Bioprocess engineer Beatrice Melinek is a postdoctoral research fellow at University College London’s Future Targeted Healthcare Manufacturing (FTHM) Hub, where she focuses on the use of cell-free protein synthesis (CFPS) as a platform for distributed production of stratified biotherapeutics. Previously Melinek specialized in purification of viral vectors and vaccines, with an engineering doctorate (EngD) in biochemical engineering and postdoctoral experience in UCL’s hematology department developing a new chromatography-based analytical method for measuring empty and full adenoassociated virus (AAV) capsids. She also worked on modifications to a polio vaccine production process at Univercells Technologies. Before that, she spent six years on a dynamic process simulations team for the Atkins Global engineering and design firm before returning to academia.

Melinek holds two master’s degrees: one in chemical engineering from Imperial College London, and the other in biochemical engineering from UCL. Last year, she was lead and corresponding author of a report on cell-free expression published in BPI’s September issue (1). I caught up with her again this spring to follow up.

Our Conversation
We’re talking about expression titers and volumetric productivity elsewhere in this supplement. How does CFPS currently compare with cell-based systems in this respect? And what’s the near and long-term future outlook? As a rule, titers from CFPS are lower than those from cell-based systems by an order of magnitude. At the limit, titers of 3 g/mL have been achieved in CFPS, whereas 10 g/mL have been achieved with Chinese hamster ovary (CHO) cells (1). Increasing CFPS titers is likely to be key to making the technology cost competitive (2).

In CFPS, the bulk protein production rate is 200‑fold slower than the in vivo rate (3). But you have the flexibility to begin that reaction at will by combining the necessary reagents — so, without that level of control, the timescale of cell-based cultures actually is much longer.

There probably is a limit to the titers that can be achieved in either type of system, but we expect the improvements going forward to be greater for CFPS than for cell-based systems. That assumption is based on

• increasing interest and investment in CFPS helping us grow our understanding
• diminishing returns with CHO-based production
• ongoing increases in maximum CFPS titers achieved (1).

Additionally, we speculate that (because in CFPS, no energy needs to be diverted to keeping cells alive) final titers achievable could be theoretically much higher in CFPS than in cell-based systems (4).

Can CFPS systems perform complex glycosylation and other posttranslational modifications? Is the process more or less controllable than cell-culture production of complex proteins? Cell extracts from some sources (e.g., CHO, insect, human K562, and plant cells) can perform glycosylation according to their native structures, with a modified preparation process to ensure that the required structures remain intact. Tinafar et al. nicely illustrate the currently available PTMs in a 2019 publication (5).
Hershewe et al. claim that tighter control of glycoproteins is possible in CFPS (6). It may be that — as with antibody–drug conjugates (ADCs) — this will be a key advantage for CFPS in the long term. It also could accelerate research on glycoproteins through faster prototyping. Similarly, for disulfide bond production, CFPS can require specific extract sources or strains and a disulfide-bond–forming enzyme (7). One complexity comes from the potential need to balance conditions for protein production and PTMs.

What biopharmaceutical proteins/peptides have been produced most successfully using CFPS so far? Have any companies made it to clinical studies with one? Sutro Biopharma is the leader in this field and working with nonnatural amino acids to improve quality control of ADCs. Sutro is in phase 1–1b trials with a number of candidates.

Even as groups such as the AltHost Consortium are working on new biological expression systems, CFPS developers are experimenting with new cell-extract sources. And what are the most promising platforms for biotherapeutic applications currently? As you say, a wide range of cell-extract sources is being considered. Each type has its pros and cons. With cost in mind, we’re working with Escherichia coli. Other researchers will be interested in specific PTMs, which could limit the source materials they can use. Enzymatic approaches can be applied, however, or may be developed. One idea I like conceptually is using blood as an extract source in what could be a truly personalized approach, although clearly a great deal of work is needed to make that viable (8).

The COVID-19 pandemic has made everyone more aware of our need for rapid development of vaccines and drugs against infectious agents. Some nascent technologies such as mRNA have moved forward much more quickly than most of us thought possible. How can CFPS help address future needs here? A number of applications of CFPS are highly relevant to fighting pandemics:

• paper-based diagnostics, which if perfected could make large-scale rapid testing much cheaper, could be particularly relevant in low- to middle-income countries (LMICs)
• diagnostics embedded in clothing, such as a facemask that could show whether its wearer is infected
• high-throughput, rapid prototyping of constructs that could be used to optimize vaccine designs before proceeding either with CFPS or cell-based production (9) and constructs for treatment (e.g., binding the coronavirus spike protein).

CFPS has the potential to enable local vaccine manufacturing with minimal equipment and (if components are lyophilized) even without the need for a cold chain. Stark, et al. propose an on-demand, point-of-care system for manufacturing conjugate vaccines (10). Adiga et al. made a manufacturing system in a suitcase for therapeutic protein treatment in hard-to-reach locations and difficult situations (11).

As with other sectors in biotechnology, a number of research groups in CFPS have pivoted to focus on the pandemic problem. We’re likely to find out how much impact this has had in the coming months and years. Some broader impact may reverberate over time: e.g., recognition of the need for localized manufacture and technology transfer between companies to meet demand, for which CFPS has potential advantages over conventional methods.

With cell extracts as a raw material, CFPS moves many traditional concerns of biologic production “upstream” rather than eliminating them. What viral safety issues come with CHO and HeLa extracts, for example? And how is raw-material consistency determined and ensured? The question of viral safety is not one I’ve considered. From what I know, it seems logical that the same risks apply to CFPS as to cell-based systems. It is possible to sterile-filter extracts (at least for those from E. coli), and some lyophilization conditions might be used to maintain extract activity while inactivating or killing viruses. This is not my area of expertise. Further, you could imagine designing an analytical method (using the cell extract itself as the engine) to detect common viral contaminants in test samples of the extract. Downstream processing for CFPS-based processes is unlikely to be very different from that for cell-based processes — including a number of steps that play a role in removing viruses.

The question of determining raw-material consistency is a big one, especially to industrialists with whom we’ve spoken on the FTHM Hub. We’re currently working on a review of this very question. One argument could be made that (as with protein therapeutics) a process controlled appropriately should produce consistent results — and that would apply to producing extract, too. Demonstrating consistency of extract, however, is very much an open question. Partial solutions include using reporter proteins, testing for ribosome activity, measuring mRNA production rates, and checking for nuclease activity. A suite of analytics probably will be required to release a batch of extract.

Do you foresee some current facilities used for biologic production transforming to manufacture cell extracts in the future? Could cell extracts ever be commoditized? I believe so. If CFPS ever is to be used for on-demand, point-of-care manufacturing, then centralized manufacturing of extracts is likely to be needed (12).

What are the major concerns in CFPS reaction process development? Are there any tricky questions regarding hardware qualification or process validation? What kinds of in-process monitoring technologies are available — or still need to be developed? Assuming that extract validation issues can be resolved, the CFPS reaction itself should be more reproducible than a cell-based reaction because it is essentially a “simple” chemical or catalytic reaction. There is some variability in key process parameters for different product types, but taking a quality by design (QbD) approach and using high-throughput methods should make it possible to assess which parameters are important for a given product/construct. That makes it clear which aspects need to be controlled most carefully and might be used to validate each run partially (13, 14). Some sort of on-line/at-line monitoring technology (e.g., Raman or mass spectrometry) also could help manufacturers monitor the concentrations of precursors, metabolites, and in some cases, product (15). That would be facilitated by uniformity of the background (the extract).

In addition, if reactions are run for mass production of therapeutics or vaccines, then the same kinds of quality control (QC) analytics used currently probably could be used for CFPS-derived products. However, if we want to use CFPS for distributed manufacturing, then the challenge of QC to regulatory standards probably would be more complex.

References
1 Melinek BJ, et al. Toward a Roadmap for Cell-Free Synthesis in Bioprocessing. BioProcess Int. 18(9) 2020: 40–52; http://lne.e92.mwp.accessdomain.com/upstream-processing/expression-platforms/toward-a-roadmap-for-cell-free-synthesis-in-bioprocessing.

2 Stamatis C, Farid S. Process Economics Evaluation of Cell-Free Synthesis for the Commercial Manufacture of Antibody Drug Conjugates. Biotechnol. J. 16(4) 2021: e2000238; https://doi.org/10.1002/biot.202000238.

3 Iskakova MB, et al. Troubleshooting Coupled In Vitro Transcription–Translation System Derived from Escherichia coli Cells: Synthesis of High-Yield Fully Active Proteins. Nucleic Acids Res. 34(19) 2006: e135; https://doi.org/10.1093%2Fnar%2Fgkl462.

4 Ranji A, et al. Chapter 15: Transforming Synthetic Biology with Cell-Free Systems. Synthetic Biology. Zhao H, Ed. Academic Press: Boston, MA, 2013.

5 Tinafar A, Jaenes K, Pardee K. Synthetic Biology Goes Cell-Free. BMC Biol. 17(1) 2019: 64; https://doi.org/10.1186/s12915-019-0685-x.

6 Hershewe J, Kightlinger W, Jewett MC. Cell-Free Systems for Accelerating Glycoprotein Expression and Biomanufacturing. J. Ind. Microbiol. Biotechnol. 47(11) 2020: 977–991; https://doi.org/10.1007/s10295-020-02321-4.

7 Dopp JL, Reuel NF. Simple, Functional, Inexpensive Cell Extract for In Vitro Prototyping of Proteins with Disulfide Bonds. Biochem. Eng. J. 164, 2020: 107790; https://doi.org/10.1016/j.bej.2020.107790.

8 Burgenson D, et al. Rapid Recombinant Protein Expression in Cell-Free Extracts from Human Blood. Sci. Rep. 8(1) 2018: 9569; https://doi.org/10.1038/s41598-018-27846-8.

9 Colant N, et al. Escherichia coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles. Vaccines (Basel) 9(3) 2021: 193; https://doi.org/10.3390%2Fvaccines9030193.

10 Stark JC, et al. On-Demand, Cell-Free Biomanufacturing of Conjugate Vaccines at the Point-of-Care. Science Adv. 7(6) 2021; eabe9444; https://doi.org/10.1101/681841.

11 Adiga R, et al. Point-of-Care Production of Therapeutic Proteins of Good-Manufacturing-Practice Quality. Nat. Biomed. Eng. 2(9) 2018: 675–686; https://doi.org/10.1038/s41551-018-0259-1.

12 Ogonah OW, Polizzi KM, Bracewell DG. Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing? Curr. Opin. Chem. Eng. 18, 2017: 77–83; https://doi.org/10.1016/j.coche.2017.10.003.

13 Colant N, et al. A Rational Approach to Improving Titer in Escherichia coli-Based Cell-Free Protein Synthesis Reactions. Biotechnol. Prog. 37(1) 2021: e3062; https://doi.org/10.1002/btpr.3062.

14 Duran-Villalobos CA, et al. Multivariate Statistical Data Analysis of Cell-Free Protein Synthesis Toward Monitoring and Control. AIChE J. 11 March 2021; https://doi.org/10.1002/aic.17257.

15 Swartz JR. Transforming Biochemical Engineering with Cell-Free Biology. AIChE J. 58(1) 2012: 5–13; https://doi.org/10.1002/aic.13701.

Further Reading
Chiba CH, et al. Cell-Free Protein Synthesis: Advances on Production Process for Biopharmaceuticals and Immunobiological Products. BioTechniques 70(2) 2021; https://doi.org/10.2144/btn-2020-0155.

Gregorio NE, Levine MZ, Oza JP. A User’s Guide to Cell-Free Protein Synthesis. Methods Protoc. 2, 2019: 24; https://doi.org/10.3390/mps2010024.

Khambhati K, et al. Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems. Front. Bioeng. Biotechnol. 7, 2019: 248; https://doi.org/10.3389/fbioe.2019.00248.

Cheryl Scott is cofounder and senior technical editor of BioProcess International, part of Informa Connect; 1-212-600-3429; cheryl.scott@informa.com.