Commercial talks include a presentation by Microchip Biotechnologies on sample preparation for microfluidic devices and Micronit Microfluidics, which will provide a tutorial on connecting micro- and nano-analysis to macro-world instrumentation.

    Ronny van ‘t Oever, Micronit CTO, will introduce a new connection platform, Fluidic Connect, which solves problems of integration and replacement of microfluidic components in human-accessible instrumentation. The modular connection platform adapts to specific analytical applications depending on the relative importance of pressure, temperature, chemical compatibility, unit cost, and low dead volume for the connection.

    According to Micronit, Fluidic Connect attaches microfluidics to standard pumps, detectors, and valves without the need to purchase new, dedicated instrumentation. Fluidic Connect will attach microfluidic devices like an HPLC or capillary electrophoresis chip to any instrument thereby saving development time and costs for developers of microfluidics-based instrumentation.

    Perhaps the major advantage of the new platform is that integration will not require a high level of engineering know-how. “Experts can easily handle existing microfluidic components, but with Fluidic Connect, anyone walking out of a supermarket will be able to do it,” says van ‘t Oever.

    Micronit’s new microfluidics integration platform serves the company’s business model well. The firm does not sell instruments or systems. Instead it provides analysis chips to companies with access to instrument expertise and markets. “The analogy with computer chips is nearly perfect,” notes van ‘t Oever. “We supply chips and sockets—the interface—to manufacturers and help them engineer these components into instruments.”

    Chromatography on the Extremes

    Frantisek Svec, Ph.D., of Lawrence Berkeley National Laboratory is no stranger to these pages, or to this meeting. At “Pittcon 2008” Dr. Svec will discuss extreme separations using monolithic stationary phases—his pet chromatography platform—from his own work and from the literature. Topics include extremely rapid protein purification and separations at the high extremes of column length.

    Since their discovery in 1968 and their commercial introduction in the late 1980s, silica and polymeric monoliths have quickly shed their academic research reputation and are now found in commercial products in a variety of separation formats (e.g., normal- and reverse-phase and ion exchange). Monoliths are easy to fabricate, readily modified with desirable surface chemistries, and provide rapid separations at relatively low pressure, even at high flow rates, and the columns are virtually indestructible.

    Early monoliths were considered too slow for practical separations. A chromatography run lasts for a long time at a flow rate of 4 mL/hr. That is one extreme that monolith aficionados are happy to leave behind. One of Dr. Svec’s early collaborators BIA Separations has demonstrated the separation of six proteins in five seconds using a monolithic disk. In New Orleans, Dr. Svec will also discuss the separation of five proteins in less than 15 seconds using 10 cm long monolithic column. “That’s the other extreme,” he observes wryly.

    Monoliths can serve extremely slow chromatography as well. One interesting extreme involves an over 12-hour proteomics separation through a long monolithic column of more than 2,400 peptides from a peptide digest of a microorganism. Even more extreme is a 4 m long silica monolithic column with more than a million theoretical plates, which is capable of separating benzene from monodeuterated benzene.

    Monolithic stationary phases are known for their durability as well as resolving power. One of Dr. Svec’s postdocs recently attempted to determine the usable lifetime of a column used in LC/MS mode. After more than 2,500 injections no degradation of performance was observed. The experiment was stopped, because the research was kicked off the mass spec by another group member.

    Dr. Svec’s relationship with BIA began in the early 1990s, when the company licensed monolith technology for manufacturing high-performance chromatography disks. Dr. Svec and colleague Tatiana Tennikova developed this technology while at the Academy of Sciences in Prague. BIA has since run with the monolith idea, commercializing it in several formats including tubular columns with volumes of up to 8 L.

    Monolithic solid phases are extremely flexible in their design potential as well, fitting just about any usable form factor including microtiter plates, syringes, centrifuge tubes, open columns, and disks. “Because the material behaves the same no matter how it’s shaped, performance is identical at every scale,” Dr. Glover claims.

    Monolithic columns’ capacity, production-worthiness, and scalability make them extremely attractive to bioprocessors. What works at analytical scale, pretty much works with multiliter columns. Dr. Glover says BIA monoliths are currently used in two commercial processes: for plasma DNA purification and a Phase I clinical-stage influenza vaccine. “We expect two other cGMP processes to adopt monoliths in 2008,” he reports.

    Against the Grain

    Every chromatographer is familiar with conventional ion-exchange chromatography using a predominantly aqueous mobile phase: negatively charged proteins separated through positively charged resin chemistries or vice versa. It is less well known that using a mostly organic mobile phase, say 65% acetonitrile, even species with the same charge as the stationary phase are held up due to hydrophilic interactions. This phenomenon, known as electrostatic repulsion hydrophilic interaction chromatography (ERLIC), will be addressed by Andrew Alpert, Ph.D., president of PolyLC.

    “ERLIC is like having an immobilized salt gradient on an ion-exchange column,” says Dr. Alpert. “It provides separations you can’t get any other way, as with the isocratic separation of nucleotides.” The obvious application here is the selective isolation of tryptic phosphopeptides for proteomics. A paper introducing ERLIC mode was published online in Analytical Chemistry late last year.

    When analyte species differ significantly in charge, repulsion effects can cause species that would otherwise be retained to elute. ERLIC thus permits the high-resolution separation in isocratic mode of complex mixtures that normally require gradients; for example, peptides, amino acids, nucleotides nucleic acids, and phosphopeptides.

    Elution is a function of both hydrophilic and electrostatic effects. What’s happening here is that at high organic solvent concentrations, hydrophilic interactions overwhelm the normally determinative electrostatic interactions between analyte and column. “This promotes retention whatever the charge of the solute, as long as it’s polar,” explains Dr. Alpert. Strong hydrophilic interactions, which are engendered by high-concentration organic solvent, force the solute to be retained despite electrostatic repulsions.

    PolyLC introduced a related technique, HILIC (hydrophilic interaction chromatography), in 1990. The American Chemical Society’s division of analytical chemistry is hosting a symposium on HILIC at “Pittcon 2008.” Dr. Alpert, who is chairing this group, will introduce ERLIC in the opening talks. Other speakers, from industry and academe, will cover HILIC-mode separations of pharmaceuticals, histones, glycoproteins, and oligosaccharides.

    Ion Analysis by LC

    Measuring inorganic ion impurities or constituents in pharmaceutical samples can be challenging. Conductivity-based ion chromatography works, but the instrumentation and reagents are not commonly available at biotech companies.
    ESA Biosciences’ HPLC marketing director, Darwin Asa, Ph.D., will present data on inorganic ion quantification using his company’s Corona® CAD® (charged aerosol detector) downstream of a HILIC-style HPLC column.

    According to Dr. Asa the combination simplifies ion analysis to a robust chromatography method that any lab with HPLC expertise can run. “And the data is much better, less costly, and simpler to acquire than with ion chromatography systems and methods.”

    ESA calls the CAD detector universal for its ability to quantify proteins, peptides, lipids, carbohydrates, and small molecules in the same analytical run. In charged-aerosol detection the mobile phase coming off the column is nebulized into small droplets and evaporated, leaving the analyte particles behind. Those particles are then charged by ionized nitrogen gas in the CAD and measured. Applications include testing for impurities, stability, product characterization, extractables/leachables, cleaning validation, and QA/QC.

    Numerous software vendors will present posters and talks at “Pittcon 2008.” Covering them all would be impossible, but we would like to point out one that takes a universalist approach to analytical informatics not bound by any particular method or instrument vendor.

    Structure-based analytical software developer Advanced Chemistry Development Labs (ACD/Labs) will showcase its programs for a range of applications and hardware through presentations on chiral HPLC, automated chromatography, IR and Raman spectroscopy, and isotope methods.

    The core components of ACD/Labs’ products are application-specific processors or kernels, and a database that interfaces with a structure-drawing program. A package for LC/MS, for example, would consist of a generic chromatography processor and an MS processor. Data imports from any instrument provided it is available in generic format.

    One of the company’s talks, on quantification of isotope ratios using LC/MS, will introduce a novel isotope software filter that isolates the peaks of interest such as 14C/13C in metabolism studies without the use of the control sample as a comparator. The technique also works with stable chlorine- or sulfur-containing compounds due to the high abundance of stable isotopes for those elements.

    The key to success in the analytical software business, says product manager, Graham McGibbon, Ph.D., is to be vendor friendly as opposed to vendor neutral. In other words, to serve as many instrument bases as possible. “We’re not neutral about anything,” he quips. “We favor working with all vendors.”



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