October 1, 2015 (Vol. 35, No. 17)

In Early Drug Discovery it’s Often Unclear Which Recombinant Proteins Will Be Affected by Changing the Host Cell

When drug developers use different cell lines for manufacturing and preclinical research, they risk generating inconsistent results, proteins with various structures and functions. Then, confounded by variability, drug developers may lavish attention on irrelevant candidates and overlook promising candidates. 

To avoid misleading themselves, drug developers must find ways to avoid or account for protein variants, which include post-translational modifications, particularly alternative glycosylations. Such variants occur all too frequently among different host cell lines, an extensive body of literature documents.

“Variability is most evident when comparisons are made between mammalian and nonmammalian cells,” says James Brady, Ph.D., vice president of technical applications and customer support at MaxCyte. “But depending on the protein that is being produced, even different mammalian cell lines, such as HEK and CHO, will exhibit substantial differences in post-translational modifications.” Differences can lead to altered protein stability, activity, or in vivo half-life.

It is often unclear during the early drug discovery process which recombinant proteins will be affected by changing the host cell. However, misleading early-stage data are associated with significant costs and extended timelines. It therefore makes sense to adopt a single host cell for all stages of the development pipeline. That is the rationale behind MaxCyte’s flow electroporation transfection platform.

Large-Scale Electroporation

Chemical transfection based on lipids or polymers are the most common alternatives to electroporation for large-scale transient transfection. However, reagent costs, lot-to-lot reagent variability, scale-up difficulties, and low transfection efficiency with certain cell types often are significant challenges of chemical transfection, particularly in biomanufacturing-relevant cells such as CHO.

Viral transfection vectors are another possibility. “While viral vectors may be more effective than chemical methods for introducing genes into certain difficult-to-transfect cell types, producing viral vectors often requires the development of packaging or producer cell lines,” Dr. Brady explains. “There are also biosafety concerns associated with some viral vectors.”

Unlike stable transfection, transient gene expression does not involve integration of the transgene into the host chromosome. Therefore, influences of the integration site on protein expression levels or other protein attributes are not evident. Rather the host cell’s genetic background, media/feed formulation, and culture conditions are the most significant factors influencing product quality, regardless of whether the protein is produced by stable or transient expression.

While high-end titers for stably transfected cells are now advancing into the low double-digit grams per liter, average titers are still in the lower single digits. Thus, the titers of 2–3 g/L that have recently been reported for transient expression via flow electroporation in nonengineered CHO cells are beginning to rival those of stable cell lines.

“So far, upper limits to titer by stable or transient expression have not been reached,” Dr. Brady tells GEN. “It is likely that innovations in vector design, advances in cell-line engineering, and improvements to cell-culture processes will lead to continued advances in both stable and transient titers.”

Monitoring Expression

Analytical methods are crucial for quantifying not only protein expression but also quality. A group at Fujifilm Diosynth Biotechnologies led by Greg Adams, Ph.D., the company’s director of analytical development, is promoting analytical techniques applicable throughout a molecule’s life cycle.

Depending on the expression system, the Fujifilm Diosynth team focuses mostly on aggregation, glycosylation, and heterogeneity. The team employs a mix of rapid and conventional analyses, for example, mass spectrometry, ultra-performance liquid chromatography (UPLC), glycan analysis with rapid 2-aminobenzamide (2-AB) labeling and normal-phase UPLC, and capillary electrophoresis (CE) techniques such as imaged CE (iCE) and the CE-sodium dodecyl sulfate (CE-SDS) method. “Our objective,” declares Dr. Adams, “is same-day quality attribute analysis for understanding what’s happening in a bioreactor while designing the upstream process.”

Note that all the aforementioned techniques are standard analysis methods. The novelty is the context in which Fujifilm Diosynth uses them. Another distinction is the company’s high-throughput approach. The company uses liquid-handling workstations with pre-loaded tips for culture purification over protein A. The 30–60-minute preparation provides purified, active, concentrated antibody that may be analyzed in a number of ways. “We are able to analyze multiple ambr™ minireactor or 2 L bioreactor samples in hours versus days,” asserts Dr. Adams.

When it is applied to cell-line development, the rapid analysis philosophy holds that the same methods should be used from early development through GMP manufacturing. In practice, this is easier with antibodies because molecules of this class lend themselves to affinity purification and rapid method optimization through design of experiment (DOE), potentially beginning with transfectant pool material.

“Hopefully, we can have a method that we don’t have to change for the lifetime of the program,” Dr. Adams says. “It certainly helps to be able to trace data back through clinical phases and not have to worry about chromatographic profile and column changes. This has been very successful in several programs using the newer techniques, where the development phase is assisted by the speed by which you can run each method.”

The next challenge is to transfer this methodology to products expressed in microbial fermentation, which Dr. Adams refers to as the “next generation” of this approach to analytics.

A scientist at Fujifilm Diosynth Biotechnologies operating an ambr250 mini-bioreactor system from Sartorius Stedim Biotech business unit TAP Biosystems.

Improving Solubility

Escherichia coli became the workhorse of recombinant protein expression because of its simple genetics, ease of culturing, scalability, rapid expression, and prodigious productivity. Negatives include a lack of eukaryotic post-translational machinery, codon usage bias, and difficulty with high-molecular-weight proteins.

Pros and cons must be weighed in terms of the target protein’s intended use. Quality and purity requirements for research-only proteins vary significantly, and may be worlds apart from therapeutic proteins. “The end application dictates to a large degree the choice of expression host, purity requirements, how you design the construct, and which tags to use,” says Keshav Vasanthavada, marketing specialist at GenScript.

A disadvantage in E. coli on par with low expression is insoluble expression, which results in aggregates (inclusion bodies). Researchers can deal with this phenomenon at the process level or molecular level. But before they embark on an improvement project, they should, Vasanthavada advises, check the literature to see if other researchers have produced the target protein in adequate yield and at acceptable quality. If so, it would be worthwhile to look at the other researchers’ methods and see if they can be reproduced.

Process-level strategies, which do not require target reengineering, include changing expression conditions, in vitro protein refolding, switching E. coli strains, adjusting media and buffers, or incorporating chaperone co-expression. Molecular-level approaches involve eliminating undesirable elements through truncations or mutations.

“The easiest approach is adoption of a fusion partner-based strategy,” Vasanthavada tells GEN. “It involves the use of a solubilizing partner upstream of the target protein to enhance target protein solubility.”

While this approach is generally beneficial, it has its drawbacks. For example, while a fusion partner will solubilize the target protein, there is no guarantee that the target protein will remain in solution once the tag is cleaved off. “Sometimes, you cannot ‘cleave off’ the fusion partner. The proteolytic enzyme won’t reach the cleavage site because of interference from itself,” Vasanthavada explains. “On other occasions, your fusion partner will start sticking to your target protein post-cleavage.”

Solving Microbial Expression Problems

Challenges associated with E. coli include lengthy purification, protein aggregation, and inefficient refolding. These drawbacks can be overcome with an expression platform from Ajinomoto Althea. This platform, which is based on the Corynebacterium glutamicum organism, is called Corynex® (for CORYNebacterium EXpression system). It synthesizes and secretes properly folded proteins or peptides directly into the extracellular fermentation broth.

Since properly folded proteins are recovered directly from the broth without cell lysis, host protein secretion is minimized. And since the expressing organism is a gram-positive bacterium, the system lacks endotoxins, which reduces the likelihood of antigenicity and simplifies purification.

Purification of E. coli-expressed proteins typically involves harvest, homogenization, centrifugation, and refolding—and then multiple chromatography and filtration steps. Because the C. glutamicum organism possesses secretory pathways, it expresses properly folded, biologically active proteins directly into the culture medium, thus reducing purification steps and increasing yields.

Up to 80% of recombinant proteins expressed in E. coli are contained in inclusion bodies, which form when the overexpressed recombinant proteins aggregate in an insoluble form in the cytoplasm or periplasm. Extracting biologically active protein from these aggregates reduces yield and is a major driver of production costs in microbial processes.

“The mechanisms driving the aggregation are still poorly understood,” explains Kristin DeFife, Ph.D., vice president of biologics at Ajinomoto Althea. “The evolutionary distance between bacteria and humans may be one factor. For example, prokaryotes do not have the systems for post-translational modification and membrane transport found in mammalian cells. In addition, different cellular microenvironments—pH, osmolarity, redox potential, cofactors, and folding mechanisms—may play a role.”

Uncontrolled Expression

Sam Ellis, vice president, Thomson Instrument, says that his company is bucking the “high control” trend in small-scale cell culture. Thomson has adapted the Erlenmeyer flask in a couple of systems—Ultra Yield™ Flasks (for microbial cultures) and Optimum Growth™ Flasks (for HEK293, CHO, and insect cell cultures).

Ultra-controlled microbioreactors and minibioreactors, Ellis says, are expensive and require the services of highly skilled operators: “If one of these devices goes down, you have to call in a technician, and if your highly trained operator leaves the company, the project shuts down and requires another trained operator. Getting production back up can take copious amounts of time.”

Compared with 125 mL–10 L bioreactors or bags, the flasks (125 mL, 250 mL, 500 mL, 1.6 L, and 5 L) are simple to use, affordable, and require no custom platforms or large amount of training, according to third-party testing conducted at Amgen, (A poster citing Amgen’s results is presented on  Thomson Instruments’ website.)

Ellis explains that industry-leading parallel microbioreactor systems are superb for bioprocess scaledown, but that is not what every cell culture project is about: “Our flasks are optimized for scalable mid-level protein production, but thanks to advances in media and cell biology, they serve a very high percentage of R&D cell culture. Most times people are interested only in whether their transient expression system works. Process manufacturing optimization is at least one step removed from our market.”

R&D settings, Ellis insists, often involve screening thousands of constructs: “You can’t run a thousand controlled vessels at once, but you can do a thousand shake flasks with good media, at good speed, with good aeration.”

In Ellis’ view, uncontrolled cell cultureware is making a comeback because of advances in media, supplementation, and cell-line selection. Rising titers, especially for transient gene expression, have substantially lowered the volumes necessary for generating early preclinical protein. The Amgen data shows the ability to express 40 mg of protein in a 100 mL working volume, a nearly 50-fold improvement over the titers available a decade ago.

“Think of our system as the common persons’ way to produce a lot of protein,” urges Ellis. “You don’t need to be in a well-funded lab, and you don’t need expensive bioprocess equipment. Just be open to improving your growth with good media, the correct Thomson vessel, the correct speed for shaking, and a good clone selection.”

Virus Quantification

The paradoxical impact of viruses as both a health threat and as a tool for addressing a growing list of diseases is accelerating. Our ability to keep pace with new trends in viral research has been limited by the lack of progressive manufacturing technologies capable of supporting the rapid increase in production needs. Most noteworthy has been the absence of real-time, in-line analytical methods.

In response to this unmet need, ViroCyt® has developed a portfolio of reagents enabling immediate, biologically-specific quantification of viruses dramatically faster than traditional methods, according to Michael Artinger, Ph.D. ([email protected]), vp, marketing & strategic partnering.

Marketed under the tradename ViroTag®, this new product category utilizes a fluorescently labeled, high-affinity antibody targeted to the virus of interest. During a one-minute sample analysis, the preparation is evaluated using the Virus Counter® 3100, an instrument developed solely for the purpose of virus quantification.

The first two members of the ViroTag family to be launched target Baculovirus (ViroTag BCVB) and Adenovirus (ViroTag ADVX), selected based upon the significant roles these viruses play in the protein expression and gene therapy fields, respectively.

“Substantial time and cost savings are possible by implementing the technology during the development and manufacture of products created either directly from viruses or indirectly using virus-based systems,” said Dr. Artinger. “These include viral vaccines, virus-based protein expression systems, antiviral drugs, gene delivery vectors, and oncolytic viruses.”

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