Cell-Free Protein Synthesis Holds Real Potential to Transform Drug Development and Manufacturing

Cell-Free Protein Synthesis Holds Real Potential to Transform Drug Development and Manufacturing

February 14, 2024PAO-02-24-NI-01

Although cell-free protein synthesis (CFPS) has been employed in fundamental biological research for decades, interest in the approach as a viable means for biologic drug product manufacturing has only emerged in recent years. The advantages that CFPS provides in terms of simplicity, flexibility, and time savings outweigh the hurdles that must be overcome to make commercial CFPS drug production a reality.

Decades of History with Sustained Progress

In vitro synthesis of proteins was first explored in the 1960s and was, in fact, used to map the human genome.1-3 Since then, significant advances have been made with respect to the systems employed, the yields that can be achieved, and the types of proteins that can be produced.1 It has found wide use in life science research, enabling the study of biological mechanisms and increasing understanding of the genetic code and which genes are linked to the expression of which proteins.

Today, many bacterial, plant, and insect-based cell extracts are used for cell-free protein synthesis (CFPS), with products including traditional proteins and monoclonal antibodies (mAbs), as well as antigens, virus-like particles (VLPs), cytokines, peptides, membrane proteins, viable bacteriophages and viruses, enzymes containing metal cofactors, and proteins containing nonnatural amino acids.1 The technology has also been expanded for the production of DNA and RNA products. While the biggest application has been research, in recent years CFPS has been investigated in large-scale biomanufacturing to produce biosensors.

Bacterial extracts from a variety of Escherichia coli strains are most commonly used, but yeast extracts from Pichia pastoris and Saccharomyces cerevisiae strains; mammalian extracts from Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, human epithelioid cervix carcinoma (HeLa) cells and blood-derived leukocytes; and plant extracts from wheat germ and tobacco are also employed.1

In addition to exploring and optimizing the production of these different extracts, CFPS systems built from the bottom up from individual components have also been developed.2 In addition, higher titers systems have been achieved in CFPS by adjusting the mix of supplements used with the extracts, which includes nucleotides, amino acids, metabolic intermediates, and salts.1 While in most cases titers are still lower than those realized with cell-based culture, work on CHO extracts, for instance, has been conducted for a much shorter time compared with the four decades of development and optimization of cell culture. In some cases, productivities and protein-folding efficiencies greater than those observed for comparable cellular systems have been reported.2 Indeed, if considered over time, progress with CFPS can be viewed as similar to the rate observed for mAb production in CHO cells.1

Furthermore, production costs are decreasing as cheaper energy sources and simpler methods for lysate production and storage are introduced.2 Advanced technologies, such as high-throughput screening and artificial intelligence / machine learning, are anticipated to accelerate further improvements in CFPS and increase its applicability for commercial deployment.

Many Advantages over Cell Culture

Although biologic drug substances continue to be manufactured commercially via cell culture or microbial fermentation in living cells, the potential benefits of switching to cell-free synthesis continue to mount.1–7 Protein production can begin immediately after cell lysates (prepared elsewhere, frozen, and stored/shipped on demand) are thawed, eliminating the need for a seed train. As importantly, there is no need to engineer cells to produce a specific product, making protein production more accessible. CFPS systems are also much easier to optimize.

In addition, CFPS allows the production of proteins that have proven impossible to manufacture using cell culture, such as proteins that are not included in natural protein production machinery, have toxic effects on living cells, or are poorly soluble. The incorporation of nonnatural amino acids, meanwhile, provides access to novel biomolecules that could provide therapeutic benefits not possible with proteins produced via cell culture.

Another advantage of CFPS is simpler purification protocols and improved protein quality, as these systems do not include unnecessary cellular components that can lead to production of undesired proteins and/or end up as impurities that arise when using living cells. More consistent quality is also made possible through the use of standardized reagents and the elimination of the cell membrane, which enables direct, real-time monitoring and control.

Furthermore, less energy resources are needed because CFPS does not require growth and viability of cells. CFPS is also amenable to many different manufacturing strategies (e.g., batch, continuous) and are readily scalable from research to GMP production, and manufacturing footprints are less complex and smaller than those involved in cell culture, making CFPS ideal for production of personalized medicines and for distributed manufacturing. Finally, biosafety risks are reduced with CFPS.

In one study, production of the recombinant glycoprotein human bone morphogenetic protein‐2 (hBMP2), which is difficult to produce via traditional cell culture, was compared in three systems: stably transfected CHO cells, transient expression in HEK cells, and CFPS in CHO cell lysates.8 The highest titers were achieved via CFPS in just three hours. These titers were comparable to those achieved in bacterial systems, but with proper and comprehensive posttranslational modifications (PTMs), an attribute that often drives the choice to use mammalian cell culture rather than bacterial fermentation.

Different Approaches to Cell-Free Protein Synthesis

In CFPS, the actions of protein biosynthesis, which include transcription of a DNA template to messenger RNA then translation of the mRNA into the sequence of amino acids that make up the protein, takes place most commonly using lysates produced after digestion and removal of the cell membrane. Cells are grown/expanded in cell culture but only to produce large quantities that can be then converted into lysates.

The choice of specific lysate is dictated by the target protein and the end-use application.9,10 Proteins that require PTMs are generally produced using lysates of mammalian cells. Certain DNA (or RNA) templates may be best suited for the protein production machinery of a specific lysate. Proteins from plant-based lysates, meanwhile, tend to less immunogenic and can be readily scaled. ALiCE® (LenioBio GmbH) is a CFPS platform derived from Nicotiana tabacum BY-2 lysate that can, if needed, introduce glycosylation, disulfide bonding, and other complex PTMs and provides as much as 3 g/L of protein up to the 10-L scale.10

Protein production can also be performed using different formats, including microfluidic devices, batch or continuous flow, continuous exchange, and others.6 Several CFPS lysate kits from companies such as Thermo Fisher Scientific and MilliporeSigma are on the market for research applications. For larger-scale protein production, however, it is typically more economical to generate lysates in-house. There is also at least one kit based on a bottom-up approach using purified components, including translation factors, polymerase, tRNA synthetases, ribosomes, pyrophosphatase, creatine kinase, myokinase, and nucleoside diphosphate kinase. The advantage of this approach is that the proteases and nucleases found in natural lysates are avoided.

Role to Play in Drug Discovery and Development

Given that CFPS systems were originally developed to facilitate investigation of the genome, it should be no surprise that they also find use in drug discovery and development. Their simplicity and flexibility (the variety of extracts and tolerance of different DNA templates and additives) make them suitable for high-throughput screening studies (e.g., protein engineering, mutagenesis studies, and enzyme screening) that are not practical using living cells,11 while their ability to produce a considerable array of different proteins makes CFPS systems widely applicable across many different development programs.2

Application to Large-Scale Production of Biologics

The first steps toward large-scale production of biologics via CFPS included ensuring reliable amino acid supply and the identification of more cost-effective energy sources.2 Efforts are underway today to develop scalable CFPS systems for commercial therapeutic manufacturing, including oncology drugs and vaccines.12 There are also examples of companies using CFPS systems for biomanufacturing.

Sutro Biopharma uses the proprietary Xpress CF+ cell-free platform to produce ADCs, bispecific antibodies, and cytokine-based therapies. It has candidates in preclinical and phase I clinical studies and collaborations with big pharma firms, such as Bristol-Myers Squibb, EMD Serono, and Merck.6 Ipsen Biopharm uses CFPS technology to minimize the risk of containment loss and worker exposure during the production of a highly toxic botulinum toxin, while GreenLight Biosciences uses its in-house GreenWorX cell-free platform to produce RNA-based vaccines, including for pandemic preparedness.

The development of a customized E. coli CFPS platform for production of the human therapeutic protein filaggrin leveraging the rare tRNA expressed host strain has also been reported.12 The efforts included improving the process for production of the crude lysate and optimization of process parameters for protein expression.

A key to success in these activities has been careful sourcing of raw materials to ensure GMP compliance, optimization of cell lysis and harvest steps during lysate production to increase both time- and cost-efficiency, avoiding residual Green Worx contamination and increasing yields.13,14 In one case, production of active proteins in a yield of 700 mg/L was achieved in 10 hours at the 100-liter scale.14


Cell-Free Synthesis of Therapeutic Proteins

Several groups are investigating the use of CFPS to produce therapeutic proteins, antibody fragments, vaccines, anti-cancer agents, human Interferon alpha 2b, enzymes, viruses, and VLPs.7 In addition, several candidates produced using CFPS platforms are in clinical trials.2

Table 2. List of therapeutic proteins produced through CFPS7

ProductCell extractTiter (mg/mL)Potential application
Single-chain antibody variable fragment against Salmonella O-antigenWheat germ extract0.013In vivo diagnostic and immunotherapeutic
Urokinase protease

E. coli K12

0.04Treatment of thrombus
Variant of human tissue-type plasminogen activatorE. coli0.06Treatment of acute ischemic stroke
38C13B lymphocyte Id scFv

E. coli

0.043Lymphoma immunotherapy
Insulin-like growth factor I

E. coli

0.4Central nervous system disorders

E. coli

0.854Stimulator of systemic antitumor immunity

E. coli

0.823Cancer immunotherapy, chronic wound healing
Human granulocyte colony-stimulating factor (hG-CSF)

E. coli

0.619Cancer therapy
Human interferon alpha 2b (hIFNa2b)

E. coli

0/692Anticancer agent
Murine scFV (Mvlvh)

E. coli

Human scFV (Hvlvh)

E. coli

Fusion protein with bacterial immunity protein 9 (Im9-hvlvh)

E. coli


E. coli


E. coli

Human consensus interferon-alpha

E. coli


E. coli

0.7Antiviral and antitumor agents

E. coli

0.03Cancer immunotherapy, chronic wound healing
Botulinum toxins

E. coli

1Treament for malignant mesothelioma
StreptokinaseHeLa and CHO cell lysates0.50Thrombolytic therapy

E. coli (ClearColi)

1Cancer therapy

Potential to Facilitate Decentralized and Personalized Medicine

The increased focus within the biopharma industry on the development of treatments for rare diseases and personalized medicines is creating a need for solutions that can enable cost-effective distributed manufacturing. A means for standardization of processes to ensure production of products with consistent quality and purity profiles is essential with decentralized manufacturing.

CFPS could be such an enabling technology.6,14 Cell lysates and the necessary reagents for CFPS (or CFPS systems produced from high-purity components) could be manufactured in a centralized location to provide standardized platforms for use at multiple sites around the world. The smaller footprint, simpler implementation, potential for automation, and ability to switch rapidly between different products also make CFPS attractive as a solution for drug manufacturing in areas of the world lacking significant pharma infrastructure and highly skilled workforces.

Furthermore, the currently higher cost of CFPS compared with cell culture at very large scales would not be an issue in a distributed manufacturing paradigm. It is, in fact, possible that CFPS may be economically favorable at very small scales.6

It is worth noting that some therapeutics, including conjugate vaccines, erythropoietin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been produced using reconstituted, lyophilized lysates in an approach that would facilitate “portable biomanufacturing.”14 In one instance, it was found that the use of maltodextrin as both a lyoprotectant and low-cost energy source in a CFPS system enabled the production of conjugate vaccines at ∼$0.50 – $1.00 per dose depending on the storage temperature (room temperature to 50 °C), with those values determined using the price of raw materials purchased on the lab scale.14

Significant Opportunities Outweigh Challenges

The use of CFPS for commercial production of drug products is in the very earliest exploration stages. Several hurdles must be overcome.4,6 Some of the biggest relate to the application of a quality-by-design approach to CFPS manufacturing and include the need to fully characterize cell lysates and determine their critical quality attributes and the need for better understanding of the correlations between specific cell lysate properties and CFPS process parameters, specific product quality attributes (such as protein folding), and CFPS platform performance. The hope is that, with appropriate monitoring and control systems, the consistency of protein production via CFPS will be similar to that of chemical reactions used to produce small molecule APIs.6

Outside of the CFPS platforms, ensuring reliable supply of GMP-grade versions of all necessary raw materials in sufficient quantities must also be established. Access to appropriately designed plasmids cannot be overlooked either, as demand for high-quality plasmids currently exceeds supply. However, cell-free plasmid synthesis may be able to help address this shortage and provide plasmids ideally suited for cell-free protein synthesis.

The cost and thermostability of CFPS systems must also be improved.14 The latter is particularly important if the potential of CFPS to facilitate decentralized therapeutic manufacturing is to be realized. Efforts must be focused on optimization of CFPE lysate formulations and process parameters to ensure the highest possible stability, yields, and product purities and the lowest possible costs.

If these issues can be addressed, CFPS will provide biopharma companies with a powerful new approach for accelerating the discovery, development, and manufacture of many different types of proteins and protein-related products, including candidates not accessible using traditional cell culture technologies.7 Such new biomolecules with novel structures could possibly lead to medicines to fight currently untreatable diseases and/or change the way some diseases are treated today. Combined its ability to enable decentralized manufacturing, CFPS could broaden access to advanced medicines in many parts of the world that currently do not benefit from cutting-edge treatments and thus could improve the lives of millions.




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