Challenging the workhorse: Comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.

For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture.

CHO genome mining for synthetic promoter design.

Synthetic promoters are an attractive alternative for use in mammalian hosts such as CHO cells as they can be designed de novo with user-defined functionalities. In this study, we describe and validate a method for bioprocess-directed design of synthetic promoters utilizing CHO genomic sequence information. We designed promoters with two objective features, (i) constitutive high-level recombinant gene transcription, and (ii) upregulated transcription under mild hypothermia or late-stage culture.

Leveraging a CHO cell line toolkit to accelerate biotherapeutics into the clinic.

The Biogen upstream platform is capable of delivering equivalent quality material throughout the cell line generation process. This allows us to rapidly deliver high-quality biopharmaceuticals to patients with unmet medical needs. The drive to reduce time-to-market led the cell engineering group to develop an expression system that can enable this strategy.

Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms.

Antibodies are an important class of therapeutics and are predominantly produced in Chinese Hamster Ovary (CHO) cell lines. While this manufacturing platform is sufficiently productive to supply patient populations of currently approved therapies, it is unclear whether or not the current CHO platform can address two significant areas of need: affordable access to biologics for patients around the globe and production of unprecedented quantities needed for very large populations of patients. Novel approaches to recombinant protein production for therapeutic biologic products may be needed, and might be enabled by non-mammalian expression systems and recent advances in bioengineering.

Genetic mutation analysis at early stages of cell line development using next generation sequencing.

A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development.

Structure of an engineered β-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation.

Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn(2+) at low µM concentration.

A protein therapeutic modality founded on molecular regulation.

The exquisite specificity of proteins is a key feature driving their application to anticancer therapies. The therapeutic potential of another fundamental property of proteins, their ability to be regulated by molecular cues in their environment, is unknown. Here, we describe a synthetic biology strategy for designing protein therapeutics that autonomously activate a therapeutic function in response to a specific cancer marker of choice.

NMR characterization of an engineered domain fusion between maltose binding protein and TEM1 beta-lactamase provides insight into its structure and allosteric mechanism.

RG13 is a 72 kDa engineered allosteric enzyme comprised of a fusion between maltose binding protein (MBP) and TEM1 beta-lactamase (BLA) for which maltose is a positive effector of BLA activity. We have used NMR spectroscopy to acquire [(15)N, (1)H]-TROSY-HSQC spectra of RG13 in the presence and absence of maltose. The RG13 chemical shift data was compared to the published chemical shift data of MBP and BLA.

As easy as flipping a switch?

Proteins that behave as switches help to establish the complex molecular logic that is central to biological systems. Aspiring to be nature's equal, researchers have successfully created protein switches of their own design; in particular, numerous and varied zinc-triggered switches have been made. Recent studies in which such switches have been readily identified from combinatorial protein libraries support the notion that proteins are primed to show allosteric behavior and that newly created ligand-binding sites will often be functionally coupled to the original activity of the protein.

Direct evidence for enzyme persulfide and disulfide intermediates during 4-thiouridine biosynthesis.

Two proposed mechanisms for 4-thiouridine generation share key cysteine persulfide and disulfide intermediates, and indirect evidence of their existence has been previously reported; chemical trapping and mass spectrometry have now provided direct and definitive evidence of these key intermediates.

A paradigm for biological sulfur transfers via persulfide groups: a persulfide-disulfide-thiol cycle in 4-thiouridine biosynthesis.

In support of the key features of sulfur transfer in the proposed mechanisms of 4-thiouridine generation, the enzyme ThiI can turn over only once in the absence of reductants of disulfide bonds, and Cys-456 of ThiI receives the sulfur transferred from the persulfide group of the sulfurtransferase IscS.