Another active area in CFS research is the biomanufacturing of therapeutics and other protein-based reagents. Natural biological systems have evolved a remarkable capacity to synthesize a variety of molecules ranging from metabolites to biopolymers. Cell-free protein expression systems allow the incorporation of such reactions into a highly controlled process that allows production of molecules as needed and in the field. Our primary focus here will be on a subset of biopolymers, namely therapeutic proteins. The ongoing work in this field rests on decades of research that have led to the productive and practical systems currently available [28, 29, 36,37,38, 40]. Recent advances in high-throughput preparation techniques [40, 45] and in the development of systems that can use more economical energy sources [64, 65] have made CFS highly accessible. Meanwhile, significant strides are being made towards resolving various protein folding issues and shortcomings in post-translational modifications [66] associated with traditional CFS. Recent advances have showcased the potential for scaling up cell-free reactions, with some having demonstrated reaction volumes reaching 100 liters [67, 68] to 1000 liters [69]. Cell-free expression has been used as a platform for the production of a wide range of potential therapeutics, some of which have been summarized in Table 1. A number of these products have been validated in animal models [49, 76].

Table 1 Examples of potential therapeutics expressed in CFS to date Full size table

Two primary modes of CFS have been pursued. The first, used by commercial efforts such as Sutro [94], focuses on large, centralized production. This approach leverages the advantages of synthesis outside of the cell for biomanufacturing. For these applications, CFS not only allow for rapid production, but also significantly speed up the drug development process [95]. Remarkably, Sutro has reportedly increased their cell-free production to an incredible 1000 liters [69], showcasing the scalability of centralized cell-free production. The second mode uses FD-CF systems to de-centralize biomanufacturing capacity for small-batch production of therapeutics, with applications in global health and emergency response [49, 73, 96, 97]. Using this mode of production, we have recently demonstrated the proof-of-concept capacity to manufacture over 50 therapeutics and lab reagents, including proteins (e.g., vaccines, antibodies, and antimicrobial peptides) and small molecules [49], with applications outside of the laboratory setting.

Cell-free biomanufacturing is particularly well-suited for vaccine production due to its potential for rapid scale-up in response to public health emergencies. Successful cell-free expression of a number of recombinant vaccines (e.g., botulinum, diphtheria, anthrax) has been demonstrated [49, 86,87,88,89,90, 98], with some having been validated in animal models, such as mice [49, 90]. Considering the low dose requirements (microgram range) for many of these therapeutics, commercialization of CFS-derived vaccines will likely see rapid growth in the coming years. Production of antibodies has also been an area of focus for the cell-free community [20, 49, 51, 74,75,76,77,78,79,80, 99, 100]. Due to their compact size and relatively high expression levels in CFS, single-domain antibodies have garnered particular attention and seem strategically well-placed to serve the emerging needs in personalized medicine, i.e., for therapeutics and diagnostics.

Antibiotic resistance has been recognized as a major threat to global health, resulting in approximately two million illnesses and 23,000 deaths in the US alone every year [101]. Accordingly, cell-free production of antimicrobial compounds, including antimicrobial peptides and small molecule drugs, has become the focus of some groups [49, 93]. A number of labs have also demonstrated the power of CFS to express phages [56, 102,103,104]. The upward trend in the reported antibiotic resistance cases has led to a resurgence in viewing phage therapy as a potentially viable alternative to current antibiotic regimens [101, 105]. The use of phages has also been evaluated as an effective treatment strategy for a number of plant diseases, with some phages now being commercially available for mass consumption [106]. CFS-based production of these non-traditional antimicrobials could play a significant role in battling the antibiotic resistance crisis and could also help improve food security around the globe.

Below, we will highlight some of the areas in which CFS have shown great potential for enhancing current methods of therapeutics development and manufacturing. These advances are rapidly transforming CFS into an integral part of the manufacturing ecosystem.

Membrane proteins

While approximately 70% of all drugs act on membrane proteins[107], working with these proteins is notoriously difficult due to their enrichment in hydrophobic surfaces. Cell-based expression of membrane proteins is often fraught with challenges, such as toxicity caused by their membrane incorporation or their incompatibility with the host’s physiology [108]. Recently, cell-free approaches have been used to tackle this challenging category of proteins, the coding sequences of which comprise 20–30% of all known genes [107]. When compared to current cell-based methods, CFS can be a powerful tool in the production of soluble active membrane proteins [109]. The ability to integrate steps that can tackle the challenging aspects of membrane protein synthesis is particularly valuable. For instance, previous efforts in cell-based systems have demonstrated that membrane mimics can be successfully used to synthesize and stabilize a wide range of membrane proteins such as G-protein-coupled receptors [110, 111], the epidermal growth factor receptor [71], hepatitis C virus membrane proteins [112], and an ATP synthase [109, 113]. These mimics include surfactants, liposomes, and nanodiscs [114,115,116] and can be added directly to CFS co-translationally or post-translationally. There is also evidence suggesting that functioning single-span membrane proteins can be synthesized simply in the presence of an oil–water interface (e.g., through the use of emulsions) [117].

Macromolecular production

Molecular research has highlighted the importance of protein–protein interactions and the resulting complexes that these interactions can generate. Whether it is for the biophysical study of these complexes or as vehicles for new therapeutic delivery (e.g., virus-like scaffolds for vaccines), there is a growing need for developing robust tools aimed at synthesis of such complexes. As in the case of membrane proteins, CFS have also demonstrated higher yields, compared to in vivo strategies, in the production of macromolecular assemblies such as virus-like particles (VLPs) [109]. Groundbreaking work by the Swartz group, demonstrating the cell-free expression of hepatitis B core antigen VLP (2 subunits) [91] in an E. coli-based cell-free system, opened the door to other researchers expressing a variety of macromolecular assemblies including the E. coli RNA polymerase (5 subunits) [118] and an ATP synthase (25 subunits) [113]. Earlier work with reticulocyte lysate had also demonstrated cell-free expression of the human T-cell receptor (7 subunits) [119]. Remarkably, a number of bacteriophages have now also been successfully expressed in CFS, including the T4 phage, which structurally contains 1500 proteins from 50 genes [56, 102,103,104] (Fig. 3).

Fig. 3 Multi-subunit protein complex synthesis in CFS. Various groups have demonstrated the production of increasingly intricate protein complexes. These have included the hepatitis B core antigen (HBc) VLP (2 subunits) [91], the E. coli RNA polymerase (5 subunits) [118], the human T-cell receptor (7 subunits) [119], an ATP synthase (25 subunits) [113], and the T4 phage (1500 subunits) [102,103,104] Full size image

Non-identical subunits of a protein complex are often referred to as hetero subunits. In some instances, such hetero subunits require co-translation to yield active complexes [120]. Thus, the ability of CFS to concurrently translate multiple mRNAs facilitates the production of active complexes composed of a number of different subunits [121]. Some CFS such as E. coli-based preparations are generally not capable of producing proteins that contain disulfide bonds, which are critical to numerous pharmaceutically relevant proteins (e.g., antibodies and many cytokines) [121]. However, recent efforts have augmented these systems to enable the production of complex proteins requiring multiple disulfide bonds [85, 99, 122], expanding the range of therapies that can be made in CFS.

Modification of proteins and codon tables

Effectiveness of many protein-based therapeutics hinges upon precise control over natural or non-natural modification of their peptide sequences. One of the most compelling uses of such modifications is in the development of antibody−drug conjugates (ADCs), which are quickly gaining favor as a new class of therapeutics against cancer. Classic conjugation techniques result in a heterogeneous mixture of labeled antibodies due to their reliance on arbitrary conjugation to multiple amino acid side chains. Recent studies, however, suggest that pharmacologic properties of ADCs could be improved through site-specific conjugation. Non-natural amino acids provide an efficient avenue for such site-specific conjugation [123]. To date, co-translational incorporation of over 100 different non-natural amino acids has been demonstrated in vivo [124], allowing for a wide range of modifications [125,126,127,128,129]. Many of these modifications have been demonstrated in the cell-free context for a variety of applications, including orientation-controlled immobilization [92, 98] and site-specific functionalization (e.g., phosphorylation [130], PEGylation [131], or drug conjugation [81]) [132,133,134].

CFS platforms circumvent some of the cell-based toxicity and permeability limitations and offer greater control and versatility in making protein modifications [109, 135]. Incorporation of non-natural amino acids in cell-based approaches has typically relied on repurposing stop codons to minimize the negative impacts of recoding on cell-viability [109]. In a cell-free system, however, the entire codon table can in theory be reprogrammed, allowing not only for the incorporation of non-natural amino acids, but also for the creation of entirely novel codon tables.

Taken to its extreme, the latter could help with the protection of intellectual property. DNA sequences could be obfuscated such that they are rendered non-functional outside of their specialized cell-free context. This obfuscated code would make proprietary designs difficult to copy. Codon obfuscation could also pose serious challenges for the detection of DNA sequences that may be employed by malevolent entities. For example, DNA synthesis companies would have a much more difficult time screening against DNA sequences that could be used for nefarious activities (e.g., bioterrorism). Recent work has shown that the size of the codon table can also be expanded by augmenting the four-letter genetic alphabet with unnatural base pairs [136, 137]. Thus, proteins made in CFS could—at least in theory—hold an unlimited number of non-natural amino acids.

CFS can also be employed for making naturally occurring modifications to proteins. An example of these is the grafting of sugars (i.e., glycans) referred to as glycosylation. Successful production of many therapeutics is often contingent upon highly efficient glycosylation, as lack of proper glycosylation can reduce the efficacy and circulation half-life of many therapeutic proteins [138]. Some CFS (e.g., insect, Chinese hamster ovary, and human K562 extract-based systems) are inherently capable of glycosylation. However, their repertoire of glycan structures tends to be limited to those naturally synthesized by their lysates’ source cell type. Additionally, glycosylation in these systems often requires recapitulation of the source cell’s protein trafficking mechanisms [109]. Thus, creation of synthetic glycosylation pathways in CFS has become an area of focus in recent years [135, 139]. Success in this domain will likely serve as a key catalyst in bringing cell-free-produced vaccines and other therapeutics to the masses. Figure 4 outlines some of the possible protein modifications in CFS.

Fig. 4 Protein modifications in CFS. Possible protein modifications include but are not limited to glycosylation, disulfide-bond formation, acetylation [140], phosphorylation [141], and PEGylation [131] (which may be accomplished through the use of non-natural amino acids). Non-natural amino acids can also be used for the conjugation of a wide range of compounds such as drugs (e.g., through click chemistry) [81] or fluorescent molecules [142]. Figure adapted from Pagel et al. [143] Full size image

Directed evolution

Directed evolution is a powerful tool for aptamer and protein engineering that uses iterative rounds of mutagenesis and selection to modify or tune specific bimolecular properties (e.g., an enzyme’s substrate activity). Utility of aptamers or proteins, in a given context, with respect to their corresponding nucleotide sequences is often described as a fitness landscape. Directed evolution provides a massively parallel method for searching through a fitness landscape to find optimal variants and their corresponding genotypes [144]. This generally requires one-to-one mapping of phenotype to genotype. Although cells have a built-in capacity for such mapping due to their compartmentalized nature, using cells to conduct directed evolution can impose limits on the size of candidate libraries screened, and restricts the type of solvents, buffers, and temperatures that can be sampled [145]. As a result, cell-free directed evolution platforms have gained favor [145], starting with the first truly cell-free systems published in the late 90s [146, 147]. More recently, connecting phenotype to genotype has been accomplished through artificial compartmentalization (e.g., using emulsion, microbeads, and liposomes) [145, 148,149,150,151]. Applications have included the design and optimization of Fab antibody fragments [77, 152], membrane proteins [151], and, as we will discuss below, enzyme discovery [52].