Beyond In Vivo, Pharmaceutical Molecule Production in Cell-Free Systems and the Use of Noncanonical Amino Acids Therein

Abstract

Throughout history, we have looked to nature to discover and copy pharmaceutical solutions to prevent and heal diseases. Due to the advances in metabolic engineering and the production of pharmaceutical proteins in different host cells, we have moved from mimicking nature to the delicate engineering of cells and proteins. We can now produce novel drug molecules, which are fusions of small chemical drugs and proteins. Currently we are at the brink of yet another step to venture beyond nature's border with the use of unnatural amino acids and manufacturing without the use of living cells using cell-free systems. In this review, we summarize the progress and limitations of the last decades in the development of pharmaceutical protein development, production in cells, and cell-free systems. We also discuss possible future directions of the field.

Figure 1

In order to convert a conventional cell into a cell that can be utilized for the production of compounds, including proteins, cells are (re)designed using the Design/Build/Test/Learn (DBTL)-cycle. Cells used for the manufacturing of compounds, including proteins have been referred to as cell factories. To produce small molecular drugs, enzymatic pathways are engineered or deleted in the cell, utilizing several rounds of the DBTL-cycle to optimize the production. Single enzymes, or a cascading pathway of enzymes, can be engineered to optimize the use of substrates to maximize the output of product. In the case of protein production, feedstocks containing carbon, nitrogen, and other essential elements needed for cell-growth/viability are considered the substrate which enters the cell, while the product is (secreted) protein.

Figure 2

Distribution of expression systems used for the production of 497 market approved pharmaceutical proteins up to 2022. The amount per period is pre-2001, 106 proteins; 2001–2006, 44 proteins; 2006–2010, 54 proteins; 2011–2015, 64 proteins, 2016–2020, 161 proteins, and 2021–2022, 50 proteins. For the classification, the first approval date is taken (USA or EU), new approvals which were a combination of already approved were not included, gene therapy and nucleic acid biopharmaceuticals (other than mRNA vaccine using cell or cell-free systems for production) and engineered cell-based products were not included. *) Including Saccharomyces cerevisiae, Komagataella pastoris (P. pastoris), Hansenula polymorpha; **) including baby hamster kidney cells (BKH), murine cells, Sp2/0 cells, V. cholera, hybridoma cells, cell-free systems for mRNA vaccines, however the percentage represents mainly mammalian cells. Other abbreviations: Chinese Hamster Ovary Cells (CHO), Escherichia coli (E. coli), and transgenic animals include chickens (product in the eggs), rabbits (product in milk), and goats (product in milk). Data was collected from several articles,^,− the Federal Drug Administration (FDA), and the European Medicines Agency (EMA) public databases. The figure is an update from Casteleijn and Richardson (2014).

Figure 3

Schematic of cell-free protein synthesis. The key component of a CFPS reaction is usually a cell lysate containing the cellular translation machinery. Substrates for translation and other related processes like amino acids and NTPs are supplemented as well as a system for energy regeneration. RNA- (in uncoupled reactions) or DNA- (in coupled reactions) templates encoding the protein of interest are then added to induce protein synthesis. DNA templates can be added either as plasmids or as linear constructs. Due to the open nature of the cell-free reaction, reaction conditions such as pH and salt concentrations can be manipulated easily. Additionally other components such as tRNA/aaRS pairs for noncanonical amino acids (NCAA) incorporation, additional enzymes, chemicals for bioconjugation, and other cofactors can be added before or during the synthesis.

Figure 4

Strategies for cotranslational incorporation of noncanonical amino acids. A) Depletion of a canonical amino acid from the growth media or cell-free reaction mix with the simultaneous supplementation of a noncanonical analogue can lead to an incorporation of the analogue instead of the original amino acid. This however, requires the corresponding aaRS to have a certain promiscuity toward its substrate, either be default or through protein engineering. This results in a protein wide replacement of the canonical amino acid by its analogon incorporation. B) Utilizing a tRNA that recognizes a stop-codon, the stop-codon can be suppressed to incorporate a NCAA. Recharging of the tRNA can be realized by the addition of a corresponding aaRS. To ensure site-specific NCAA incorporation at the stop-codon position, the tRNA and aaRS pair must be orthogonal to the host system, meaning that there is no inteference between endogenous tRNA/aaRS and the newly introduced pair. C) By depleting or deleting certain endogenous tRNA species, vacant codons are created. These codons can be reassigned to the NCAA using orthogonal tRNA/aaRS pairs. D) By the introduction of quadruplet codons the genetic code can be expanded further; this requires tRNAs recognizing these quadruplets specifically as well as corresponding orthogonal aaRS. Together with the use of orthogonal mRNAs and ribosome, this enables the introduction of several new codons in one template.