Cell-Free Protein Synthesis Reactor Formats: A Brief History and Analysis

Abstract

Cell-free protein synthesis (CFPS) has transformed protein production capabilities by eliminating cellular constraints, enabling the rapid expression of difficult-to-produce proteins in an open, customizable environment. As CFPS applications expand from fundamental research to industrial production, therapeutic manufacturing, and point-of-care diagnostics, the diverse array of reactor formats has become increasingly important yet challenging to navigate. This review examines the evolution and characteristics of thirteen major CFPS reactor formats, from traditional batch systems to advanced platforms. The historical development of CFPS reactors from the 1960s to present day is presented. Additionally, for each format, operational principles, advantages, limitations, and notable applications are evaluated. The review concludes with a comparative assessment of reactor performance across critical parameters, including productivity, scalability, technical complexity, environmental stability, and application suitability. To our knowledge this structured analysis is the first to focus predominantly on the various reactor formats of cell-free systems and to provide a guide to assist researchers in choosing the reactor type that best fits their specific applications.

1. Introduction

Cell-free protein synthesis bypasses the constraints of traditional cell-based expression systems using cellular extracts that contain the molecular machinery necessary for transcription and translation, but without the cell walls. These extracts, derived from a variety of organisms including prokaryotes (e.g., E. coli) and eukaryotes (e.g., CHO cells, wheat germ), include ribosomes, aminoacyl-tRNA synthetases, translation factors, and associated enzymes [1]. In this open, cell-free environment, proteins can be produced directly from DNA templates (see Figure 1). The CFPS process requires the addition of other fundamental components, including amino acids as building blocks, tRNA molecules for amino acid delivery, mRNA or DNA templates encoding target proteins, and energy substrates such as ATP and GTP to drive translation reactions. Energy regeneration constitutes a critical aspect of CFPS design, typically employing phosphoenolpyruvate-based systems or alternative metabolic pathways to maintain ATP levels throughout extended synthesis periods [1,2]. This approach offers advantages including rapid protein production (hours versus days), the ability to express toxic proteins that would otherwise prevent cell growth, detailed control over reaction conditions, and the capacity to easily incorporate non-canonical amino acids for specialized functionalities [3].

In recent years, CFPS technology has experienced transformative advancements, evolving from rudimentary laboratory demonstrations to sophisticated systems capable of industrial-scale applications [4,5,6]. The field has now matured to enable both pathway prototyping and scalable production of diverse natural products across industrial biotechnology applications [5]. Researchers have significantly enhanced CFPS yield and efficiency through optimization of energy regeneration pathways [7], improved extract preparation methods [8], and novel reactor designs [9]. The technology has enabled the production of diverse proteins, including full-length antibodies [10,11,12], complex proteins with up to 17 disulfide bonds [13], virus-like particles [14,15,16], cytotoxic proteins [17,18,19], and cancer therapeutics [4,20,21]. Cell-free systems have also expanded beyond basic protein production to facilitate synthetic biology applications [22,23], vaccine development [24,25], and progress toward artificial cells [26,27]. Point-of-care diagnostics utilizing CFPS are capable of detecting minute quantities of various targets, including amino acids [28], Zika virus [29], Ebola [30], SARS-CoV-2 [31], and endocrine disrupting chemicals [32]. With continued miniaturization, automation, and integration with microfluidic platforms, CFPS systems now offer rapid high-throughput screening for protein engineering and directed evolution [33,34,35], providing an increasingly important tool for both fundamental research and biotechnological innovation.

As the field continues to mature, a diverse array of reactor formats has emerged, each offering distinct advantages for specific applications. These formats range from simple batch systems to complex continuous-flow bioreactors, and from high-throughput microplate configurations to portable paper-based and wearable platforms. The selection of an appropriate reactor format is crucial for optimizing protein production and depends on factors such as scale requirements, resource constraints, and intended application. Understanding the strengths and limitations of each format is therefore important for researchers and engineers working in this rapidly evolving field.

This review explores the evolution of CFPS reactor formats and provides an analysis of the advantages, disadvantages, and applications of each type. By examining the technological progression and comparative performance of these diverse systems, we aim to provide a practical resource for both newcomers and experienced practitioners in the field of cell-free synthetic biology.

2. Historical Evolution of CFPS Reactor Types

Cell-free protein synthesis has evolved dramatically (see Figure 2) since Nirenberg and Matthaei’s groundbreaking research deciphering the genetic code in the early 1960s [36]. The earliest CFPS systems (1961) used simple batch reactions where all components were mixed in a single vessel. These first-generation formats were limited by short reaction lifetimes (<1 h) and low protein yields [37] due to rapid depletion of high-energy compounds and accumulation of inhibitory byproducts [1,38].

To overcome these limitations, Spirin and colleagues introduced the continuous-flow cell-free (CFCF) translation system in 1988 [9]. This approach provided a constant supply of energy sources while removing reaction byproducts using an ultrafiltration membrane, extending reaction times to 20 h and increasing yields by two orders of magnitude [9,37]. However, operational complexities limited CFCF’s practical application. By the mid-1990s, the continuous-exchange cell-free (CECF) method emerged, utilizing passive exchange through a dialysis membrane instead of active flow [1,37]. This semi-continuous approach maintained CFCF’s extended reaction lifetime advantages while simplifying equipment requirements. In 2002, Endo and colleagues developed an efficient bilayer diffusion system that eliminated membranes entirely [43]. The reaction mixture, placed beneath a feeding buffer, allowed the diffusion of substrates and removal of inhibitory byproducts across the interface [37,43], simplifying operations while producing over 10 times more protein than batch-mode reactions.

Concurrently, in the early 2000s, Swartz and colleagues improved batch reaction energetics by developing more efficient and economical alternatives to traditional ATP and GTP regeneration systems [7,38,47]. These developments extended reaction duration and demonstrated that optimized batch reactors could approach yields of one milligram per milliliter [1,37], making them viable for many applications. Other advances have adjusted the reaction energetics by incorporating complex carbohydrate sources [48,49].

A significant advancement came with the Protein synthesis Using Recombinant Elements (PURE) system in 2001 [50]. Unlike extract-based systems, PURE combines individually purified enzymes and cofactors for transcription and translation in defined quantities, offering advantages such as precise composition, the absence of contaminating proteases, and customizable reaction conditions [51]. The PURE system enables facile genetic code expansion and precise control over the translational machinery [50], though it generally produces lower yields (~100 μg/mL) compared to extract-based CFPS systems [2].

By 2011, Zawada, Swartz and colleagues demonstrated CFPS at the manufacturing scale (100-L reactions), showing industrial potential while maintaining productivity [4,37]. This scale-up represented a million-fold volume increase from laboratory-scale reactions, achieving yields of 700 mg/L even for complex proteins with multiple disulfide bonds [4].

From 2014 onward, there have been many innovations focused on field applications and point-of-care diagnostics through lyophilization techniques. Though initial attempts demonstrated that activity decreases to ~20% after 90 days at room temperature [52], in 2019, Wilding and colleagues utilized lyoprotectants to developed thermostable CFPS systems that could maintain significant activity after several months of storage at elevated temperatures [53]. These stabilized systems enabled portable, on-demand manufacturing independent of cold-chain storage requirements or laboratory infrastructure, representing a significant step toward distributed therapeutic production.

In 2020, Borkowski and colleagues addressed lysate variability using artificial intelligence for CFPS optimization [54]. Their active learning algorithms explored approximately four million possible buffer compositions, achieving 34-fold yield increases while testing only 1017 formulations. This breakthrough has shifted the field toward data-driven strategies, offering solutions to batch-to-batch variability across applications from diagnostics to industrial production [54].

Having examined the historical development of cell-free protein synthesis systems, we now turn to a detailed analysis of each reactor format that has emerged over the past several decades. In the following sections, we systematically evaluate the various CFPS reactor technologies—from fundamental batch systems to sophisticated microfluidic and wearable platforms. For each reactor type, we explore its fundamental principles, key advantages and limitations, and noteworthy applications in research and industry. This analysis provides a framework for understanding how different reactor formats address specific challenges in protein production and helps guide selection of the most appropriate system for particular applications.

3. Types of CFPS Reactors

Here, we present a detailed description of the various reactor types for cell-free systems. Schematics for the function of each type can be seen in Figure 3. Additionally, although beyond the scope of this review, it is worth noting that each reactor format is compatible with both eukaryotic and prokaryotic cell lysates, though the majority of the cited literature utilizes prokaryotic systems. Eukaryotic lysates allow for some eukaryote-specific post-translational modifications, but they come at the cost of significantly reduced protein yields, so additional care must be taken when selecting a reactor format with this type of cell [2].

3.1. Batch Reactors

Batch reactors represent the most fundamental and widely used format for CFPS. In batch mode, all components required for transcription and translation—including cell extract, energy sources, cofactors, nucleotides, amino acids, and DNA templates—are combined in a single reaction vessel without further addition of reagents during the reaction period [2].

3.1.1. Advantages

Batch reactions are widely adopted because they are fast, simple, and inexpensive to perform; all components are combined in a single vessel [2]. The open nature of batch systems offers direct access to reaction components, making it easier to monitor and control various parameters affecting protein synthesis [1]. Batch CFPS systems provide efficient substrate utilization compared to continuous systems [60,61]. The facile format enables parallel protein production for high-throughput screening applications, and the straightforward setup allows for rapid process and product development [62]. Batch formats have also proven to be the most compatible with scale-up for larger reactions over 100 L in size [4,63]. Protein yields in batch cell-free systems vary widely, typically ranging from 0.02 mg/mL to 1.7 mg/mL, depending on the source of the cell extract, the target protein, and the extent of reaction optimization [1,64].

3.1.2. Disadvantages

CFPS in batch mode is typically characterized by short reaction times due to the depletion of ATP and GTP, degradation of mRNA, and accumulation of inhibitory byproducts [9,44,60,62]. Kim and Swartz [60] identified that phosphoenolpyruvate (PEP), commonly used as a secondary energy source, and several amino acids (particularly arginine, cysteine, and tryptophan) are rapidly degraded during batch CFPS reactions. Accumulating phosphate that sequesters magnesium ions significantly contributes to the early cessation of protein synthesis in batch formats [62]. Voloshin and Swartz [61] reported that target protein yields decrease dramatically in batch mode at reaction scales above 15 μL when using conventional test tube formats. The primary causes of yield reduction during scale-up include a decreased surface-to-volume ratio, limiting gas transfer, and reduced availability of hydrophobic surfaces that benefit protein expression and folding [61].

To address scale-up limitations, several innovations have been developed. Voloshin and Swartz [61] developed a “thin film” approach for batch CFPS scale-up, maintaining consistent protein yields across reaction volumes ranging from microscale (15 μL) to larger laboratory scale (500 μL), demonstrating scalability without compromising performance. For industrial applications, Dopp and Reuel [65] optimized scalable processes for E. coli extract preparation using continuous-flow French press for cell lysis and pilot-scale lyophilization for preservation. Zawada et al. [4] documented successful industrial translation of these techniques, achieving functional CFPS at the 100 L manufacturing scale, demonstrating the commercial viability of cell-free systems for biopharmaceutical production.

3.1.3. Applications

Batch CFPS, with its inexpensive, simple setup, is particularly suited to optimization work as well as preparative protein production on lab and industrial scales. Batch formats can be utilized for personalized medicine applications, as demonstrated by Kanter et al. [66], who synthesized cytokine-fused single chain antibody fragments (scFv) targeting B-cell lymphoma, reducing production time from months to just days. Zawada et al. [4] demonstrated the scalability of traditional batch formats by producing 700 mg/L of protein in a 100 L reactor. Scalability has also been shown with cell-free RNA production being performed in batches from 50 μL to 150 L with similar production per volume [63].

3.2. Well Plate Reactors

Well plate reactors utilize standard microtiter plates as reaction vessels for CFPS [67], offering a balance between throughput and reagent conservation.

3.2.1. Advantages

Well plate reactors operate at microliters scale (10–100 μL per well), making them ideal for screening applications while preserving materials. These systems require only standard laboratory equipment like plate readers and multichannel pipettes, resulting in low capital costs. Multiple reaction conditions can be screened in parallel, enabling the simultaneous testing of numerous variables. Well plates are easily compatible with automation [68]. Standard well plates can be adapted for CECF configurations with dialysis membranes to improve performance by removing inhibitors and replenishing depleted components [67].

3.2.2. Disadvantages

Product recovery is straightforward, but the small scale of well plate reactors limits total production capacity compared to larger reactors, making these systems unsuitable for preparative protein production; moreover, typical yields are relatively low, generally ranging from 0.01 mg/mL to 0.42 mg/mL [67,68]. These reactors typically rely on passive diffusion for mixing, though orbital shaking can improve mass transfer. Standard well plate reactors typically operate as batch systems, limiting reaction duration due to inhibitory byproduct accumulation [67].

3.2.3. Applications

Well plate reactors have become the workhorse platform for high-throughput applications in CFPS, enabling rapid screening, optimization, and small-scale production of proteins for analytical and functional studies. This format enables protein array construction and antibody validation using 384-well plates [69]. High-throughput screening of mutant enzyme libraries for improved properties can be efficiently conducted in well plate formats [39,70]. Sawasaki et al. [68] conducted large-scale biochemical annotation of proteins using 96-well plates. Well plates enable the systematic optimization of reaction conditions across multiple variables [55]. For complex proteins requiring co-factors or specialized folding conditions, well plate CFPS can be modified with additives like protein disulfide isomerase or oxidized glutathione to facilitate proper folding [39,70].

3.3. Fed-Batch Reactors

Fed-batch systems improve upon the standard batch format by adding additional reagents to the batch after the reaction has already started. By replenishing crucial substrates in this way, fed-batch methods significantly extend reaction duration and improve protein yields [22,38].

3.3.1. Advantages

Fed-batch systems significantly prolong protein synthesis by periodically replenishing critical substrates. This approach has been shown to double the reaction time in ribosome construction systems [22], with Kim et al. [38] reporting extended reaction duration up to 6 h. Fed-batch systems have demonstrated significant yield improvements, including a 75% increase in protein yield in ribosome construction systems [22] and a 72% improvement in therapeutic protein expression [56]. Typical protein yields using this method range from 0.7 mg/mL to 6 mg/mL [38,71]. Fed-batch approaches mitigate the inhibitory effects of accumulated byproducts such as inorganic phosphate and enable more efficient use of costly reaction components by supporting longer reactions from the same initial extract volume [22]. Fed-batch CFPS can be conducted across scales from microliters to liters, making it versatile for different production needs [71,72].

3.3.2. Disadvantages

Fed-batch systems require more complex experimental setups and operational protocols [22]. Automated equipment may be required to fully optimize the system. Implementation often requires specialized equipment for controlled substrate addition and monitoring, increasing initial investment costs [72]. Repeated substrate addition can dilute reaction components, affecting protein synthesis rates [56]. This also requires the consumption of more reagents per reaction than typical batch reactors. The optimal feeding strategy varies significantly depending on the protein target, extract source, and energy regeneration system employed.

3.3.3. Applications

Fed-batch CFPS has shown promise for producing larger protein yields than traditional batch formats while only introducing moderate complexity to the setup and procedure. It has been demonstrated to produce FDA-approved therapeutics like L-asparaginase with improved yields, enabling potential on-demand, point-of-care synthesis [56]. The method has improved the integrated synthesis, assembly, and translation of ribosomes in vitro, facilitating more efficient ribosome construction for synthetic biology applications [22]. Kim et al. [62] developed a highly productive fed-batch CFPS system that produced more than 1 mg/mL of recombinant proteins in just two hours. Integration with miniaturized fluid array devices has enabled high-throughput protein expression with up to 463-fold improvement compared to conventional batch formats [72].

3.4. Tube-in-Tube Reactors

Tube-in-tube reactors consist of a semi-permeable inner tube encased within a gas-impermeable outer tube. All CFPS components flow through the inner tube while oxygen gas flows through the volume between the two tubes, creating a well-defined interface between gas and liquid phases for enhanced gas-liquid mass transfer [57,73].

3.4.1. Advantages

Oxygen can penetrate and saturate the reaction liquid within approximately 30 s due to the short mass transfer distance (less than 0.3 mm) and large surface area to volume ratio (3000–10,000 m²/m³) [73]. The system allows for exact control of gas concentration in the liquid phase, enabling studies on the effects of different gas levels on protein synthesis [40]. Volumetric mass transfer coefficients are 0.1–1.0 s⁻¹, which is much higher than in conventional reactors [73]. This efficient gas transfer leads to faster reaction kinetics and higher yields than traditional batch reactors, with demonstrated 40% increased green fluorescent protein (GFP) yields in half the time [73]. Typical protein yields in tube-in-tube reactors range from 0.5 mg/mL to 1 mg/mL, reflecting their enhanced performance relative to other formats [74]. When integrated with analytical instruments, these reactors enable the continuous monitoring of reaction parameters, facilitating process optimization and kinetic studies [73].

3.4.2. Disadvantages

The specialized materials and construction required for tube-in-tube reactors increase complexity compared to simpler batch systems. The inner tube typically requires specialized materials like Teflon AF-2400 [73], which can be expensive and has specific handling requirements. Maintaining optimal flow rates in the gas phase requires carefully calibrated control systems. While effective at laboratory scale, scaling up tube-in-tube reactors for larger-volume production presents engineering challenges. The specialized nature of these reactors means fewer commercial options compared to standard reaction vessels.

3.4.3. Applications

Tube-in-tube reactors offer increased control over gaseous environments in CFPS, enabling researchers to optimize reaction conditions and investigate how specific gas concentrations affect protein production efficiency and quality. Lin et al. [40] used a tube-in-tube reactor to investigate the effect of oxygen concentration on protein synthesis machinery in Escherichia coli-based cell-free systems, discovering that 21% oxygen yielded optimal protein production. The precise gas control makes these reactors valuable for optimizing production conditions for oxygen-sensitive therapeutic proteins. The ability to rapidly achieve gas saturation enables detailed studies of reaction kinetics under precisely controlled gas environments [73]. The system could be adapted to study the effects of various gases on protein synthesis beyond oxygen, including hydrogen and carbon dioxide, which can also pass through the semipermeable inner tube [73]. Tube-in-tube reactors provide an excellent platform for process development and optimization before scaling up to larger production systems.

3.5. Foamed Batch Reactors

The foamed batch format pumps air into the batch reaction, creating a scaffolding of bubbles. This hydrofoam increases efficiency by providing increased surface area for oxygen transfer, thus overcoming one of the major limitations of batch reaction scale-up [41,61,75].

3.5.1. Advantages

Nelson et al. [41] demonstrated that implementing foam structures in CFPS reactions can substantially enhance protein production. Typical protein yields in foam reactors ranged from 0.3 mg/mL to 1.4 mg/mL. Their studies revealed yield improvements of nearly double compared to standard methods. A significant benefit of this technique is its simplicity—it requires only basic laboratory equipment and standard pipetting procedures. While traditional cell cultures often suffer damage from foam formation, CFPS systems do not contain intact cells and thus remain functional in foamed environments. This characteristic provides a unique opportunity to leverage foam’s beneficial properties without its typical drawbacks [41].

3.5.2. Disadvantages

The research revealed several limitations of the hydrofoam approach. While proteins with simple structural arrangements (like GFP) maintained their functionality, Nelson’s team observed that more complex proteins with quaternary structures experienced performance issues, likely due to the foam making it difficult for proper folding to take place [41]. For example, their work with Erwinase showed approximately two-fifths reduction in specific activity when produced in foam-based systems. The temporary nature of foam presents another challenge—observations showed foam volume diminishes over time, retaining only about 60% of its initial volume after 90 min. This degradation might necessitate periodic re-foaming during extended production runs [41]. Additional challenges include achieving consistent foam characteristics between batches and developing effective large-scale implementation strategies.

3.5.3. Applications

Foamed batch CFPS reactors show potential in the ability to generate higher yields than typical batch reactions without any need for complicated equipment. This approach shows particular promise when combined with shelf-stable reagent preparations, potentially enabling protein therapeutic production in areas with limited resources or infrastructure. Beyond therapeutics, the improved oxygen transfer capability could benefit various other CFPS applications, including biosensor development with enhanced sensitivity and biocatalytic processes involving oxygen-dependent enzymes [41]. Thus far, researchers have applied this technology to manufacture Erwinase, a therapeutic protein used in cancer treatment. Despite the reduction in specific activity, the substantially higher production volume resulted in greater overall therapeutic potential [41].

3.6. Emulsion-Based Reactors

Emulsion-based CFPS combines cell-free protein synthesis with emulsion technology to compartmentalize reactions in water-in-oil or more complex emulsion formats, offering advantages in miniaturization and high-throughput screening.

3.6.1. Advantages

By compartmentalizing reactions in microdroplets, researchers can dramatically reduce reaction volumes from microliters to picoliters, enabling substantial cost savings particularly when using expensive reagents like the PURE system [58]. Microfluidic devices can generate monodispersed droplets at rates of thousands per second, enabling the screening of vast libraries in short timeframes. Holstein et al. [34] developed a cell-free directed evolution platform in microdroplets that operates at “ultrahigh throughput,” allowing the rapid screening of millions of enzyme variants. Emulsion-based CFPS systems offer unprecedented control over reaction conditions at the individual droplet level, allowing researchers to conduct just one experiment that includes a wide variety of reagent concentrations [58]. The uniform size and composition of modern microfluidic droplets enhance experimental reproducibility compared to conventional emulsion techniques [76]. Water-in-oil emulsions allow the coupling of phenotype and genotype, where DNA molecules and their corresponding proteins can be confined exclusively within isolated compartments [77]. This compartmentalization is particularly valuable for evolving proteases and other proteins that might be harmful to host cells [34].

3.6.2. Disadvantages

The stability of emulsion droplets plays a crucial role in the success of CFPS reactions. Single emulsions (water-in-oil) are often insufficient for longer reactions, particularly when high salt concentrations are present [58]. Despite advances in emulsion formation technologies, there remains some heterogeneity in droplet composition because of the inherent randomness of encapsulation, particularly pronounced at nanoscale volumes [78]. Due to the inherent randomness of droplet formation and their intentionally small size, achieving consistent and high protein yields is challenging, typical concentrations range from 0.2 μM to 2.4 μM [26,76,78]. When CFPS occurs in cell-sized compartments, the surface-to-volume ratio significantly impacts gene expression patterns, with the compartment’s surface introducing regulatory effects on gene expression [79,80]. Creating stable emulsions with consistent droplet sizes requires specialized equipment and expertise, including microfluidic devices with precise flow control systems [58]. In small-volume droplets, resource depletion can occur rapidly, limiting the duration of protein production. Energy sources and building blocks for protein synthesis cannot be readily replenished as in continuous-flow systems. A significant challenge in emulsion-based CFPS is the recovery of synthesized proteins from droplets. The extraction process often involves breaking the emulsion, which can lead to product loss or denaturation.

3.6.3. Applications

Emulsion-based CFPS systems excel in extremely high-throughput screening, which has applications for process optimization and directed evolution of biological materials. Emulsion-based CFPS systems also play a crucial role in synthetic biology efforts to create minimal cells.

Holstein et al. [34] took advantage of emulsions’ high-throughput capability to direct the evolution of a protease that is toxic to E. coli and identified an enzyme variant that functions five-fold faster. Gan et al. [33] similarly utilized emulsions to rapidly create, analyze, and optimize genetic elements controlling protein expression. Gan et al. [77] performed both PCR and CFPS inside of emulsions before displaying proteins on microbeads, achieving a 917-fold enrichment of targeted sequences in one round of screening.

In the realm of synthetic biology, Caschera et al. [26] successfully demonstrated protein synthesis within liposomal, double-emulsion compartments. Nuti et al. [81] demonstrated simultaneous production and screening of antimicrobial peptides, enabling the evaluation of membrane specificity for developing new antimicrobials. Lu et al. [82] developed an innovative system combining Pickering emulsions stabilized by enzyme-polymer conjugates with CFPS for cascade biotransformation, resulting in significantly improved catalytic performance.

3.7. Continuous Reactors

CFCF formats overcome the limitations of batch systems by establishing ongoing exchange of substrates and removal of byproducts, enabling extended reaction times and higher total protein yields. Substrates are continuously pumped into the reactor, while an ultrafiltration membrane allows the product and byproducts to flow out [9].

3.7.1. Advantages

The CFCF system allows protein synthesis to continue for 40 h or longer, which is a significant extension over batch systems [9]. Continuous removal of inhibitory byproducts prevents their accumulation and the associated inhibition of protein synthesis [9]. This extended reaction time often increases total protein yields compared to batch systems, with some systems demonstrating two orders of magnitude improvement [9,37]. Continuous-flow reactors consistently achieve protein yields around 1 mg/mL, reflecting their enhanced productivity over other formats [37].

3.7.2. Disadvantages

The productivity of CFCF systems are inferior to batch systems on a per-time basis, primarily because the ultrafiltration membrane can cause leakage of valuable translation components or can retain synthesized product in the reactor [44,83,84]. This reduced reaction rate can be so severe that even the longer reaction time does not always result in a significant increase of production over simple batch systems [44]. It has been shown that cell-free reaction components can precipitate and thus clog filtration membranes [85], which could impair efficient substrate exchange in continuous systems. CFCF systems require complex apparatuses that make them impractical for routine laboratory use [37]. These systems generally involve higher complexity and cost compared to batch systems. CFCF systems have a strict requirement for solubility of protein product and are not applicable to high-molecular-weight protein production due to the fear that a membrane which allows large products to pass through would also allow the cellular machinery to escape. To address this issue, Lamla et al. [84] designed a semi-continuous-flow system in which the product is retained in an affinity column and only unwanted byproducts flow out through the ultrafiltration membrane.

3.7.3. Applications

Continuous reactors represent a significant advancement in CFPS technology, enabling prolonged reaction times, higher protein yields, and integrated purification capabilities for industrial applications. However, practical applications are severely limited by the cost and complexity of such reactors. The principles of continuous flow have been applied to larger-scale systems, contributing to the development of industrial CFPS processes. Lamla et al. [84] integrated an affinity-tag purification system directly into the CFPS reactor, allowing continuous removal of synthesized protein from the reaction chamber. Yamamoto et al. [86] developed a hollow fiber membrane CFCF reactor with a large filtration membrane surface area for increased condensation of the reaction mixture, with 10-fold condensation raising protein production 20-fold.

3.8. Microfluidic Reactors

Microfluidic CFPS systems operate at nanoliter-scale volumes, utilizing networks of small channels and chambers to offer advantages in reagent conservation, the precise manipulation of reaction conditions, and integration capabilities for various applications [87].

3.8.1. Advantages

Microfluidic CFPS systems significantly reduce reagent consumption while maintaining high-throughput capabilities compared to traditional bench-scale reactions. The microfluidic environment improves CFPS productivity through better heat and mass transfer characteristics [87], with systems producing proteins at high concentrations (e.g., 700 ng/μL of GFP) in relatively short timeframes [88]. Microfluidic formats provide increased control over reaction conditions, including temperature, residence time, and reagent concentrations, enabling optimization of complex reactions [45,89]. Unlike batch reactions, microfluidic devices maintain steady-state conditions through continuous-flow or controlled reagent exchange, enabling sustained protein production over extended periods [90]. Microfluidic CFPS systems are particularly suitable for point-of-care production due to compatibility with lyophilized starting materials, which remain stable at typical temperatures, eliminating the need for cold storage [88].

3.8.2. Disadvantages

Microfluidic CFPS systems require specialized equipment and expertise for fabrication and operation, potentially limiting accessibility compared to simpler batch reactions. The high surface-to-volume ratio in microfluidic channels can lead to non-specific protein adsorption to channel surfaces, particularly in polydimethylsiloxane-based systems, resulting in reduced yields and requiring surface modifications to mitigate [45]. While excellent for small-scale applications, scaling up microfluidic systems for larger production volumes presents significant engineering challenges. Small channel dimensions make microfluidic systems susceptible to clogging from particulates or protein aggregates. While parallelization is possible, the volumetric production of individual microfluidic channels is inherently limited by their small dimensions.

3.8.3. Applications

Microfluidic CFPS systems allow the rapid testing of different conditions and genetic constructs with minimal reagent consumption, enabling the efficient optimization of expression parameters. Their small scale has also shown promise for applications in on-site therapeutic production [88]. Niederholtmayer et al. [90] demonstrated the potential for efficient optimization by individually regulating each step of protein production. Microfluidic platforms enable on-demand, point-of-care production of therapeutic proteins at clinically relevant quantities (e.g., antimicrobial peptides at 63 ng/μL with 92% purity), eliminating distribution challenges [88]. Timm et al. utilized an extended channel microfluidic reactor to extend reaction times and increase the reaction volume sufficiently to produce a whole dose of a therapeutic on their microfluidic chip [89]. These platforms can integrate protein purification directly into the production workflow, outperforming conventional methods with higher recovery rates (54.6% vs. 39.6%) and shorter processing times (<40 min vs. 1–1.5 h) [88]. By spatially separating protein synthesis and modification processes, microfluidic systems enable precise control over complex pathways like glycosylation [87]. The ability to rapidly test multiple conditions in parallel makes microfluidic systems valuable for screening applications in drug discovery and protein engineering.

3.9. Dialyzed Reactors

Dialyzed CFPS, also known as continuous-exchange cell-free, employs a semi-permeable membrane to separate the reaction mixture and feeding mixture, providing continuous nutrient supply while removing inhibitory byproducts [44].

3.9.1. Advantages

The constant addition of necessary substrates significantly extends reaction times. Additionally, the continuous removal of inhibitory byproducts through the dialysis membrane prevents their accumulation and the associated inhibition of protein synthesis. Dialyzed systems achieve yields between 0.4 and 10 mg/mL, significantly higher than standard batch reactions [72,91]. This reactor format provides these enhanced yields without being as complicated to setup and run as continuous-flow reactors. Kigawa et al. [59] demonstrated yields of 6 mg/mL, while Takeda et al. [91] reached 5–10 mg/mL using a bilayer-dialysis method. Kim and Choi [44], initial users of this system, achieved continuous synthesis for at least 14 h compared to the much shorter duration of batch reactions. Jackson et al. [72] demonstrated that optimizing important factors, such as the volume ratio of feeding solution to reaction mixture, the interface surface area, and the height difference between feeding solution and reaction mixture, resulted in a yield increase up to 463-fold. The approach can be adapted for high-throughput formats [92].

3.9.2. Disadvantages

Precipitation of reaction components can lead to membrane clogging, reducing efficiency over time, though vertically oriented membranes can help mitigate this issue [85]. The rate of exchange across the membrane can become limiting for high-yield reactions, requiring optimization of membrane properties and reaction conditions. The dialysis setup is more complex and expensive than simple batch reactions, requiring additional components and more careful assembly. Maintaining an optimal feeding-to-reaction volume ratio (at least 20:1) is necessary for maximum expression, which can increase the overall system cost and the quantity of reagents required [72].

3.9.3. Applications

CECF excels at extending reaction times to produce large quantities of protein (up to 10 mg/mL) without the complexities of continuous CPFS, although they are significantly more complicated than batch [91]. Dialyzed systems also demonstrate good scalability and throughput capabilities. The dialysis format has become valuable for structural studies, with Kigawa et al. [59] demonstrating stable isotope labeling for NMR spectroscopy. Shimono et al. [93] expressed functional bacteriorhodopsin, while Periasamy et al. [94] achieved co-translational insertion of membrane proteins into liposomes during wheat germ CFPS. Takeda et al. [91] produced high-quality monoclonal antibodies against G-protein-coupled receptors using cell-free synthesized antigens. Aoki et al. [92] developed automated systems completing synthesis to purification within 14 h for up to 96 proteins simultaneously.

3.10. Bilayer Reactors

Bilayer systems offer a significantly simplified setup compared to continuous-flow or dialysis methods as the reaction mixture is simply layered below a feeding solution without requiring a separating membrane [43,95]. This batch design allows for continuous protein synthesis through passive diffusion of substrates and removal of byproducts along the concentration gradient between layers [43,95].

3.10.1. Advantages

Bilayer reactors represent a simplified alternative to continuous-flow CFPS systems that eliminates the need for a semi-permeable membrane while maintaining high productivity through a diffusion principle [43]. The bilayer approach demonstrates remarkably extended reaction duration, functioning more than 10 times longer than typical batch reactions [43]. Production yields are substantially higher than batch methods, ranging from 0.01 mg/mL to 8.5 mg/mL [43,91,96]. The method offers excellent scalability, working effectively in formats ranging from 96-well microtiter plates for high-throughput screening to larger wells for increased protein production [95]. Simplified automation is possible due to the absence of semi-permeable membranes, with robotic systems like “GenDecoder” and “Protemist” enabling parallel small-scale and large-scale synthesis, respectively [95]. The bilayer method is more efficient than the batch mode and simultaneously easier to perform than dialysis-based methods, offering a balance between complexity and productivity [43].

3.10.2. Disadvantages

The efficiency of bilayer formats may be affected by the container shape and the ratio of volume to contacting surface area, which affects substrate exchange rates [95]. The wheat germ extract used in many bilayer systems is relatively expensive since it often requires manual selection of wheat embryo particles by human operators to ensure extract quality [95]. While the bilayer method improves reaction duration compared to batch systems, it still cannot match the sustained production capability of dialyzed or continuous-flow systems for very long reactions [9,43,95].

3.10.3. Applications

Bilayer systems have primarily been utilized with wheat-germ based cell-free protein synthesis and have been very successful in increasing the yields from such systems [43,68,95,96,97]. The ease of setup and lack of sophisticated equipment means bilayer systems excel at high-throughput applications. The ability to use wheat-germ extracts to produce significant yields also enables some unique applications, such as protein structural studies and the production of proteins at higher solubility levels [95].

Researchers have demonstrated the potential of bilayer systems for high-throughput screening by successfully synthesizing 12,996 proteins from randomly chosen 13,364 human proteins [98]. The bilayer cell-free system has been utilized to successfully express functional plant solute transporters and reconstitute them into liposomes, creating proteoliposomes that enabled detailed functional analysis of these membrane proteins [97]. For biochemical characterization, the bilayer approach has enabled parallel synthesis and functional validation of proteins, as demonstrated with 439 Arabidopsis thaliana protein kinases that were tested for substrate specificity after translation [68]. The bilayer method has also been successfully applied to synthesize challenging G-protein coupled receptors like the human dopamine D2 receptor long isoform using wheat germ-based cell-free systems [96].

3.11. Lyophilized Reactors

Lyophilized (freeze-dried) CFPS systems offer significantly enhanced stability and storage capabilities, making cell-free protein synthesis accessible in field settings without cold-chain requirements. The entire reaction can be lyophilized together or as individual components, and it can be rehydrated and recombined to initiate CFPS when or wherever it is needed.

3.11.1. Advantages

While aqueous extracts lose nearly all activity within one week at room temperature, properly formulated lyophilized extracts can retain full activity after 30 days [99]. At refrigeration temperatures (4 °C), the trend is the same, with lyophilized extracts maintaining more than 70% activity for at least 90 days compared to aqueous extracts’ 30-day stability [52]. Some formulations can actually improve productivity beyond the original aqueous extract, with certain formulations demonstrating productivity increases up to 195% compared to the benchmark [100]. Protein yields from lyophilized systems can range from 0 to 1 mg/mL, depending on storage temperature and duration [52]. Lyophilization enables higher density storage by reducing extract volume and mass by a factor of 2 and 9, respectively [52]. Bacterial contamination is significantly reduced in lyophilized extracts, which contain no bacterial colonies after 30 days at room temperature, while aqueous extracts become heavily contaminated [52]. The enhanced stability enables deployment in resource-limited settings without cold-chain infrastructure.

3.11.2. Disadvantages

Most lyophilized extracts initially maintain approximately 85% of fresh aqueous extracts’ protein synthesis capacity [52], with yields continuing to decrease after extended storage at elevated temperatures [99]. The most effective stabilizing formulations can involve complex mixtures of additives, increasing costs and preparation complexity [53]. The stability of high-energy compounds such as nucleotide triphosphates remains challenging, with alternative energy systems based on more stable components showing significantly lower productivity [52]. Optimization of lyoprotectants and cryoprotectants should be determined for each CFPS system due to differing results across the literature [99]. While lyophilized CFPS systems show tolerance to many organic solvents, this tolerance varies by solvent type and CFPS formulation [101].

3.11.3. Applications

Lyophilized CFPS enables simple rehydration protocols for field applications, particularly in terms of biosensors. Single-pot formulations containing both extract and energy components maintain most of its initial productivity compared to conventional systems after two weeks at room temperature [100]. Paper-based systems with lyophilized CFPS on paper discs allow for modular assembly [99]. Modular systems combine lyophilized extracts with separately stored powdered energy systems, amino acids, and DNA templates [52].

On-demand therapeutic production applications include endotoxin-free FDA-approved enzymes with full activity [53], conjugate vaccines produced for as little as ~USD 0.50 per dose after storage at 37 °C for 4 weeks [24], cancer therapeutics that can be stored for a year at temperatures above freezing [17], and peptide hormones for point-of-care production [102].

Biosensing applications include cost-effective RNase inhibitor production for CFPS-based biosensors [103], paper-based synthetic gene networks for virus detection [30], improved sensor performance through optimized lyophilization-to-rehydration ratios [104], and wearable COVID-19 detection systems [31].

3.12. Wearable Reactors

Wearable CFPS systems leverage flexible materials and freeze-dried reactions to create portable biosensors and biomanufacturing capabilities for point-of-use applications.

3.12.1. Advantages

Wearable CFPS systems utilize lyophilization techniques that preserve biomolecular components in a stable format on porous substrates. Nguyen et al. [31] displayed that this allowed for the system to be stored, transported, and used without cold-chain or other specialized conditions. Lyophilized cell-free reactions can be activated by rehydration, permitting long-term storage and on-demand use. Once rehydrated, wearable CFPS systems function autonomously without requiring continuous monitoring or external input, making them valuable for diagnostic applications where user intervention should be minimized. The flexible materials used in wearable formats allow for comfortable integration with personal items like face masks and clothing. These systems can operate under typical environmental conditions, including common humidity levels and temperatures [31].

3.12.2. Disadvantages

Current CFPS systems, especially those based on purified components like the PURE system, can be costly. Extract-based systems may offer more economical alternatives for wearable platforms but typically provide less precise manipulation of reaction conditions [105]. While suitable for sensing applications, wearable formats typically have limited capacity for protein production compared to conventional reactors. Wearable CFPS biosensors are limited by their one-time usability [31]. Despite improvements, environmental factors like extreme temperatures and humidity can still impact performance. Creating intuitive signal readout mechanisms for non-specialists remains challenging. Combining biological components with wearable materials while maintaining functionality requires careful design and materials selection.

3.12.3. Applications

Wearable CFPS reactors are primarily suited for point-of-care diagnostics, environmental monitoring, and personalized healthcare applications where decentralized, real-time detection capabilities are essential. These systems transform conventional laboratory-based bioassays into portable, user-friendly formats that can be deployed in resource-limited settings or integrated into everyday items for continuous monitoring. By bringing biological sensing capabilities directly to the point of need, wearable CFPS platforms enable rapid decision-making without requiring laboratory infrastructure. Nguyen et al. [31] embedded genetic circuits into flexible elastomer-contained cellulose substrates to create biosensors that can detect various targets, including anhydrotetracycline, Ebola virus RNA, and small molecules like theophylline. Such wearable sensors incorporate hydrophilic threads to immobilize reagents, hydrophobic elastomers to contain the reaction system, and polymeric optical fibers for verification of proper function, detecting fluorescent and luminescent outputs with high sensitivity. Wearable CFPS sensors utilizing CRISPR-Cas systems demonstrate femtomolar detection limits for nucleic acids, integrated into applications such as face masks capable of detecting SARS-CoV-2 from respiratory aerosols with sensitivity similar to laboratory PCR methods [31].

3.13. Paper-Based Reactors

Paper-based platforms use various cellulose substrates as matrices for CFPS, offering advantages in portability, stability, and ease of use compared to traditional liquid-phase reactions.

3.13.1. Advantages

Lyophilized cell-free reactions on paper discs can be stored at room temperature while maintaining functionality [99]. Optimized formulations with β-lactose and maltodextrin can achieve 100% preservation of activity at room temperature for up to one month [99]. Hunt et al. [106] reported that at room temperature, lyophilized paper-based CFPS biosensors are stable for up to 7 weeks. Paper formats enable simple “just-add-sample” applications where the sample liquid rehydrates the lyophilized reaction components. Paper-based systems can be manufactured at very low cost, with some diagnostic tests costing less than USD 0.50 per test [106].

3.13.2. Disadvantages

Paper matrices can only hold a finite volume of liquid, limiting the total reaction volume and potential yield. Mass transfer in paper matrices relies primarily on diffusion, which can be slower than in well-mixed liquid systems. Paper fibers can potentially bind to some reaction components, affecting their availability. Different paper types and treatments result in different wetting properties, which can affect reaction uniformity [99,106]. Detecting results from paper-based reactions may require optimization due to background signals or absorbance from the paper substrate [107].

3.13.3. Applications

Paper-based reactors have emerged as versatile platforms for cell-free protein synthesis applications, particularly in the development of accessible, low-cost biosensors for environmental monitoring, medical diagnostics, and public health surveillance. Zhang et al. [108] developed a paper-based biosensor employing allosteric transcription factors for the detection of heavy metals in water, detecting mercury at concentrations as low as 0.5 nM and lead at 0.1 nM. Gräwe et al. [109] developed a paper-based biosensor system capable of detecting harmful substances like mercury and date rape drugs such as gamma-hydroxybutyrate (GHB), along with a two-filter system compatible with conventional smartphones to detect fluorescence signals, eliminating the need for expensive specialized equipment. Cao et al. [110] created a paper-based cell-free system to detect and identify respiratory syncytial virus (RSV) subgroups using colorimetric toehold switch sensors. Hunt et al. [106] developed a paper-based CFPS toehold switch biosensor for SARS-CoV-2 RNA detection in saliva that gives off a bioluminescent signal in just seven minutes after rehydration. Free et al. [111] presented simple, inexpensive methods for diluting and filtering blood samples so that blood tests could be conducted at home on a paper-based biosensor.

4. Comparative Analysis

Figure 4 provides a comparative overview of the different reactor types discussed in this review, qualitatively highlighting their relative performance across key metrics, including scale-up potential, throughput capability, cost efficiency, shelf stability, operational complexity, product recovery, and process control.

4.1. Operational Complexity vs. Performance

Traditional batch systems represent the foundation of CFPS technology, offering unparalleled operational simplicity and accessibility. These systems require minimal specialized equipment and technical expertise, making them ideal entry points for new researchers, laboratories with limited resources, and educational kits. However, batch reactions face fundamental limitations in reaction duration due to rapid depletion of energy sources and accumulation of inhibitory byproducts. Recent innovations have addressed these constraints, with improved energy mixes and optimized extract preparation protocols significantly enhancing batch reaction performance while maintaining simplicity [38,65]. Similarly, well plate formats extend this accessibility to high-throughput applications, enabling efficient screening for protein engineering and optimization studies [70].

4.2. Advanced Exchange Systems

More sophisticated reactor configurations—including fed-batch, continuous, bilayer, and dialysis systems—overcome the inherent limitations of batch reactions through controlled substrate exchange mechanisms. By continuously or periodically replenishing critical components while removing or diluting inhibitory byproducts, these systems substantially extend reaction durations from just a few hours to 20+ hours [9]. This extension translates directly to dramatically improved protein yields, with continuous and dialyzed systems demonstrating up to 100-fold increases in productivity compared to batch formats [9,72]. The bilayer approach offers a particularly elegant compromise, achieving much of this improvement without requiring complex membrane systems. However, these performance gains come at the cost of increased technical complexity, requiring specialized equipment, more sophisticated setup procedures, and greater expertise to implement effectively.

4.3. Miniaturized High-Throughput Systems

Microfluidic and emulsion-based CFPS formats represent a paradigm shift in reaction scale and throughput capability. These systems dramatically reduce reaction volumes from microliters to nano- or even picoliters, enabling substantial reagent conservation while simultaneously increasing experimental capacity. Microfluidic platforms leverage precise fluid handling and enhanced mass transfer to improve productivity, with some systems achieving protein concentrations of 700 ng/μL in compact devices [88]. Emulsion-based approaches compartmentalize reactions in water-in-oil droplets, enabling the screening of vast libraries with millions of variants in single experiments [34]. This unprecedented throughput has revolutionized directed evolution applications, allowing researchers to evolve enzymes that would be toxic to living cells. While these miniaturized systems require specialized equipment and expertise to implement, they unlock applications that would be impossible with conventional formats, particularly for protein engineering and high-throughput screening.

4.4. Field-Deployable Systems

Paper-based and wearable CFPS platforms incorporating lyophilized components represent the frontier of portability and environmental stability in cell-free technology. These systems preserve the complex biochemical machinery of CFPS in a dormant, dehydrated state that can be stored at ambient temperatures for extended periods—with up to 100% activity retention after one month at room temperature with optimized formulations [99]. Upon simple rehydration, these systems rapidly activate to produce proteins or generate diagnostic signals. Paper substrates enable “just-add-sample” applications costing less than USD 0.50 per test [24], while integration with wearable items like face masks creates continuous monitoring capabilities [31]. These formats operate effectively across variable environmental conditions, eliminating cold-chain requirements and specialized equipment needs. This revolutionary stability and simplicity have enabled applications from point-of-care therapeutic production to environmental sensing in resource-limited settings, expanding CFPS technology beyond traditional laboratory environments to address global challenges in health care access and environmental monitoring.

5. Remaining Limitations in Cell-Free Protein Synthesis Reactors

Despite the diverse array of reactor formats developed for cell-free protein synthesis and their individual advantages, several fundamental limitations persist across the field that continue to hinder optimal performance and widespread adoption.

5.1. Cross-Format Compatibility and Standardization

A major challenge facing the CFPS field is the lack of standardization across different reactor formats and extract preparations. Research has been conducted on multiple different cell-free extract sources, genetic strains, and microorganisms (including E. coli, wheat germ, yeast, rabbit, HeLa, CHO, and human blood), leading to variability that affects reactor performance and product consistency [74]. This diversity, while beneficial for specialized applications, creates difficulties in comparing results across studies and translating optimized conditions between reactor formats. Additionally, even within the same strain, various components in the cell extract can have different activities depending on the growth media used and at what stage of growth cells were harvested [74]. Contradictions in results across the literature suggest that optimization of reaction components, particularly lyoprotectants and cryoprotectants, need to be determined individually for each CFPS system [99], preventing the development of universal protocols that could accelerate reactor development.

5.2. Fundamental Energy and Substrate Limitations

While various reactor formats have addressed duration limitations through different exchange mechanisms, underlying issues with energy metabolism remain problematic across all systems. The high-energy molecules that fuel CFPS reactions, such as ATP and GTP, are the single most expensive part of producing proteins in a cell-free environment [112]. The temperature stability of high-energy compounds such as nucleotide triphosphates remains challenging, with alternative energy systems based on more stable components showing significantly lower productivity [52].

These energy limitations create a fundamental trade-off between reaction duration, cost, and performance that affects all reactor types. Even advanced continuous systems that successfully extend reaction times through substrate replenishment face economic constraints that limit their practical implementation at industrial scales.

5.3. Process Monitoring and Control Gaps

CFPS systems require precise external control of multiple parameters simultaneously, such as temperature [20] and oxygen availability [73]. Due to the utilization of cell extracts without an exact knowledge of all the components and concentrations in the lysate, controlling and predicting the biological activity of the synthesized proteins remains difficult [74]. This limitation is compounded by the open nature of cell-free systems, which allows for direct manipulation but also requires careful control of multiple variables including redox potential, energy substrate levels, pH. and protein folding conditions.

Current reactor systems across all formats often lack sophisticated real-time monitoring capabilities that would enable optimal process control. The need for monitoring protein folding, energy metabolism, and product formation simultaneously requires advanced analytical capabilities that are not yet fully integrated into most reactor designs, regardless of their specific format or scale.

5.4. Regulatory and Infrastructure Barriers

While CFPS systems avoid certain regulatory hurdles associated with genetically modified organisms, they encounter distinct challenges in meeting Good Manufacturing Practice (GMP) standards. Key issues include defining critical process parameters and ensuring production aligns with GMP requirements. Additionally, compliance with International Council for Harmonisation (ICH) guidelines (specifically ICH Q5, Q8, Q9, and Q10) is essential for gaining the market approval in regions such as the USA, Europe, Japan, Brazil, Canada, China, and others [112].

The infrastructure requirements vary dramatically across reactor formats, from simple batch systems requiring minimal equipment to sophisticated microfluidic and continuous systems demanding specialized facilities. This variability makes it difficult to establish standardized manufacturing protocols and regulatory pathways that could facilitate the broader adoption of CFPS technology.

5.5. Technology Transfer and Scalability Gaps

A persistent challenge across the CFPS field is the difficulty in translating promising laboratory-scale results to practical applications. Current cell-based technologies are only suited for large-scale protein production in costly industrial facilities [112]. CFPS systems have the potential to overcome this limitation of cell-based formats by offering solutions across multiple scales. However, despite advancements in efficiency, CFPS continues to face unique challenges at each scale transition [113].

The gap between proof-of-concept demonstrations and practical implementation is particularly pronounced for advanced reactor formats like microfluidics and wearable systems, where laboratory successes have not yet translated to robust, field-deployable platforms. This technology transfer challenge is exacerbated by the need for specialized expertise and equipment that may not be readily available in industrial settings.

5.6. Future

Several promising developments suggest these limitations may be addressable. Data-driven approaches using artificial intelligence have demonstrated remarkable optimization potential, achieving significant yield increases while testing a limited number of formulations from approximately millions of possible compositions [54]. The integration of such computational approaches with the diverse reactor formats already developed may provide pathways to overcome current standardization and optimization challenges.

The field’s strength lies in its diversity of reactor formats, each addressing specific applications and constraints. However, realizing the full potential of CFPS technology will require addressing these cross-cutting limitations through coordinated efforts in standardization, process control, and regulatory framework development.

6. Conclusions

This review presents a comprehensive comparative analysis of CFPS reactor formats and their intended applications. The evolution of CFPS reactors from basic research tools to versatile biomanufacturing platforms highlights their significant potential in addressing complex challenges in biotechnology and health care. Our analysis reveals a fundamental trade-off between reaction yield and operational complexity across all reactor formats. While simple batch systems offer unparalleled accessibility and ease of use, they are limited by short reaction durations and modest yields. Conversely, advanced systems like continuous-flow and dialyzed reactors can achieve dramatically higher yields (up to 100-fold improvements) but require specialized equipment and technical expertise. Additionally, oxygen transfer limitations persist across multiple reactor formats, particularly affecting scale-up potential, while temperature control remains critical yet often under-addressed, with most reactor designs requiring careful thermal management that adds operational complexity and limits deployment flexibility.

Looking forward, we identify two critical research directions that could address the most pressing challenges in CFPS reactor development. First, systematic investigation of reactor material composition and surface properties is a promising but under-explored field, as the choice of reactor materials significantly affects protein and small molecule adhesion, impacting reaction efficiency, product recovery, and system reproducibility. Understanding and optimizing these material–biomolecule interactions could lead to substantial improvements in reactor performance while maintaining operational simplicity. Second, developing adaptive, intelligent reactor systems that automatically balance the complexity–yield trade-off represents a transformative opportunity. Such systems would integrate real-time monitoring with automated control mechanisms to optimize reaction conditions dynamically, potentially achieving high yields while maintaining operational simplicity through machine learning algorithms and modular designs.

By selecting appropriate reactor formats for specific applications and pursuing these critical research directions, researchers can further expand the capabilities and accessibility of this powerful technology. The future success of CFPS will depend on developing integrated solutions that address the fundamental challenges of complexity, scalability, and ease of use while maintaining high performance for demanding biotechnology and medical applications.