February 15, 2015 (Vol. 35, No. 4)

Engineers Are Retooling To Keep Up With Surging Demand

The field of protein engineering is booming. It represents a market that is projected to reach $168 billion by 2017.

This projection comes from a report produced by MarketsandMarkets, which divides the protein engineering market into three application areas: biotherapeutics, diagnostics, and research. Relevant technologies cited in the report include sequence modification/glycosylation, pegylation, display technologies, humanization technologies, hybrid technologies, and transgenic mice.

Similar issues were discussed at two recent conferences, CHI’s PepTalk (held in San Diego) and GTCbio’s Protein Discovery Summit (held in Boston). These events featured cutting-edge research and highlighted current challenges and evolving solutions in the field. Presentations included novel ways of utilizing mass spectrometry, new strategies to engineer and express proteins in yeast and baculovirus systems, how to better validate biosimilar drugs, and improved instrumentation to assess protein aggregation.

The ability to characterize proteins from initial sequence to final conformation is critically important for delineating the safety and efficacy of protein drugs. “Biological mass spectrometry (MS) provides a variety of approaches to study structural properties not only at the quality control step, but also throughout the process of discovery and design,” noted Igor A. Kaltashov, Ph.D., professor and graduate program director, department of chemistry, University of Massachusetts Amherst.

An indispensable tool of molecular biophysics, MS stands out for characterizing the higher order structure of protein. “The amazing success enjoyed by MS is largely because of the introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). ESI-MS is particularly well suited for characterizing intact proteins. Despite this, the technique has been much less applied to pharmaceutical work. But that is beginning to change.”

According to Dr. Kaltashov, MS is enjoying dramatic growth in popularity as a tool for assessing conformation and dynamics of protein drugs. “Although one of the shortcomings of ESI-MS is that studies need to be done in solutions compatible with the overall process (often volatile electrolytes such as ammonium acetate or ammonium bicarbonate), there are other approaches.

“For example, when the use of ‘ESI-friendly’ solvent systems is not appropriate, one can use hydrogen/deuterium exchange (HDX) coupled with MS, which provides an elegant way to assess conformation and stability by targeting labile hydrogen atoms.”

Many labile hydrogen atoms, however, do not readily undergo HDX. Some of these recalcitrant hydrogen atoms are involved in hydrogen bonding; others are sequestered in the protein interior. Still, they may be amenable to other approaches.

One such approach, suggested Dr. Kaltashov, is a simple coupling of technologies: “Native ESI-MS provides a convenient way to assess protein-ligand interaction (such as protein drugs with their therapeutic targets). When combined with size-exclusion chromatography, it provides an elegant means for characterizing quaternary structure of complex proteins. There is no ‘one size fits all’ when it comes to MS.”

Dr. Kaltashov proposed that MS is steadily making its way into mainstream pharma. “MS-based methods are being increasingly applied to examine the pharmacokinetics of biopharmaceutical products,” he said. “Although traditionally done using immunoassays, MS can reveal details that cannot be derived by other experimental techniques. As we solve issues of enhancing throughput and assuring a robust, sensitive technique for quantification, MS will continue to advance by leaps and bounds.”

Mass spectroscopy plays a critical role in characterizing the higher order structure of proteins. [angelblue1/iStock]

Assessing Biosimilars

As multibillion dollar blockbuster and other drugs come off patent, the scramble to produce biosimilars ensues. “Biosimilars are one of the fastest growing segments of the pharmaceutical industry today,” noted Paul Belcher, Ph.D., market development leader, GE Healthcare Life Sciences.

As opposed to generics, which are copycat versions of small molecule drugs, biosimilars refer to biological drugs (such as therapeutic antibodies) that are, according to the FDA, “highly similar” to the originator molecule. However, “the process to make the innovator molecule is often unknown and differences in attributes like glycosylation can give structural and functional differences.”

To confirm biosimilarity to the originator molecule, Dr. Belcher said the more technologies employed to evaluate it, the better: “The focus is on extended characterization efforts to show similarity to the reference medicine. Characterization and quality control can be significantly improved with robust, information-rich analytics like surface plasmon resonance that provide more data, more rapidly or in real time.”

To illustrate the high-throughput process development of a biosimilar molecule, Dr. Belcher cited the example of interferon alpha-2a. Comparability studies of this biosimilar relied on a molecular interaction analysis system, the Biacore™ T200, and a new, fully integrated Western blot platform, the Amersham™ WB.

“Biacore calibration-free concentration analysis was used to find the optimal refolding conditions by parallel monitoring of folded and unfolded protein. It was also used to select the most appropriate chromatographic media and conditions during process development,” Dr. Belcher said. “Finally, kinetic characterization was used to assess comparability to the originator molecule.”

Purity is another key attribute in this process. For that, Dr. Belcher’s team employed the Amersham WB system.

“We needed to assess purity and confirm the identity of the biosimilar in the many different purification steps,” explained Dr. Belcher. “This system is fully integrated and standardizes electrophoresis, scanning, transfer, and probing with minimal assay variability allowing the reproducible assessment of purity of the biosimilar throughout process development as well as confirmation of the identity.”

Dr. Belcher observed that in this very competitive market, the design of the production process can impact on the ability to produce the drug at a competitive price and techniques such as surface plasmon resonance and modern chromatography media, “can give an all-important competitive edge by providing fast and reliable information on product quality, helping save time and lower production cost.”

“Awesome Power of Yeast”

Transmembrane proteins play critical roles such as serving as transporters and receptors that transduce the binding of extracellular ligands into intracellular signals. They constitute 20–30% of the protein coding potential of most genomes. But, transmembrane proteins also present challenges for characterizing and analysis related to expression, solubilization, and purification, especially if derived from eukaryotes.

“The difficulties encountered in working with transmembrane proteins constitute a significant barrier to fully understanding their mechanisms,” said Mark E. Dumont, Ph.D., professor of biochemistry and biophysics, University of Rochester Medical Center. Dr. Dumont’s laboratory focuses on eukaryotic transmembrane proteins and employs baker’s yeast systems for expression.

“Many crystallographers prefer to express proteins in bacteria,” noted Dr. Dumont. “For transmembrane proteins, however, this approach is often unsuccessful for reasons that are not well understood.

“Additionally, although nearly 100,000 structures of soluble proteins have been solved, there are only about 500 structures of transmembrane proteins and a few dozen structures of transmembrane proteins from eukaryotic organisms. We focus on expression in yeast. They grow very quickly, provide good expression, are economical, and share many of the genes from humans.”

As an example, Dr. Dumont’s team, working with collaborators in the laboratories of Michael Wiener, Ph.D., from the University of Virginia, and Michael Malkowski, Ph.D., from the Hauptman Woodward Medical Research Institute, in Buffalo, solved the three-dimensional crystal structure of Ste24p, an integral membrane protein—more particularly, a CAAX protease. Ste24p is a homolog of the human zinc metalloprotease ZMPSTE24.

These proteases are critically involved in the post-translational maturation of the yeast mating pheromone a-factor and human lamin A, respectively. Mutations that affect proteolytic cleavage by ZMPSTE24 in humans lead to premature aging disorders in children, such as Hutchinson–Guilford Progeria Syndrome.

“In our project, which is funded by the Protein Structure Initiative of the National Institute for General Medical Sciences, we employed 20 different yeast strains for this work and found one, Saccharomyces mikatae, that worked the best to provide a protein we could crystallize,” explained Dr. Dumont. “Screening of membrane proteins derived from different strains of yeast can help determine the most suitable one for subsequent studies.”

For the future, Dr. Dumont said that he expects to continue employing the “awesome power of yeast!” He added some of the severe side-effects of HIV protease inhibitors may arise from off-target effects of the drugs on the CAAX protease. “We hope to engineer yeast so that they more exactly replicate expression in humans for this and other projects.”

Prenylated Protein Challenges

Many proteins considered important by pharmaceutical companies are prenylated in vivo. This type of post-translational modification adds lipid-like, hydrophobic molecules to the carboxyl terminus to facilitate attachment to cell membranes.

What’s straightforward for Mother Nature has proven a challenge to replicate in some artificially produced systems. “Expression and isolation of homogenous recombinant proteins can be very difficult because of the heterogeneous nature of post-translational processing,” said William K. Gillette, Ph.D., senior scientist, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research.

Dr. Gillette’s work is part of a project at the Frederick National Laboratory, funded by the National Cancer Institute, to study the RAS family, a large group of GTPases involved in growth, differentiation, and survival. “We focus on KRAS4B, a key player involved in the signal transduction pathways that goes haywire in cancer,” Dr. Gillette pointed out. “Our interest is to develop a system for the expression and purification of large amounts of this protein for further study. We are an open-source player that collaborates worldwide with academia and industry in this area.”

Although expression in baculoviral systems may not always recapitulate post-translational modifications produced by human cells, Dr. Gillette’s team engineered the system and succeeded. “We modified the viral genome to co-express specific human enzymes that could achieve prenylation of the protein, recalled Dr. Gillette. “We were pleasantly surprised by the results when mass spectrometry and other analyses verified that prenylation of the protein was exactly the same as in humans. Further, we could purify milligram quantities.”

The group will now work to optimize the system and enhance efficiency. According to Dr. Gillette, such yield and purity from a baculoviral system is new to the field.

“It’s rather exciting as it demonstrates the usefulness of this system as an alternative source, in addition to the standard Chinese hamster ovary and human embryonic kidney 293 cells, for making human enzymes,” asserted Dr. Gillette. “With the high-yield expression in insect cells coupled with today’s ability to easily modify genomes, the baculovirus system presents a very useful tool.”

Protein Aggregation

Regardless of which system is utilized to express proteins, one key issue in the final product is aggregation. The significant presence of different forms of aggregates may influence not only quality, but also immunogenicity, adversely affecting patients.

“The amount and size of aggregates, if excessive, can wreak havoc on biological therapeutics,” said David F. Nicoli, Ph.D., vice president of research and development, Particle Sizing Systems. “Our company develops strategies to better quantify multimeric aggregates in biopharmaceuticals.”

One strategy is the company’s focused beam, light extinction/light scattering technology. According to Dr. Nicoli, this technology—single particle optical sizing (SPOS)—provides high sensitivity and the highest (single-particle) resolution for detecting the concentration and size distribution of aggregates of proteins and other macromolecules over a very large size range.

“We initially developed a new version of SPOS, based on combining the existing, traditional methods of light extinction (LE) and light scattering (LS),” explained Dr. Nicoli. “The resulting ‘LE + LS’ combined signal allows particles over a wide size range, 0.5 to 400 microns, to be counted and sized.”

Initially, the technology presented a signal disadvantage: the need to dilute, often extensively, the starting sample. This difficulty, however, was lessened by subsequent development.

“We introduced a radical change in SPOS technology by developing a focused laser beam sensor, first based on focused extinction (FX) and then based on focused scattering (FX-Nano),” pointed out Dr. Nicoli. “This focused-beam approach helps solve two problems.

“First, it provides higher sensitivity (lower size limit) of particle detection, down to 0.6 microns for FX and 0.15 microns for FX-Nano. Second, it requires much less dilution of the starting sample. The resulting size distributions have much better statistical accuracy than results obtained from other counting methods, such as nanoparticle tracking analysis, resonant mass measurement, resistive pulse sensing, and microfluid imaging.”

For the future, the company plans to continue refining and optimizing instrumentation to drive particle aggregate detection to even lower size levels and higher concentrations.

The AccuSizer FX Nano SIS system from Particle Sizing Systems is specifically designed to measure protein aggregation. The system consists of the SIS sampler, modified to be able to accurately deliver sample volumes as small as 250 µL, and two sensors, the LE400 and FX Nano sensors. These sensors can quantify the amount and size of protein aggregates from 0.15 to 200 microns.

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