Recombinant proteins are at the heart of the pharmaceutical and synthetic biology sectors, driving innovations in drug development, diagnostics, industrial biotechnology, and basic biological research. The reliable and scalable production of proteins has fueled the rise of novel therapeutics, while also deepening our understanding of enzymes and biological pathways.
For nearly fifty years, cell-based protein synthesis (CBPS) has been the standard for protein production in R&D and manufacturing. However, as biology research has become increasingly data-driven, cell-free protein synthesis (CFPS) has emerged as a catalyst of the next generation of discoveries. The open format of CFPS lends itself to flexibility, control, and speed that are complementary to traditional high-throughput screening and emerging machine learning applications. With continual refinements and emerging applications, CFPS is evolving from a research tool into a practical complement and, in some cases, a replacement for CBPS across discovery and manufacturing.
This article compares cell-free and cell-based protein synthesis methods in the context of today’s rapidly evolving R&D landscape.
Evolution of protein synthesis: coupled histories
Cell-based protein synthesis
In the late 1970s, Genentech achieved the overproduction of recombinant human insulin in E. coli. Shortly thereafter recombinant insulin entered the clinic as a lifesaving treatment for diabetic patients, marking the first commercial success of cell-based protein synthesis (CBPS)(Figure 1).
Genentech’s milestone achievement was the culmination of a decade of rapid advancements in recombinant DNA technology. Beginning in the early 1970s with the discovery of enzymes like exonucleases and ligases—tools that allowed scientists to cut and paste DNA—these innovations enabled the development of engineered systems capable of over-expressing foreign genes in host cells to produce proteins[1]. CBPS quickly became the cornerstone of the biotechnology industry, transforming how we produce a wide range of essential proteins, including monoclonal antibodies, hormones, vaccines, and enzymes.
The process of CBPS begins with the selection of an appropriate host cell such as a microbial strain or derived cell line (e.g., insect or mammalian)[2]. The desired DNA is then cloned into a vector and introduced into the host. Cells bearing the gene of interest are then grown up to a critical density followed by induction of protein expression and additional incubation time for protein synthesis. The final step involves purifying the proteins from the lysed cells or culture broth[3](Figure 2).
Since its first commercial triumph, CBPS secured a dominant role in both research and industrial manufacturing. This position was further solidified by ongoing advancements in genetic engineering, fermentation technology, and bioprocessing, which continue to support its vital role in the biotechnology and pharmaceutical industries today.
Figure 1. Milestones in cell-based and cell-free protein synthesis.
Cell-free protein synthesis
Cell-free protein synthesis (CFPS) has evolved from a mid-20th-century research tool into a powerful platform for modern biotechnology. Initially critical for understanding protein synthesis and the genetic code, CFPS systems have now become indispensable for various applications, including those that complement traditional CBPS (Figure 1).
CFPS operates in an open reaction format, synthesizing proteins using essential components for transcription and translation—such as RNA polymerase, ribosomes, tRNAs, amino acids, and energy systems[4,5]. These components are typically derived from E. coli lysates but can also be sourced from other prokaryotes, eukaryotic cell lines, or archaea, depending on the protein’s complexity and origin. For specific applications requiring particularly clean backgrounds, CFPS systems can even be prepared from purified recombinant proteins.
The CFPS workflow is straightforward yet powerful: it begins with selecting and preparing the appropriate transcription-translation system, including any necessary supplements like cofactors and folding agents. The DNA template is then introduced, the reaction is incubated, and the process is followed by downstream processing or direct assay[4](Figure 2).
The 1970s marked a turning point for CFPS as it transitioned from a fundamental research tool to a viable protein production platform. This shift saw the development of more robust DNA-based transcription-translation systems, moving beyond the initial mRNA-based translation-only methods[1,6]. Most innovations to date have occurred in E. coli-based systems.
A significant leap in transcriptional efficiency came in the 1980s with the introduction of phage promoters and RNA polymerases into these systems[1]. This period also witnessed the advent of continuous CFPS systems, which used flow cells or buffer exchange to remove inhibitory byproducts and replenish substrates, dramatically increasing productivity from microgram to milligram scales.
In the early 2000s, the implementation of energy regeneration strategies paved the way for the first proof-of-concept industrial scale-up of a therapeutically relevant protein by Sutro Biopharma in 2009. Renewed interest in CFPS has also driven advancements in eukaryote-derived systems, better suited for producing complex proteins. Recently, CFPS has emerged as a game-changing tool for AI-driven biology, enabling the rapid generation of rich datasets crucial for refining machine learning models used in polypeptide engineering and de novo design[7,8].
Figure 2. Comparison of cell-based and cell-free protein synthesis workflows.
Comparative analysis
Cell-free and cell-based approaches to recombinant protein production each offer unique advantages depending on the application. This section outlines the key attributes and applications that set these two methods apart (Figure 3).
Speed
Workflows for CFPS typically take hours to days, compared to the days to weeks and sometimes months required for CBPS. Once the lysate is prepared, CFPS is a straightforward process of adding DNA and reagents. In contrast, CBPS involves multiple complex steps: molecular cloning, transferring DNA into a host, growing the host cells, inducing protein expression, and carrying out labor-intensive downstream processing[3].
Modifications in CFPS are easily achieved by adding specific reagents, while similar changes in CBPS often require extensive strain engineering. The primary challenge in CFPS is lysate preparation, which often demands extensive initial optimization and careful attention to detail to ensure reproducibility. However, once prepared, lysates can be batch-produced and stored stably, either frozen or lyophilized[4].
On the other hand, CBPS benefits from the natural regulatory mechanisms and metabolism of living cells, which generally offer robust reproducibility with less upfront effort[3]. The inherent stability of CBPS makes it a reliable option for many applications, despite its longer and more complex workflows.
Stability
CBPS systems are under evolutionary pressure and susceptible to genetic instability and other forms of contamination that could lead to decreases in protein production efficiency with propagation. In contrast, CFPS systems do not require autonomous replication for protein synthesis and thus are not subject to genetic instability. Nonetheless, stable CBPS systems can be easier to maintain than CFPS systems due to the latter’s requirement for complex user inputs.
Scalability
CBPS remains the industry standard for protein production, largely due to the inherent scalability of living cells and well-established manufacturing practices. While CFPS has the potential to achieve higher protein yields—thanks to the absence of energy demands for biomass production and its lower susceptibility to contamination—it is often constrained by the costs of system maintenance[4,5].
Despite the status quo of CBPS in manufacturing, significant progress has been made in scaling CFPS for industrial use, with innovations in cofactor and energy regeneration, lysate preparation, and process design. Additionally, the production of certain proteins, such as those incorporating non-standard amino acids or cytotoxic proteins, may only be possible using CFPS[5].
Cost
CBPS is a well-established method for large-scale protein production, benefiting from economies of scale and mature technologies. While CFPS has seen prototype scale-ups, it has yet to prove itself as a cost-effective manufacturing technique[4,5]. Unlike CFPS, where lysate preparation is costly, living cells in CBPS naturally produce the necessary enzymes and cofactors at virtually no expense. Although recent advances in energy regeneration and continuous processes are narrowing the cost gap, CFPS still needs significant yield improvements to compete with traditional cell-based methods[6].
While CBPS may continue to prove the more cost-effective method for traditional applications, CFPS holds distinct advantages for emerging applications, such as on-demand protein manufacturing for personalized medicine, multiplexed protein production, and large-scale rapid prototyping. The portability of CFPS also makes it an appealing strategy for potentially cost saving distributed manufacturing[5].
Flexibility
CFPS’s open and flexible format allows for real-time modifications of reaction conditions, such as the addition of chemical folding agents, non-proteinogenic amino acids to expand the repertoire of protein properties, and stable isotope probes for NMR structural characterization[5,6].
While CBPS can also be used to troubleshoot protein expression and incorporate unnatural building blocks, it does so with less control and often requires time-consuming strain engineering, which may still result in a heterogeneous product[6].
Protein purification
Protein purification from cell-free reactions is simplified by the upfront engineering and processing of the lysate, which removes cellular debris, genomic DNA, endogenous mRNAs, nucleases, and proteases. In contrast, CBPS involves isolating the target protein from a complex lysate or broth matrix, often requiring extensive processing to achieve high purity[3,6].
High-throughput screening
The open interface of CFPS makes it exceptionally well-suited for testing multiple reaction conditions in parallel. Its flexibility and speed are ideal for high-throughput screening applications, such as protein engineering, functional genomics, and de novo protein design, enabling direct protein characterization much like DNA microarrays for gene expression.
While CBPS remains the default for functional screening, it is limited by library size and amplification bias due to the need for cellular DNA uptake. In contrast, the open format of cell-free systems allows for the systematic and controlled addition of DNA and reagents, simplifying workflows and eliminating biases[9].
Synthesizing complex proteins
Some complex proteins are better suited to cell-free systems, while others are more effectively produced in cellular environments. CFPS’s ability to function under conditions that would be harmful to living cells makes it ideal for expressing cytotoxic proteins, membrane proteins, and proteins that form inclusion bodies[6].
However, CFPS faces challenges with complex post-translational modifications (PTMs), such as disulfide bridges and glycosylation, which are crucial for the activity of many therapeutic proteins. In contrast, eukaryotic cell-based systems naturally support these modifications through their organelles[9].
Despite these challenges, advancements in CFPS—such as fine-tuning redox conditions and using native or introduced microsomes that mimic the oxidizing environment of the endoplasmic reticulum—have enabled the formation of disulfide bridges and glycosylation in cell-free systems, bridging the gap with CBPS for producing complex proteins.
Compatibility with machine learning and artificial intelligence applications
CFPS systems offer distinct advantages for integrating machine learning in synthetic biology. Unlike cellular environments, CFPS allows for precise control over system composition, including enzyme and cofactor concentrations[10,11]. This level of control enables accurate computational simulations of pathway kinetics, supporting model-driven optimization and characterization. The open nature of CFPS also allows for real-time adjustments and immediate feedback, making it ideal for iterative testing[11].
The synergy between CFPS and machine learning has already led to the successful development of novel antimicrobial peptides and proteins, showcasing the potential of this powerful combination[7,8].
Figure 3. Comparing key attributes of cell-based and cell-free protein synthesis.
The future of recombinant proteins
While CBPS remains the industry standard for protein production, CFPS is rapidly gaining ground in R&D and making inroads into manufacturing. The combination of cell-based and cell-free methods promises to accelerate advancements in biotechnology.
The true potential of CFPS lies in emerging, data-driven applications, where its flexibility enables rapid parallel testing of protein variants and offers endless opportunities for fine-tuning parameters and refining computational models.
CBPS was developed in a pre-genomic era when data was sparse, and DNA synthesis was expensive. Now, in the age of data-driven generative biology and more accessible DNA synthesis, an agile technology like CFPS is poised to take center stage.
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