March 15, 2011 (Vol. 31, No. 6)

Streamlined Methods Stray from Classic Hosts to Produce Higher Yields in Fewer Steps

The challenge to produce more protein faster is leading to the development of new, engineered cell hosts and more tightly targeted ways of introducing genetic material to those hosts, as well as strategies that exert more precise control even at microtiter scale, according to speakers at the “Recombinant Protein Production” conference held in Vienna last month.

These advances are taking recombinant protein-expression systems beyond their traditional bacterial hosts to new, streamlined production systems that yield large quantities of high-quality protein faster and with fewer steps. The result is a more economical production system that also may help speed the overall project toward commercialization.

Oxyrane has developed a system to express biologics in the glyco-engineered yeast Yarrowia lipolytica. Wouter Vervecken, Ph.D., head of molecular biology, explained that the system was developed to express antibodies and lysosomal proteins with the correct glycosylation. “The latter are used for enzyme-replacement therapy,” he said. “The glycans on lysosomal proteins are important for targeting locations (lysosomes) within cells.”

“Typically, lysosomal proteins are produced in CHO cells,” which have many challenges and a low yield. “Perhaps 5% produce particles with the desired glycan structure,” Dr. Vervecken said. Using Oxyrane’s approach, more than 85% of the particles produced have the desired glycan structure. And, because they are produced in yeast, they avoid many of the issues associated with mammalian cell-based protein production.

“The benefits are improved yields and timelines, and decreased capital costs. These lysosomal proteins also outperform those that come out of other systems. We’re aiming for clinical benefit.” At dosages of 20 mg/kg of body weight, “we hope to decrease doses and complications.” He claimed that by precisely targeting a specific area of a cell, dosages can be reduced along with side effects, infusion time, and costs.

Preclinical work is promising, showing no significant problems, according to Dr. Vervecken. “We hope to have this in the clinic by early 2013.”

Engineered Strains

c-LEcta is engineering strains of Bacillus, Escherichia coli, and Pichia pastoris to produce enzymes that are superior to those produced using traditional strains, according to Stefan Schönert, Ph.D., head of strain and process development. Target proteins can be either produced in the cytoplasm or secreted into growth media.

Some of the latest work produces the Serratia nuclease enzyme from Serratia marcescens. That enzyme is used to remove all nucleic acids and thereby reduce viscosity during vaccine and biopharmaceutical production.

Engineered strains are used, Dr. Schönert explained, because they “have reduced extracellular protease activity and an enhanced secretion capability” compared to traditional strains. Therefore, “they are being used in bioreactors for the extracellular production of enzymes,” he said. They have a production rate of up to 10 grams per liter.

“The strains were produced by chemical and targeted mutagenesis. In the first round, a Bacillus strain was treated with chemicals, and a strain with a better secretion rate was selected. Finally, some proteases were disrupted by double homologous recombination.”

Glucose Control

By controlling the amount of glucose present over time, BioSilta’s EnBase cell cultivation kit has achieved “a 20-fold increase in cell density and a 10-fold increase in protein production compared to standard lab cultivations,” according to Craig Fuller, Ph.D., business development and product manager.

The system is composed of two elements—nutrients dissolved in the liquid medium containing both the substrate and salts to support growth, and the enzyme, which is the biocatalyst responsible for releasing the glucose in a controlled manner. Precisely controlling the release of glucose avoids the problems of over- or underfed bacteria, which, in turn, affects the oxygenation transfer rate, Dr. Fuller said.

EnBase can be utilized in volumes ranging from 0.15 mL in 96-well plates to liters used in single-use bioreactors. It is especially beneficial when working with small volumes, Dr. Fuller said, where optimal control of glucose levels in the milliliter scale becomes challenging.

Three versions of the product are available. The original EnBase was gel-based, and the polysaccharide was embedded in the gel layer. In that version, the enzymes are added to the liquid media and facilitate the degradation of the polysaccharide, thus releasing glucose over time.

Today, the most widely used EnBase product is a liquid media. “It requires users only to add the bacteria and enzymes and incubate the solution. Customers then can regulate the amount of glucose simply by adding additional enzymes.”

The most recent version, introduced last autumn, is a tablet designed for 50 mL cultures. “Drop it in sterile water and add bacteria and an enzyme.” The various EnBase versions are effective in temperature ranges from about 10°C to 42°C.

Site-Specific Integration

Regeneron has developed a site-specific integration cell-line platform with optimized host cell, vector, and medium to maximize cell productivity from single-gene cell lines, according to James Fandl, Ph.D., vp, protein-expression sciences. Dr. Fandl explained that traditional methods of causing a cell line to produce enough protein to conduct an experiment are imprecise, and the results are generally not predictable.

“When using random integration methods, for example, the genetic material integrates into different locations in the cell genome each time, producing different results that are neither predictable nor reproducible.” In contrast, a site-specific cell line is both predictable and reproducible because the genetic material is inserted precisely at the same spot each time. “A site-specific cell line is easy to make, reproducible, and efficient,” he said, noting that a single researcher can produce more than 20 cell lines within four to six weeks.

Regeneron’s more targeted approach uses site-specific recombination sites—flp/frt or cre/lox, for example—inserted into a transcriptional hotspot. Regeneron has had consistent results in its decade of experience with site-specific cell-line development.

Isolating site-specific integrants by flow cytometry further increases throughput, and bioreactor titers currently range from 1.5 to 2.5 g/L, Dr. Fandl said. “We can, therefore, screen a lot more antibodies up front, purified from what will become the production cell line. That saves a tremendous amount of time.”

Regeneron has developed a site-specific integration cell-line platform with optimized host cell, vector, and medium to maximize cell productivity from single-gene cell lines.

Streamlined Production

Ajinomoto’s Corynex™ expression system is a gram-positive, fast-growing soil bacterium that produces a high yield of recombinant protein secreted directly into the culture supernatant. The process is streamlined and generates fewer impurities than the traditional E. coli production system, according to the company.

“Many experiments that hardly produced protein in many traditional methods could be efficiently secreted in the Corynex,” reported staff scientist Yoshimi Kikuchi, Ph.D. “Furthermore, a protein that has complex structure, such as many disulfide bonds, homodimer, heterodimer, and multimer structures could be secreted as correctly folded structures possessing biological activity.” Therefore, refolding is not required.

Dr. Kikuchi said the purification process is simple. “Since there are minimal amounts of original secreted proteins in the culture supernatant of Corynebacterium glutamicum, the purity of the target protein secreted in the Corynex is very high.” Purification requires only six steps, according to Ajinomoto’s literature, whereas the production process using E. coli listed 12 steps.

As an example, Dr. Kikuchi elaborated, human IGF-1 could be produced efficiently using either the Corynex system or E. coli as a host strain. Using Corynex, it is produced in its correctly folded form and purified in only one-step chromatography.

“Using E. coli as the host strain, IGF-1 accumulates in the cytoplasm as an inclusion body, for which a re-folding process is required to obtain the active form. Furthermore, the re-folded IGF-1 is purified in four-step chromatography.”

Tests, in which living MCF-7 cells were measured four days after being stimulated by human epidermal growth factor produced naturally or by the Corynex system, showed nearly identical results.

Ajinomoto’s Corynex expression system harnesses the benefits of Corynebacterium to express active, correctly folded proteins directly into the medium.

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