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

Gail Dutton

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.