June 15, 2016 (Vol. 36, No. 12)

Kathy Liszewski

Before Any Biopharma Claims Can Be Staked, Tools and Techniques Need To Be Optimized For Biotherapeutic Recovery

In the early 1900s, a Russian-Italian botanist named Mikhail Semyonovich Tswett was investigating plant pigments when he hit upon a curiously effective separation technique.

At the time, just two plant pigments were known, but several more came to light after Tswett’s ministrations. He used a mixture of ether and ethanol to extract pigments from ground-up plants, and he decanted the resulting tinctures to a vertical glass tube that contained powdered calcium carbonate.

As the pigment-laden mixture, the mobile phase, was washed downward through the calcium carbonate, the immobile phase, as many as 10 bands of color appeared. Tswett surmised that each band corresponded to a different pigment, and that each pigment passed through the immobile phase at a different rate, presumably because the immobile phase was more or less adsorbent with respect to the different pigments.

When Tswett discovered this phenomenon—separation by means of differential adsorption—he called it “chromatography,” after the Greek words meaning color and writing. Chromatography has become more sophisticated since then. For example, demanding chromatography applications have become commonplace in the biotechnology and pharmaceutical industries.

Although it is a mainstay of many laboratories, chromatography continues to be refined. It was an important part of the program at the recent Pittcon Conference and Expo, which took place in Atlanta. Pittcon’s chromatography coverage included the selection of chromatographic media, the fine-tuning purity analysis, and the rigorous identification of harmful genotoxins. Chromatography, Pittcon’s presenters made clear, can still become much more efficient and economical.

Genotoxic Impurities

Genotoxic impurities (GTIs) can develop during many stages of drug development, for example, during synthesis, formulation, or storage. They may represent byproducts or result from degradation. According to Margaret Maziarz, a senior scientist at Waters, GTIs are considered unusually toxic: “They have potential to react with DNA and induce genetic mutation, which may consequently lead to tumor development. Therefore, GTIs must be controlled at low levels to ensure quality of the pharmaceutical product and patient safety.”

Rigorous identification and quantification of these impurities early in the drug development process requires reliable and highly sensitive methods for accurate determination of both drug substance and drug products. “This is one area in particular where leveraging optical detection (UV), mass spectrometry (MS), and ultra-high-performance liquid chromatography (UHPLC)-based technologies can have a big impact,” Maziarz stated. “It’s something Waters and many analytical vendors are working hard to improve.” For their part, Maziarz and colleagues set out to develop a sensitive and robust UHPLC method with dual detection for routine monitoring of genotoxic impurities in pharmaceuticals.

“By closely integrating UV and mass detection using a Waters Acquity QDa detector, we enabled correct peak identification while minimizing the need for multiple standard runs to confirm the identity of peaks by retention times,” Maziarz explained. “Using both UV and MS spectral data, we were able to quickly confirm spectral homogeneity and method specificity of each analyte in the active pharmaceutical ingredient matrix using Empower 3 Software.”

Maziarz asserted that employing mass detection provides greater sensitivity for the identification of low-level GTIs in pharmaceutical samples. Further, she emphasized that high-performance technologies are more accessible than many people realize.

“UHPLC, column chemistries, MS, and informatics have evolved to a point where they are becoming mainstream,” Maziarz said. “In addition, we see an increasing need to transfer methods and technology from older high-performance liquid chromatography (HPLC), MS, and UV instruments to platforms that are capable of analyzing increasingly potent (and low dosage) medicines, combination therapeutics, novel formulations, and biotherapeutics that present an entirely unique set of challenges often while continuing to address the challenges of managing legacy methods.

“These technologies need to be implemented across the product development pipeline. Regulatory agencies expect adoption of new tools to address challenges, such as GTIs, faced in the pharmaceutical industry today and in the future.”

A Waters team integrated UV and mass detection using an Acquity QDa detector to enable correct peak identification. The team reported that this approach also minimizes the need for multiple standard runs to confirm the identity of peaks by retention times.

Methods Transfer

The transfer of methods from conventional HPLC to UHPLC can present significant challenges. At Agilent Technologies, Gregory Hunlen, an applications engineer, and Michael Woodman, an application scientist, reported that they are tackling this problem head on. “We are observing an acceleration of interest in this transition,” Hunlen said. “We wanted to demonstrate how emulation technology may be used for bridging the gap between performances in the multivendor LC environment of the laboratory.”

The notion of having one powerful UHPLC system that can perform like the varieties of other systems in our laboratories is a unique concept, suggested Hunlen. “Agilent introduced Intelligent System Emulation Technology (ISET) in 2011,” he continued. “This allows the 1290 Infinity LC to be a truly universal system capable of duplicating the chromatographic results of Agilent and non-Agilent LC systems without having to make method or instrument changes.

“The important point to make is that not only delay volumes are being emulated but the complete mixing behavior of the targeted system.”

According to Hunlen, the Agilent system was challenged to perform a gradient method transfer from HPLC to UHPLC where different column geometries were explored and modifications of method parameters were necessary. “The investigation was performed on the Agilent 1290 Infinity II LC system in emulation mode to duplicate results from the targeted system, an Agilent 1200 Series RRLC,” he summarized. “The basic chromatographic calculations for injection volumes, flow rates, and gradient slope were simplified by using the Agilent Method Translator.

“The comparative analysis, using the same chromatographic conditions on the two systems, resulted in excellent agreement in chromatography and compound retention times. This means all operations took place on just one system—running the legacy method, optimizing the new method, and finally, running the new UHPLC method.  Intelligent System Emulation Technology allows system adaptability for nearly universal instrument to instrument transfer methods.”

When the Agilent 1290 Infinity II LC is augmented with Intelligent System Emulation Technology (ISET), it can emulate other systems and achieve a transfer of methods between LCs, regardless of brand.

Media Selection

Different phases in the processing of a biopharmaceutical product require specific types of chromatographic media. Carefully selecting the best type can help shorten development time and reduce overall cost.

Takashi Sato, deputy manager of sales and marketing at YMC, said that the development of efficient, economical, and selective separation methods is especially important for intermediate purification and polishing steps. When these steps are carried out, high-resolution ion exchange chromatography is often used. A key factor, he noted, is salt concentration.

“Normally, a scientist must first include a desalting step in processing,” Sato continued. “But this can also lead to loss of sample and an increase in the time needed for processing. Further, most current types of commercially available products have low mechanical stability and lower binding capacity/resolution when the flow rate and salt concentration are increased.”

YMC has developed and introduced a novel high-binding-capacity media that can tolerate high salt effectively, thus eliminating the need for a desalting step. “Our BioPro media with 10 and 30 micron particles is designed to have an optimal degree of cross-linkage,” he pointed out. “This provides maximum rigidity to tolerate high flow rates and maintain as large a surface area as possible. The new media has a balanced mechanical stability, binding capacity, and separation ability. Also, a sample that contains high salt can be directly loaded onto the resin.”

The size of the particle utilized is also important. According to Sato, small particle resins in the range of 10–30 microns can more efficiently isolate impurities. “Smaller particles are widely used in high-throughput purifications,” he explained, “because they suppress interparticle sample dispersion and thus provide better resolution.”

Sato recommends scientists carefully consider their media selection especially with regard to tolerability of salt concentrations. “High-binding-capacity media that can handle higher salt without reducing the flow rate,” he concluded, “will increase productivity.”

Novel Amino Acid Analysis

Amino acid analysis is used for identifying and determining the purity of complex and simple biopharmaceutical active ingredients (APIs) and excipients. Appropriate tests are governed by regulatory agencies such as the United States Pharmacopeial Convention, the European Pharmacopoeia, the Indian Pharmacopoeia, and the Japanese Pharmacopoeia.

“Since these pharmacopoeias outline the analytical amino acid methods that can be used,” reported Natalia Belikova, Ph.D., analytical services director, SGS Life Science Services, “our goal was to develop a liquid chromatography method for simultaneous determination of ~20 commonly present amino acids in simple and complex mixtures that complied with requirements.”

Dr. Belikova said that her approach differs from using traditional thin-layer chromatography, which does not allow analysis of multiple amino acid analysis in complex mixtures. “Our method,” she detailed, “employs an ion-exchange HPLC system with a solvent delivery system, an autosampler, a dual wavelength detector, and data collection module.”

“Three mobile phases with different pH levels (Mobile Phases A, B, and C), a mobile phase that contained a regeneration solution (Mobile Phase D), and a shared column temperature gradient program were used in order to separate amino acids in complex mixtures,” she continued. “After separation, the eluent underwent post-column ninhydrin derivatization with subsequent detection at 570 nm and 440 nm for amino and imino acids, respectively (Pinnacle PCX Post-Column Derivatization Instrument).”

According to Dr. Belikova, the SGS method was optimized to comply with system suitability requirements per compendial monographs. Further, the SGS method provides several benefits.

“The post-column ninhydrin derivatization step has a big advantage over pre-column derivatizations in that it provides a much more complete reaction and thus more accurate quantification especially when the main API peak is in excess compared to the quantity of impurities present,” she asserted. “Also, the method does not require use of relatively expensive compendial standards.

“For example, per European Pharmacopoeia compendia, the quantitation of ninhydrin-positive compounds present in the amino acid sample is always performed relative to the original sample preparation. It does not require European Pharmacopoeia standards.”

Mobile-Phase Effects

Optimizing conditions for the separation of proteins, in particular, monoclonal antibodies (mAbs) during development or biomarker research, is like aiming at a moving target. During chromatographic purification, nonspecific protein adsorption onto an HPLC column can affect protein recovery, peak shape, and resolution.

“Monoclonal antibodies are much more complex molecules than traditional drugs,” advised Hillel K. Brandes, Ph.D., supervisor, principal R&D chemist, Analytical Biomacromolecule Separations, MilliporeSigma. “They require several different analytical approaches for structural and chemical characterization, be it in the discovery stage or monitoring of production processes or in quality control.”

Dr. Brandes and colleagues examined the effect of secondary mobile-phase modifiers on protein recovery and performance as a function of temperature using reversed-phase chromatography of mAbs. He utilized low levels of small primary alcohols.

“To get more complete recovery and improve chromatographic performance, a mobile phase must be selected that reduces the interaction between protein and the accessible surface area of the column,” Dr. Brandes explained. “Our overall approach was to examine the effects of secondary mobile-phase modifiers. We compared 1-butanol with 1-propanol, 2-butanol, and 2,4-butanediol as a function of temperature.”

Dr. Brandes’ studies found some, but not all aliphatic primary alcohols could dramatically reduce the temperature needed for optimum recovery and chromatography of mAbs. “We demonstrated that with inclusion of 1-butanol, up to 5% in the mobile phase can reduce the optimum temperature by about 20°C,” he summarized. “However, we also observed that 1-propanol wasn’t nearly as effective as predicted, and that 2-butanol was not as effective as 1-butanol.

“These findings were remarkable—not anything we would have predicted. To our greatest surprise, 2,4-butanediol was virtually ineffective! I believe the results of our study call into question the proposed explanation of the mechanism of the how 1-butanol positively affects the reversed-phase chromatography of mAbs.”

According to Dr. Brandes, the take-home message of his studies is that the mechanisms of how mobile-phase modifiers operate are not entirely straightforward. “My advice to researchers working on difficult chromatographic issues with biotherapeutics is to be diligent about keeping up with the literature,” he insisted. “Stay in touch with column manufacturers and inquire about any insights or work on collaborative solutions. And think outside the box!”

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