James Zawada

Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production—A New Approach for Shortening Protein Production Development Timelines

Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell‐based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli‐based open cell‐free synthesis (OCFS) system that is optimized for predictable high‐yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high‐level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte‐macrophage colony‐stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide‐bonded biotherapeutic proteins. Biotechnol. Bioeng. 2011; 108:1570–1578.

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.

Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell‐Free Protein Synthesis

Escherichia coli extracts activate cell‐free protein synthesis systems by providing the catalysts for translation and other supporting reactions. Recent results suggest that high‐density fermentations can be used to provide the source cells, but the subsequent cell extract preparation procedure requires multiple centrifugation and dialysis steps as well as an expensive runoff reaction. In the work reported here, the extract preparation protocol duration was reduced by nearly 50% by significantly shortening several steps. In addition, by optimizing the runoff incubation, overall reagent costs were reduced by 70%. Nonetheless, extracts produced from the shorter, less expensive procedure were equally active. Crucial steps were further examined to indicate minimal ribosome loss during the standard 30 000g centrifugations. Furthermore, sucrose density centrifugation analysis indicated that although an incubation step significantly activates the extract, ribosome/polysome dissociation is not required. These insights suggest that consistent cell extract can be produced more quickly and with considerably less expense for large‐scale cell‐free protein production, especially when combined with high‐density fermentation protocols.

A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell‐free protein synthesis systems

Cell‐free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell‐free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re‐examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell‐free system with a 95% reduction in reagent costs (excluding cell‐extract, plasmid, and T7 RNA polymerase made in‐house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze‐thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high‐throughput screening, and biotherapeutics.

Abstract 67: Characterization and preclinical development of STRO-001, a novel CD74-targeting antibody-drug conjugate (ADC) for the treatment of B-cell malignancies

CD74 is a type II transmembrane glycoprotein involved in the formation and transport of MHC class II protein. CD74 is highly expressed in many B-cell malignancies with limited expression in normal tissues (Stein R. et al., CCR 2007). STRO-001 is a novel CD74-targeting ADC containing an anti-CD74 aglycosylated human IgG1 antibody (SP7219) conjugated to a non-cleavable dibenzocyclooctyne (DBCO)-maytansinoid linker-warhead. SP7219 was discovered from a Fab ribosome display library based on Sutro’s Xpress CFTM technology. Highly efficient site-specific conjugation enabled by our cell-free antibody production and click chemistry results in a well-defined homogeneous ADC drug product with a drug-antibody ratio (DAR) of 2. Conjugation sites were selected based on highest stability both in vitro and in vivo, thereby limiting loss of drug moiety from STRO-001 in circulation. Due to its limited cell permeability, the major catabolite released by STRO-001 has 1000X lower cell killing activity on CD74 positive and negative cells compared to the reference cytotoxic maytansine. In vitro cytotoxicity assays show potent activity of STRO-001 in a diverse panel of B-cell tumor lines including 4 multiple myeloma (MM), 9 germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL), 3 activated B-cell (ABC) DLBCL, and 3 mantle cell lymphoma (MCL) cell lines with IC50 ranging from 0.17-20 nM. CD74 cell surface expression is required for STRO-001 cytotoxic activity but expression level, as measured by antibody-binding capacity, does not correlate with in vitro potency (R2=0.4640). STRO-001 inhibits the formation of visceral tumors (p 90 days). STRO-001 exhibits dose-dependent tumor growth inhibition in SU-DHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. The combination of bendamustine/rituximab (BR) + STRO-001 further improves tumor suppression in SU-DHL-6 xenografts compared to vehicle (p = 0.002) or BR alone (p = 0.02). Preliminary studies with a MCL xenograft model, Jeko-1, demonstrate potent anti-tumor activity compared to vehicle (p Citation Format: Cristina Abrahams, Xiaofan Li, Venita DeAlmeida, Millicent Embry, Abigail Yu, Stellanie Krim, Heidi Hoffmann, James Zawada, Maureen Bruhns, Shannon Matheny, Stuart Bussell, Toni Kline, Alice Yam, Ryan Stafford, Trevor Hallam, Mark Lupher, Arturo Molina. Characterization and preclinical development of STRO-001, a novel CD74-targeting antibody-drug conjugate (ADC) for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 67. doi:10.1158/1538-7445.AM2017-67