April 1, 2008 (Vol. 28, No. 7)

Designing Better Systems to Energize Therapeutic Pipelines
The expression of proteins for characterization, therapeutics, and diagnostics continues to be a challenging and complex task, requiring much time and untold expense. The payoffs, though, are well worth it. “Biomanufacturing is a multibillion dollar burgeoning industry,” noted Carlos Miguez, project leader, microbial and enzymatic technology group, National Research Council Biotechnology Research Institute in Canada. New data emerges every day that provides unique insights into methods for producing these proteins.

CHI’s “PepTalk 2008” conference in San Diego earlier this year explored challenges with protein expression, peptide and protein-based therapeutics, and mining the plasma proteome. Various solutions were offered for problems and bottlenecks.

Versabodies as Antibody Copycats

Antibody mimetics derived from nonhuman protein scaffolds offer an alternative to antibodies and small molecules. Volker Schellenberger, Ph.D., cofounder of Amunix, described his company’s microprotein-based Versabody™ platform and the philosophy that has driven it.

“Versabodies offer the superior affinity, specificity, half-life, and safety of whole antibodies combined with the superior stability, size, delivery, and nonimmunogenicity of microproteins. Since different targets have different requirements, format flexibility is critical,” Dr. Schellenberger added.

Amunix’ protocols utilize binding modules and spacer modules to create a variety of product formats that optimally fit each target’s biology. “The binding modules are microprotein domains of 20–35 AA including 2–4 disulfide bonds, which make them extremely stable and minimize immunogenicity.”

“A major limitation of many biopharmaceuticals is their short serum half-life requiring frequent injections,” Dr. Schellenberger continued. “Chemical PEGylation is frequently used to improve half-life and reduce protein immmunogenicity. However, PEGylation significantly complicates the manufacturing process and frequently results in difficult to separate and characterize mixtures.

“Amunix’ rPEG technology is based on protein sequences with PEG-like properties that are genetically fused to biopharmaceuticals, avoiding the extra chemical conjugation step. rPEGs have an increased hydrodynamic radius and show an apparent molecular weight that is about 15-fold their actual molecular weight, mimicking the way PEGylation achieves a long serum half-life.

“By alternating multiple microprotein domains with multiple rPEG linkers, we can create a single-protein product that has high potency due to multivalency and/or multispecificity, a long serum half-life, and is easy to manufacture in E. coli,” Dr. Schellenberger explained.

Versabodies are typically 20–60 kD and are expressed at high levels in soluble form in the E. coli cytoplasm. “The high disulfide density of microproteins makes our proteins extremely stable, enabling the use of differential heat-denaturation/precipitation as a one-step purification process,” he said.

To minimize the possibility of an immune response that cross reacts with native human proteins, Dr. Schellenberger reported that his group uses nonhuman microproteins. “Using directed evolution, we engineer the epitope content and protease sensitivity of these domains to minimize their immunogenicity.”

The resulting freedom to completely change the product’s amino acid sequence allows Amunix to optimize products for all desired properties such as stability, expression, formulation; whatever the therapeutic area calls for. “The product format is also designed to support optimal patent strategies,” Dr. Schellenberger concluded.

Novel E. coli Expression System

“You look at the number of microbial expression systems out there and wonder why there are so many of them,” Miguez pointed out. “The short answer to that is that, while an expression platform for protein x may work beautifully, when you move to protein y, it doesn’t work quite as well.”

Miguez and his group developed a gene-expression system for E. coli that could replace the IPTG-based platforms currently available. “The new system is capable of higher product yields compared to IPTG and uses p-cumate, a nontoxic, easy-to-handle, and cost-competitive inducer that can be used with popular strains of E. coli,” Miguez explained. “Applications include production of biopharmaceutical proteins, production of industrial enzymes, and basic and applied use in universities and research institutes.”

This E. coli gene-expression system is based on the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using cumate as an inducer. It includes a specific expression vector, pNEW, that contains a partial T5 phage promoter fused to a repressor binding DNA fragment (operator) and the repressor gene cymR driven weakly by a kanamycin promoter PKm designed to express the repressor gene constitutively in the E. coli host strain.

“The exogenous inducer p-cumate is necessary for the induction of transcription,” Miguez explained. “The very tightly cumate-regulated expression system can be used with popular E. coli strains and potentially produce any gene that can be expressed using IPTG-inducible systems.”

Miguez noted that his E. coli gene-expression system is capable of high induction of transcription and low basal expression, surpassing the yields of IPTG-based gene expression systems. “The expression vector can be used with popular strains such as BL-21 (DE3), enabling those currently using IPTG-based systems to adopt the novel E. coli gene-expression system.”

One key feature Miguez commented on was that this expression vector is a viable alternative to ITPG-based systems that do not express some genes well, leading to insoluble or incorrectly folded proteins. Miguez’ E. coli gene-expression system may be an avenue for the production of these types of genes. “There is a lot of interest, and several companies want to try it out. Expression is gene dependent, which is why there are so many expression systems.”

Optimization in Baculoviral Expression System

Jim King, Ph.D., principal scientist for Boehringer-Ingleheim, and his group use the baculoviral expression system for optimization of proteins for crystallography. He calls this expression system the workhorse platform in both industry and academia for the last 15 years. “Until about five years ago,” Dr. King added, “it was really just a production system. In the last five years, it’s really advanced to screening a much larger number of constructs.”

Dr. King’s group has optimized and automated a high-throughput, small-scale baculoviral expression process to quickly and effectively assess protein expression. After one-step affinity purification, the resulting protein is analyzed using a Caliper Life Sciences’ LabChip 90 and is suitable for follow-up characterization to prioritize large-scale expression.

“We have applied this technique to many projects to scan domain boundary variants and point mutants. We actually do parallel small-scale screening, which allows us to use 96 constructs in parallel during one screen, and that screen takes about 10 days to get through all the steps. With the help of Caliper, we’ve created an automated system that does all the steps for us. The system is up and running, working out well, and performing exactly as we had hoped.”

The end game is to make systems easier to use and more accurate, making it easier for scientists to do their jobs, commented Professor Linda King, founder of Oxford Expression Technologies. “People who use high-throughput systems want them to be precise, so they can be used in conjunction with robotic technologies. Also, increasing the quality of the protein is key—as purity goes up, specific activity should also increase.”

Dr. King’s presentation highlighted recent advances in high-throughput, robotic technologies for the production of proteins in insect cells. The new advances include modifications to the baculovirus expression vector, flashBAC, to delete nonessential genes that result in enhanced yield and quality of many proteins. “The development of baculovirus-expression vector systems has accompanied a rapid expansion of our knowledge about the genes, their function, and regulation in insect cells,” said Dr. King.

Baculovirus gene expression occurs in an ordered cascade, regulated by early, late, and very late gene promoters. It has also been realized that the insect host cell has innate defenses against baculoviruses in the form of an apoptotic response to virus invasion. “Baculoviruses counter this by encoding apoptotic-suppressors, which also appear to have a role in determining the host range of the virus,” Dr. King stated. “Also of importance to our understanding of baculovirus expression systems is how the virus can accumulate mutations within genes that affect recombinant protein yield in cell culture.”

Protein Engineering for Diagnostics

“The work we do is not directly related to protein therapeutic applications, but we do research protein-expression technologies for diagnostic purposes,” stated Bob Wolfert, Ph.D., CSO for diaDexus. “What our team has done is optimize methods at high secretion rates. Our first product, the PLAC test, is a simple blood test that measures levels of an inflammatory enzyme that represents a new risk factor for coronary heart disease and stroke.”

Shaoqiu Zhuo, Ph.D., presented diaDexus’ work on the human embryo kidney-293 (HEK-293) cell line, which has been widely used to produce recombinant proteins. Many human proteins are cell-type dependent in expression and post-translational processing. “We have developed protein engineering methods for the expression and secretion of recombinant proteins in HEK-293 cells,” said Dr. Wolfert.

“One of the case studies we showed in the presentation is to enhance the expression and secretion of the mature macrophage inhibitory cytokine-1. We have identified one of the rate-limiting steps for the folding of the protein based on a novel glycosylation mechanism and are able to significantly increase the secretion of the mature dimeric form of the protein. This mutation and expression process can be easily adapted into 510K GMP production for our diagnostic products.”

Diagnostic kits for colon cancer, ovarian cancer, and breast cancer are currently in the works, added Dr. Wolfert. In creating immunoassay systems he emphasizes that he wants proteins that are native in structure yet amplified in nature. “We want to actually reflect proteins that are released in the blood stream.”

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