Biotechnol Bioeng. 2011 Jul;108(7):1570-8. doi: 10.1002/bit.23103. Epub 2011 Mar 31.

Microscale to manufacturing scale-up of cell-free cytokine production–a new approach for shortening protein production development timelines.

Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, Gold DS, Heinsohn HG, Murray CJ.

Abstract

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.

 

PMID:    21337337

PMCID:    PMC3128707

DOI:    10.1002/bit.23103

Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21337337/

 

 

MAbs. 2012 Mar-Apr;4(2):217-25. doi: 10.4161/mabs.4.2.19202. Epub 2012 Mar 1.

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

Yin G, Garces ED, Yang J, Zhang J, Tran C, Steiner AR, Roos C, Bajad S, Hudak S, Penta K, Zawada J, Pollitt S1, Murray CJ.

Abstract

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 (IgG 1) 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.

 

PMID:    22377750

PMCID:    PMC3361657

DOI:   10.4161/mabs.4.2.19202

Full text:   https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22377750/

 

 

Bioconjug Chem. 2014 Feb 19;25(2):351-61. doi: 10.1021/bc400490z. Epub 2014 Jan 29.

Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system.

Zimmerman ES, Heibeck TH, Gill A, Li X, Murray CJ, Madlansacay MR, Tran C, Uter NT, Yin G, Rivers PJ, Yam AY, Wang WD, Steiner AR, Bajad SU, Penta K, Yang W, Hallam TJ, Thanos CD, Sato AK.

Abstract

Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.

 

PMID:    24437342

DOI:   10.1021/bc400490z

Full text:  https://dx.doi.org/10.1021/bc400490z

 

Protein Eng Des Sel. 2014 Apr;27(4):97-109. doi: 10.1093/protein/gzu002. Epub 2014 Feb 28.

In vitro Fab display: a cell-free system for IgG discovery.

Stafford RL, Matsumoto ML, Yin G, Cai Q, Fung JJ, Stephenson H, Gill A, You M, Lin SH, Wang WD, Masikat MR, Li X, Penta K, Steiner AR, Baliga R, Murray CJ, Thanos CD, Hallam TJ, Sato AK.

Abstract

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

 

PMID:    24586053

PMCID:    PMC3966677

DOI:   10.1093/protein/gzu002

Full text:  https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24586053/

 

 

MAbs. 2014 May-Jun;6(3):671-8. doi: 10.4161/mabs.28172. Epub 2014 Feb 11.

Engineering toward a bacterial “endoplasmic reticulum” for the rapid expression of immunoglobulin proteins.

Groff D, Armstrong S, Rivers PJ, Zhang J, Yang J, Green E, Rozzelle J, Liang S, Kittle JD Jr, Steiner AR, Baliga R, Thanos CD, Hallam TJ, Sato AK, Yam AY.

Abstract

Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies. However, given the ease and manipulability of bacteria, antibody discovery efforts often employ bacterial expression systems despite their limitations in generating high titers of functional antibody. Open-Cell Free Synthesis (OCFS) is a coupled transcription-translation system that has the advantages of prokaryotic systems while achieving high titers of antibody expression. Due to the open nature of OCFS, it is easily modified by chemical or protein additives to improve the folding of select proteins. As such, we undertook a protein additive screen to identify chaperone proteins that improve the folding and assembly of trastuzumab in OCFS. From the screen, we identified the disulfide isomerase DsbC and the prolyl isomerase FkpA as important positive effectors of IgG folding. These periplasmic chaperones function synergistically for the folding and assembly of IgG, and, when present in sufficient quantities, gram per liter IgG titers can be produced. This technological advancement allows the rapid development and manufacturing of immunoglobulin proteins and pushes OCFS to the forefront of production technologies for biologics.

 

PMID:    24517929

PMCID:    PMC4011911

DOI:   10.4161/mabs.28172

Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24517929/

 

 

Biotechnol Prog. 2015 May-Jun;31(3):823-31. doi: 10.1002/btpr.2082. Epub 2015 Apr 18.

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

Cai Q, Hanson JA, Steiner AR, Tran C, Masikat MR, Chen R, Zawada JF, Sato AK, Hallam TJ, Yin G

Abstract

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.

 

PMID:    25826247

PMCID:    PMC5029582

DOI:   10.1002/btpr.2082

Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25826247/

 

 

 

Mol Pharm. 2015 Jun 1;12(6):1848-62. doi: 10.1021/acs.molpharmaceut.5b00082. Epub 2015 May 21.

Antibody conjugates with unnatural amino acids.

Hallam TJ, Wold E, Wahl A, Smider VV.

Abstract

Antibody conjugates are important in many areas of medicine and biological research, and antibody-drug conjugates (ADCs) are becoming an important next generation class of therapeutics for cancer treatment. Early conjugation technologies relied upon random conjugation to multiple amino acid side chains, resulting in heterogeneous mixtures of labeled antibody. Recent studies, however, strongly support the notion that site-specific conjugation produces a homogeneous population of antibody conjugates with improved pharmacologic properties over randomly coupled molecules. Genetically incorporated unnatural amino acids (uAAs) allow unique orthogonal coupling strategies compared to those used for the 20 naturally occurring amino acids. Thus, uAAs provide a novel paradigm for creation of next generation ADCs. Additionally, uAA-based site-specific conjugation could also empower creation of additional multifunctional conjugates important as biopharmaceuticals, diagnostics, or reagents.

 

PMID:    25898256

DOI:   10.1021/acs.molpharmaceut.5b00082

Full text: https://dx.doi.org/10.1021/acs.molpharmaceut.5b00082

 

 

MAbs. 2015;7(1):231-42. doi: 10.4161/19420862.2015.989013.

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system.

Xu Y, Lee J, Tran C, Heibeck TH, Wang WD, Yang J, Stafford RL, Steiner AR, Sato AK, Hallam TJ, Yin G.

Abstract

Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF+™) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF+™ in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

 

PMID:    25427258

PMCID:    PMC4623329

DOI:   10.4161/19420862.2015.989013

Full text:   https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25427258/

 

 

Pharm Res. 2015 Nov;32(11):3480-93. doi: 10.1007/s11095-014-1596-8. Epub 2014 Dec 16.

Methods to Make Homogenous Antibody Drug Conjugates.

Kline T, Steiner AR, Penta K, Sato AK, Hallam TJ, Yin G.

Abstract

Antibody drug conjugates (ADCs) have progressed from hypothesis to approved therapeutics in less than 30 years, and the technologies available to modify both the antibodies and the cytotoxic drugs are expanding rapidly. For reasons well reviewed previously, the field is trending strongly toward homogeneous, defined antibody conjugation. In this review we present the antibody and small molecule chemistries that are currently used and being explored to develop specific, homogenous ADCs.

 

PMID:    25511917

PMCID:    PMC4596908

DOI:   10.1007/s11095-014-1596-8

Full text:  https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25511917/

 

 

Organic Process Research & Development. 2016 Jun 8;20(6):1034-43.

RP-HPLC DAR characterization of site-specific antibody drug conjugates produced in a cell-free expression system

Xu Y, Jiang G, Tran C, Li X, Heibeck TH, Masikat MR, Cai Q, Steiner AR, Sato AK, Hallam TJ, Yin G

Abstract

Antibody drug conjugates (ADCs) harness the target specificity of a monoclonal antibody (mAb) and the high cytotoxicity of a small molecule, enabling improved delivery of a potent antitumor agent compared to traditional chemotherapy for cancer therapy. Only two ADCs have been marketed, both of which are produced via nonsite-specific conjugation of the cytotoxic drug to either interchain cysteine (Adcetris) or lysine (Kadcyla). A growing body of evidence suggests that site-specific ADCs, because of their payload homogeneity, will improve pharmacokinetics and have wider therapeutic windows when compared to heterogeneous ADCs. Previously, we have demonstrated the use of a cell free expression system (Xpress CF+™) for rapid production of site-specific ADCs. Here we report the generation of a variety of ADCs via conjugation between a site-specific incorporated non-natural amino acid (nnAA), para-azidomethyl-l-phenylalanine (pAMF), and dibenzocyclooctyl-(polyethylene glycol)4 (DBCO-(PEG)4) linked payloads using this platform. We developed a reversed phase HPLC method for drug to antibody ratio (DAR) characterization, which is applicable to both reduced and intact ADCs. We demonstrate that these ADCs are of near complete conjugation and exhibit potent cell killing activity and in vitro plasma stability. Moreover, we generated an ADC conjugated at both light and heavy chains, resulting in a DAR close to 4. With the increased number of payloads, the resultant DAR 4 ADC is potentially more efficacious than its DAR 2 counterparts, which could further improve its therapeutic index. These studies have demonstrated the competency of Xpress CF+™ for site-specific ADC production and improved our understanding of the site-specific ADCs in general.

 

DOI: 10.1021/acs.oprd.6b00072

Full text:  https://pubs.acs.org/doi/full/10.1021/acs.oprd.6b00072

 

 

Sci Rep. 2017 Jun 8;7(1):3026. doi: 10.1038/s41598-017-03192-z.

RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates.

Yin G, Stephenson HT, Yang J, Li X, Armstrong SM, Heibeck TH, Tran C, Masikat MR, Zhou S, Stafford RL, Yam AY, Lee J, Steiner AR, Gill A2, Penta K, Pollitt S, Baliga R, Murray CJ, Thanos CD, McEvoy LM, Sato AK, Hallam TJ

Abstract

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

 

PMID:    28596531

PMCID:    PMC5465077

DOI:   10.1038/s41598-017-03192-z

Full text:   https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/28596531/