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

Revolutionizing Protein Expression in R&D Arena

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.”

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