While-you-wait proteins? Producing biomolecules at the point of need

KEYWORDS:

• Cell-free protein synthesis
• microfluidics
• personalized medicine
• metabolic engineering
• biologics

The functional diversity of proteins is astounding. Proteins can selectively organize, bind, or catalyze a vast array of substrates. Living systems are enabled by these capabilities and various applications benefit from their engineered production. For example, protein-based pharmaceuticals, or biologics, have grown in popularity since the development of biosynthetic human insulin in 1978. Currently, biologics include some of the most highly prescribed drugs, including Humira for treating autoimmune diseases and Epogen/Procrit for addressing anemia resulting from renal failure and chemotherapy. Biologics are the fastest growing sector of the pharmaceutical industry [1] and their advantages over small molecule drugs ensure continued development. These advantages include: (i) unmatched potency, (ii) potentially lower toxicity, and (iii) a broader diversity of targets for which they are selective [2].

Advances in genetically engineered organisms allow economical production of biologics. Often, microbial systems are employed due to their fast growth and genetic tractability. Currently, over a third of biologics are produced from genetically modified microbes such as S. cerevisiae and E. coli [3]. Further, modern genetic construction and cloning techniques facilitate the discovery and development of strategies for the biosynthesis of novel biologics [2]. These genetically engineered microbial processes can be scaled-up to large batch cultures capable of manufacturing biologics with yields up to 7 g/L [4]. As optimization tools for genetic and metabolic engineering progress, the time and costs associated with developing new biologics are reduced.

Producing and distributing biologics comes with challenges. Unlike many small molecule drugs, biologics can be expensive to manufacture and temperature-sensitive, causing a reduced shelf life. While storage and distribution rarely present problems for advanced medical facilities, use in under-developed communities and remote environments is challenged by the need for uninterrupted cold storage. Maintaining protein activity until use may demand alternative approaches for preparing biologics. For example, if the material can be manufactured at its point of need, storage and distribution requirements can be minimized. Living cells are well suited for large-scale production. However, maintaining cell viability until use is not practical. In contrast, cell-free approaches for preparing biomolecules can overcome this limitation. This technique utilizes the protein machinery extracted from cells to carry out complex biosynthesis processes. For example, the transcription and translation machinery can be used to synthesize a chosen protein, or metabolic capabilities can be activated, or created, to prepare a metabolic compound. Potentially, cell extracts can be stored as universal reagents capable of transforming simpler reagents into a diverse range of biologics. Through selection and addition of a DNA sequence, and a mixture of shelf-stable reagents, potentially any desired protein can be created. While approaches for maintaining optimal activity of cell extracts require further investigation, transitioning to a universal reagent set would maintain versatility and dramatically reduce needs for cold storage. Further, use of lyophilized cell extracts has been demonstrated [5], which could facilitate long-term reagent stability.

The usefulness of cell-free methods matches other emerging needs in medicine. As DNA sequencing costs drop, a paradigm shift in medicine is occurring with the goal of tailoring treatments to a patient’s genetic make up. Individualized biologics, personalized to the unique needs of the patient, have been proposed for treating disease [6]. While large-batch industrial production of biologics has been sufficient for generally applicable pharmaceuticals, a flexible production strategy will be necessary to accommodate the rapid personalization of medicine. Feasibly, the quantity of a custom biologic needed for a single dose can be prepared by cell-free protein synthesis (CFPS) in a portable format allowing remote, personal production of pharmaceuticals for specialized and emergency situations.

To advance CFPS for rapid, on-demand production of biologics, the product yield of the reaction must be improved. Reaction performance depends on the cell extract that is chosen. Cell extracts can be isolated in numerous ways, and from a variety of organisms. At the highest level, these extract preparation methods can be separated into two categories: purified and crude extracts. Purified extracts include one-pot reaction cascades carried out by combinations of purified enzymes and cellular machinery [7,8]. These well-defined extracts reduce the number of potential side products and can simplify product clean-up steps; however, the upfront enzyme purification steps may render these extracts too costly for purposes other than research and development. In contrast, crude extracts are made from the lysis of whole cells. Native nucleic acids, membranes, and metabolites are removed and the remaining cellular machinery is resuspended in a solution of salts, buffers, and cofactors necessary to carry out bioprocesses. Extracts from both eukaryotic [9] and prokaryotic cell lines [10,11] have been used for carrying out in vitro bioprocesses. Prokaryotic systems, namely E. coli, are the most advanced, and ongoing efforts in strain engineering and energy regeneration systems have improved protein synthesis yields [12,13]. Typically, protein synthesis reactions are complete within a few hours. Bacterial extracts, however, cannot carry out all of the steps necessary to synthesize functional eukaryote-derived and post-translationally modified proteins without additional enzymatic processing. In contrast, common eukaryotic wheat-germ, rabbit reticulocyte, and insect cell-free expression systems are capable of particular protein modifications; however, they lack the strain engineering tools and high protein yields offered by E. coli extracts. Continued efforts in genome wide engineering of cell extract source strains through clustered regularly interspaced short palindromic repeats (CRISPR), multiplex automated genomic engineering (MAGE), and trackable multiplex recombineering (TRMR) have increased mRNA and protein stability and enabled new functions [14]. The development of robust cell-free protein expression in other cell extracts, or appropriate modifications of bacterial extracts, is needed for effective production of biologics.

The potential for producing therapeutically useful amounts of proteins in a portable format is becoming increasingly feasible. Typical requirements for therapeutic protein dosages range between 0.01 to several milligrams per day [15]. For comparison, highly productive E. coli strains lead to CFPS yields, in batch mode, of more than 2 mg/mL [16]. Further, it has been demonstrated that protein synthesis in microfluidic formats can improve yields by more than sixfold compared to traditional batch reactors [15]. Miniaturization efforts are critical to enabling portability, and when combined with high-yield extracts, can make remote production of useful amounts of biologics possible. However, similar to cell-based bioprocesses, proteins derived from cell-free systems need additional purification in order to be usable in a therapeutic context. Cell-free synthesis approaches alleviate the need for cell lysis and related processing steps allowing the reaction products to be directly applied to chromatography columns. Microfluidic versions of common protein separation techniques have also been demonstrated [17]. Modular combinations of purification procedures, such as affinity binding, anion exchange, and protein desalting, can potentially be configured to isolate a variety of desired products in a miniature format. Complementary tools for assessing protein quantity, activity, and purity at the microscale are needed. Development of these tools is promising as they share many of the same challenges as point-of-care diagnostics, which have realized steady improvements in the analysis of proteins, nucleic acids, and complex biological samples [18]. Nevertheless, the tools and technology for rapid, portable production of proteins are bringing on-demand biologics closer to reality.

With advances in on-demand biologics, other technical applications become feasible. Cell-free approaches can also produce enzymatically active proteins. Functional metabolic pathways can be reconstructed from purified enzyme products, or possibly produced in situ by CFPS, to prepare valuable commercial and therapeutic small molecules (e.g. antibiotics, vitamins, dyes, and pest-control compounds) [19]. Beyond use in remote- or resource-challenged settings, cell-free biosynthesis is capable of facilitating various avenues of basic research; miniaturized and high-throughput proteomics for diagnostics, prototyping of biosynthetic pathways for eventual scale-up, or analysis of genetic networks for synthetic biology can be considered [20]. Further, preparation of any of the large number of unknown proteins identified by genome sequencing efforts can facilitate understanding of their functional roles [21] and can benefit from microfluidic-based approaches to determine their binding partners [22].

The ease of implementing protein synthesis and purification at reduced scales, coupled with the constantly decreasing costs of gene synthesis, will expand the reach of cell-free technologies and facilitate its use by small businesses, schools, and enterprising individuals, similar to the now-widespread adoption of additive manufacturing in the Maker community. Comparable to 3D printing, cell-free techniques can accommodate niche and diverse markets due to low start-up costs and decreasing barriers to entry compared to traditional large-scale bioprocesses. As with most democratized technology, there are issues that will need to be addressed regarding regulations, intellectual property, and biosafety; but, many of these issues are already under consideration by the scientific and regulatory communities with regard to synthetic biology.

CFPS exploits the efficiency and versatility of biological processes without the limitations that come with managing living organisms. With the aid of micro- and nano-fluidic technologies, this technique can produce diverse biomolecules at therapeutically relevant scales. Furthermore, the absence of requirements for cell viability makes this technique ideal for the on-demand and customized synthesis of biologics at the point-of-care. Continued development and integration of the underlying technologies can enable rapid, convenient access to the functional diversity of proteins.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

The authors acknowledge research support by the Genomic Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Area (http://pmi.ornl.gov). ST Retterer and MJ Doktycz are supported in part by the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the US Department of Energy under Contract no. DEAC05-00OR22725.