WO2025153819A1

WO2025153819A1 - Protein manufacturing process additives

Info

Publication number: WO2025153819A1
Application number: PCT/GB2025/050072
Authority: WO
Inventors: Jason RUTT; Tim Bourne
Original assignee: Azadyne Ltd
Current assignee: Azadyne Ltd
Priority date: 2024-01-16
Filing date: 2025-01-16
Publication date: 2025-07-24
Anticipated expiration: 2026-07-16

Abstract

The present invention provides an additive which is queuine or its analogues to improve mammalian cell viability and the yield during the bio-manufacture of proteins expressed by the cells.

Description

Protein Manufacturing Process additives

Field

The invention relates to an additive to improve the bio-manufacture of proteins.

Background to the Invention

Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of relevant cell systems such that the protein of interest is produced in artificially high quantities. Protein production systems (also known as expression systems) have multiple applications in medicine from production of proteins for vaccinations, to bioactive factors such as growth factors and hormones e.g. human insulin to treat diabetes, to therapeutics such as fusion proteins and antibodies.

There are also significant applications for expression systems in industrial fermentation to manufacture e.g. enzymes.

Protein production systems can be optimised in many different ways. Approaches to do this include the use of different conditions to optimise growth of cells, use of different cells (species they are derived from and type of cell), through to the design and optimisation of the expression constructs used. There is also an evolving field of cell- free expression systems. But fundamentally they rely on the same process of transcription of the recombinant or native DNA to messenger RNA (mRNA) and the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

Understanding of this process continues to develop but is much more complex than originally thought. There is increasing evidence that protein translation is not a linear process and that many factors can influence the final protein product, both in terms of amount and quality (i.e. ability to perform its intended function).

The present invention relates to the queuine-tRNA ribosyltransferase pathway (also known as the ‘TGT’ pathway),

Queuine (chemical name: 2-amino-5-[[[(lS,4S,52?)-4,5-dihydroxycyclopent-2-en-l- yl]amino]methyI]-3,7-dihydropyrrolo[2,3-d]pyrimidin-4-one) is a substrate for the TGT enzyme (tRNA guanine transglycosylase) a complex made of two proteins known as queuine tRNA-ribosyltransf erase 1, and the partner protein QTRTD1 (queuine tRNA transglycosylase domain containing 1), also referred to as QTRT2 or Qvl. Queuosine (7-({[(lS,4S,5R)-4,5-Dihydroxycyclopent-2-en-l-yl]amino}methyl)- 7-carbaguanosine), systematic IUPAC name 2-Amino-5-({[(lS,4S,5R)-4,5- dihydroxycyclopent-2-en-l-yl]amino}methyl)-7-[(2R,3R,4S,5R)-3,4-dihydroxy-5- (hydroxymethyl)oxolan-2-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, is the nucleoside containing the nucleobase queuine.

The TGT pathway, also known as the queuine tRNA ribosyltransferase (QTRT) enzyme pathway was first elucidated in a 2009 publication (Boland et al., J. Biol. Chem. 2009. 3; 284(27): 18218-27).

The TGT enzyme is known to insert the natural product ‘queuine’ into tRNA^asp, tRNA^asn, tRNA¹¹¹⁵ and tRNA^tvr, and only these tRNAs in every cell in the body.

These tRNA are specific for four amino acids and these amino acids are encoded by two synonymous codons each. These synonymous codons end in either a uridine or a cytidine.

Insertion of queuine into the relevant anti-codon for these codons affects the processing speed of these codons by the ribosome leading to changes in the relative levels, and potentially quality, of the proteins being translated. This has the potential to affect protein production at multiple different levels.

The pathway has been exploited to provide treatment for diseases. WO 2016/050804, WO 2016/050806, WO2022/023438 & WO2022/023433 describe queuine mimetic compounds that act via the TGT pathway suitable for use in the treatment of autoimmune diseases, especially multiple sclerosis (MS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and diabetes.

Queuine and its presence or absence from tRNA in a cell is known to have an effect upon the rate and ability of cells to proliferate or differentiate.

Recent work suggests that the metabolism of cells and the machinery needed are modified, to support high level and effective protein production. Therefore, modulation of these can also affect protein production.

Huge efforts have been made to optimise culture conditions for optimum cell growth and protein expression. In addition, a key aspect has been the drive to replace animal- derived materials in the culture conditions and in particular moving to serum-free media. Interestingly, queuine is a key component of many sera. When the move from serum supplementation for protein production was made, culture conditions were reoptimised to offset the reduction in production seen without serum, but without routinely using synthetic queuine to achieve this. This means that the potential benefits of adding synthetic queuine or queuosine to culture media for protein expression have not been targeted.

The ability to influence tRNA affinity for one codon over another provides a new, previously unrecognised variable to influence protein synthesis. Using the TGT pathway its also possible to modify tRNA by incorporating an analogue of queuine at the position on a tRNA where a queuine would ordinarily be bound. The effect of the queuine analogue will skew the modified tRNA to favour one codon over another, allowing the speeding or slowing of protein translation. In addition to increased yields and faster production of protein, the rate of translation also affects protein folding, which is a key component of protein activity.

Summary of the Invention

The present invention provides for the use of queuine, queuosine, and queuine analogues as an additive in protein expression systems to facilitate protein production. In one aspect, the invention is suitable for commercial methods of protein synthesis, such as expression of commercial proteins e.g. biologic drug products from commercial cell lines.

Detailed Description of the Invention

The present invention provides the use of queuine, queuosine, and queuine analogues or salts or solvates thereof in protein synthesis as set out herein. Other features of the invention will be apparent from the dependent claims, and the description that follows. The invention also provides for the use of queuine, queuosine, queuine analogues or a salt or solvate thereof in cell expressed methods of protein synthesis.

The invention also provides for the use of queuine, queuosine, and queuine analogues or a salt or solvate thereof as an additive to cell fermentation media.

The invention also provides queuine, queuosine, and queuine analogues or a salt or solvate thereof as an additive to serum free media for use in cell expressed methods of protein synthesis.

In a particularly suitable embodiment, the cells from which the protein is expressed are actively proliferating, or would benefit from enhanced cell proliferation.

The invention also provides for the use of queuine, queuosine, and queuine analogues or a salt or solvate thereof to optimise protein expression.

Suitable proteins include biologic molecules made for medical purposes such as antibodies, vaccines or hormones. A key task of all living cells is to produce protein. To do so via an imperfect system is to strain the cell. By allowing cells to incorporate queuine, queuosine, a queuine analogues or a salt or solvate thereof has a positive effect upon the cell, increasing the lifespan and improving the function of the cell. Accordingly, the invention also provides for the use of queuine, queuosine, and queuine analogues or a salt or solvate thereof for increasing cell viability.

Queuine analogues which are molecules capable of acting as a queuine-tRNA ribosyltransferase enzyme complex substrate (also known as TGT substrate) may be identified by use of a TGT tRNA displacement assay as described below:

Production of [8-¹⁴C] Guanine labeled tRNA (tRNA*)

Components added in the order listed in Table 1. Before adding the 8-[¹⁴C] guanine solution to the reaction the solution was neutralised with an equal volume (vol/vol) 0.01 M NaOH, as the [8- ¹⁴C] Guanine is supplied in 0.01 M HC1 aqueous solution. In vitro synthesised human tyrosyl tRNA was prepared by T7 transcription in ultrapure nuclease-free water as described previously (Alqasem et al., 2020). Recombinant human QTRT1 enzyme containing an TV-terminal polyhistidine tag and human QTRT2 containing a C-terminal TE V- Strep -Tag®II tag were produced in BL21(DE3) Z t::Km_r cells as described previously (Alqasem et al., 2020).

Table 1 Components of [8-¹⁴C] Guanine tRNA labeling reaction

Component Volume (pL) Final cone.

1 M Tris-HCl pH 7.5 7.5 50 mM

5 M NaCl 0.6 20 mM

I M MgCh 0.75 5 mM

1 M DTT 0.3 2 mM

Human tRNA^Tyr 30 10 pM

RNase free H2O Up to 469 pL

QTRT:QTRT2 21 700 nM

[8-¹⁴C] guanine 10 200 nM

The reaction was incubated for 1 h at 37 °C. The reaction mixture was extracted by the addition of an of equal volume (500 pL) of Acid Phenol: chloroform (5: 1; pH 4.5) and centrifuged at 16,000 x g for 5 min. The upper aqueous phase was transferred to a new 1.5 mL tube. The radiolabelled tRNA with [8-¹⁴C] guanine in the third position of the anticodon loop (tRNA*) was precipitated by the addition of 0.1 volume (50 pL) of 3 M sodium acetate (aq.) and 2 volumes of ethanol (1 mL) and incubated overnight at - 20 °C. The tRNA* was pelleted by centrifugation at 16,000 x g for 20 min at 4 °C. The pellet was washed with 1 mL of ice-cold 70 % ethanol, without disturbing the pellet. The tRNA* pellet was resuspended in 30 pL nuclease-free water and the concentration measured spectrophotometrically at A260. Displacement assays

Each reaction was set up in triplicate and incubated for 30 mins at 37 °C. Each of the components in the reaction were added in the order shown in Table 2, with the tRNA* added last to initiate the reaction. ‘Compound’ refers to the molecules that are under investigation.

Table 2 Components of [8-¹⁴C] guanine displacement assays

After 30 mins the reactions were quenched by mixing with 2.5 mL ice-cold 10 % trichloroacetic acid (TCA) and placed on ice for one hour to precipitate the tRNA. The RNA precipitate was collected using vacuum filtration onto GF/C 2.4-cm glass fiber disks (set up in a Millipore Polymeric Vacuum Filter manifold). Each disk was rinsed with 40 mL ice-cold 5 % TCA. The filters were vacuum dried by rinsing them with 5 mL of freshly-made, ice-cold 95 % ethanol. The vacuum manifold was disassembled, the filters recovered and dried again at room temperature before being placed in scintillation vials containing 10 mL of Ecoscint A and radioactivity levels measured by scintillation counting.

In this assay, maximum displacement by 50 pM queuine base, the natural substrate of the queuine-tRNA ribosyltransferase enzyme complex, is >98% of the [¹⁴C] guanine. Background displacement values were < 2%. Therefore, a displacement of >5% is considered a positive substrate for TGT.

Suitable queuine analogues for use in the present invention are positive substrates for the TGT enzyme as determined by the TGT tRNA displacement assay. Suitable queuine analogues have a displacement of at least 5% in the assay.

Queuine analogues are also described in WO2022/023438, WO2022/023433 & WO20 16/050804. These compounds are suitable for use in the present invention. The compounds include the following genus and individual examples: a) compounds of formula (I):

Or pharmaceutically acceptable salt thereof

Wherein: R¹ is selected from H and CH3

R² is selected from C4H9 alkyl C6H13 alkyl and CsHe-phenyl, said phenyl optionally substituted by OH or OCH3

X is O or S; and

Y is CH, N or S. b) A compound of formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein: Y is selected from C or N;

X is selected from O or S;

R¹ is selected from hydrogen or methyl;

Z is CR²R³, wherein R² and R³ are each independently selected from hydrogen, (1 -5C)alkyl, or R² and R³ are taken together to form a cyclopropyl or cyclobutyl ring; Z¹ is selected from CR⁴R⁵, O, S, C(O) or NR⁶, wherein R⁴ and R⁵ are each independently selected from hydrogen, (1-5C)alkyl, phenyl, or R⁴ and R⁵ are taken together to form a cyclopropyl ring, and R⁶ is selected from hydrogen and (1 -5C)alkyl, provided that one, but not both, of Z and Z¹ is CH2 ; c) A compound of formula (I): or pharmaceutically acceptable salt or solvate thereof, wherein:

Y is selected from C or N;

X is selected from O or S; bond a is a single or double bond; x is 1 when a is a single bond and x is 0 when a is a double bond;

Ri is selected from hydrogen and methyl;

R2 (when present) is selected from hydrogen and methyl;

R3 is selected from hydrogen, (1-6C)alkyl and (1-6C)alkyl-Ph, wherein said Ph is optionally substituted by 1-3 substituents each independently selected from OH, O(1-6C)alkyl, (1 -6C)alkyl, CI & F.

Further suitable compounds include

Compounds can be synthesised using the general methods described in WO20 16/050804 and may be obtained from WuXi Apptec (Hong Kong) Ltd., (Unit C, 20/F., OfficePlus @ Mong Kok, No. 998 Canton Road, Kowloon, Hong Kong). Further suitable alternative compounds such as the one below are also available from Wuxi.

In another embodiment suitable compounds are (example 2, WO2016/050804) The molecule is available from Wuxi and its synthesis is described in WO2016/050804; and Compound 2 The molecule is available from Wuxi and its synthesis is described in example 4, WO2022/023433

In a particularly suitable embodiment, the cells from which the protein is expressed are actively proliferating or would benefit from enhanced cell proliferation.

The invention provides for the use of queuine, queuosine, a queuine analogue or a salt or solvate thereof to optimise protein expression.

The invention provides for the use of queuine analogues to optimise protein expression.

The invention provides for the use of queuine, queuosine or a salt or solvate thereof to optimise protein expression. The invention provides for the use of queuine, queuosine, a queuine analogue or a salt or solvate thereof to increase protein expression.

The invention provides for the use of queuine analogues to increase protein expression. The invention provides for the use of queuine, queuosine or a salt or solvate thereof to increase protein expression.

The invention provides for the use of queuine, queuosine, a queuine analogue or a salt or solvate thereof to increase the quality of protein expressed.

The invention provides for the use of queuine analogues to increase the quality of protein expressed.

The invention provides for the use of queuine, queuosine or a salt or solvate thereof to increase the quality of protein expressed.

The invention provides for the use of queuine, queuosine, a queuine analogue or a salt or solvate thereof in systems where protein is poorly expressed in serum free media. The invention provides for the use of queuine analogues in systems where protein is poorly expressed in serum free media.

The invention provides for the use of queuine, queuosine or a salt or solvate thereof in systems where protein is poorly expressed in serum free media.

Another issue in protein expression is maintaining the viability of the cells. Multiple methods such as optimising feeding of cells and modulating oxygen levels within the fermentation vessel have been used to maintain the viability of the cells. Surprisingly the present invention has been shown to increase the viability of the cells. This allows cells to continue to express high quality protein for longer, with greater yields and savings in costs of reagents, time and money.

The invention provides for the use of queuine, queuosine, a queuine analogue or a salt or solvate thereof for increasing cell viability.

The invention provides for the use of queuine analogues for increasing cell viability.

The invention provides for the use of queuine, queuosine, or a salt or solvate thereof for increasing cell viability.

In one embodiment the present invention is suitable for use for maintaining cell viability in a protein expression process that takes >3 days. In one embodiment the present invention is suitable for use for maintaining cell viability in a protein expression process that takes >7 days.

In one embodiment the present invention is suitable for use for maintaining cell viability in a protein expression process that takes >10 days.

In one embodiment the present invention is suitable for use for maintaining cell viability in a protein expression process that takes >12 days.

Unless otherwise stated, the following terms used in the specification and claims have the meanings set out below.

The term “comprising” or “comprises” means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of’ or “consists essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. The term “consisting of’ or “consists of’ means including the components specified but excluding other components.

Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of’ or “consisting essentially of’, and may also be taken to include the meaning “consists of’ or “consisting of’.

The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments.

Suitable protein manufacture methods include those derived from bacteria, yeast, baculovirus/insect, mammalian cells or filamentous fungi and non-cell based methods.

The oldest and most widely used expression systems are cell-based and may be defined as the "combination of an expression vector, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level" . Overexpression is an abnormally and excessively high level of gene expression which produces a pronounced gene-related phenotype. The skilled person is aware of multiple ways to introduce foreign DNA to a cell for expression.

Different host cells may be used for expression.

For example, common hosts are bacteria (such as E.coli, B.subtilis), yeast (such as S.cerevisiae) or eukaryotic cell lines. Common DNA sources and delivery mechanisms are viruses (such as baculovirus, retrovirus, adenovirus), plasmids, artificial chromosomes and bacteriophage (such as lambda).

The best expression system depends on the gene involved, for example S.cerevisiae is often preferred for proteins that require significant post-translational modification. Insect or mammalian cell lines are used when human-like splicing of mRNA is required.

Because bacteria are prokaryotes, they are not equipped with the full enzymatic machinery to accomplish the required post-translational modifications or molecular folding. Hence, multi-domain eukaryotic proteins expressed in bacteria often are nonfunctional. Also, many proteins become insoluble as inclusion bodies that are difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding. To address these concerns, expression systems using multiple eukaryotic cells have been developed for applications requiring the proteins be conformed as in, or closer to eukaryotic organisms. For this, cells of plants (i.e. tobacco), of insects or mammals (i.e. hamsters) are transfected with genes and cultured in suspension and even as tissues or whole organisms, to produce fully folded proteins. Mammalian expression systems have however low yield and other limitations (time-consuming, toxicity to host cells). To combine the high yield/productivity and scalable protein features of bacteria and yeast, and advanced epigenetic features of plants, insects, mammalian systems and other protein production systems may also be developed using unicellular eukaryotes (i.e. non-pathogenic ‘ Leishmania’’ cells).

Suitable protein manufacture methods include those utilising bacterial cells.

Escherichia coli is one of the most widely used bacterial expression hosts, and DNA is normally introduced in a plasmid expression vector. The techniques for overexpression in E. coli are well developed and work by increasing the number of copies of the gene or increasing the binding strength of the promoter region so assisting transcription. For example, a DNA sequence for a protein of interest could be cloned or subcloned into a high copy-number plasmid containing the lac (often LacUVC) promoter, which is then transformed into the bacterium E. coli. Addition of IPTG (a lactose analog) activates the lac promoter and causes the bacteria to express the protein of interest. E. coli strain BL21 and BL21(DE3) are two strains commonly used for protein production. As members of the B lineage, they lack Ion and OmpT proteases, protecting the produced proteins from degradation. The DE3 prophage found in BL21(DE3) provides T7 RNA polymerase (driven by the LacUV5 promoter), allowing for vectors with the T7 promoter to be used instead.

Non-pathogenic species of the gram-positive Corynebacterium are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine components of human food, animal feed and pharmaceutical products.

Expression of functionally active human epidermal growth factor has been done in C. glutamicum, thus demonstrating a potential for industrial-scale production of human proteins. Expressed proteins can be targeted for secretion through either the general, secretory pathway (Sec) or the twin-arginine translocation pathway (Tat).

Unlike gram negative bacteria, the gram-positive Corynebacterium lack lipopolysaccharides that function as antigenic endotoxins in humans.

The non-pathogenic and gram-negative bacteria, Pseudomonas fluorescens, is used for high level production of recombinant proteins; commonly for the development of biotherapeutics and vaccines. P. fluorescens is a metabolically versatile organism, allowing for high throughput screening and rapid development of complex proteins. P. fluorescens is most well known for its ability to rapidly and successfully produce high titers of active, soluble protein.

Suitable protein manufacture methods include those utilising yeast cells.

Expression systems using either S. cerevisiae or Pichia pastoris allow stable and lasting production of proteins that are processed similarly to mammalian cells, at high yield, in chemically defined media.

Suitable protein manufacture methods include those utilising filamentous fungi.

Filamentous fungi, especially Aspergillus and Trichoderma, but also more recently Myceliophthora thermophilia Cl have been developed into expression platforms for screening and production of diverse industrial enzymes. The expression system Cl shows a low viscosity morphology in submerged culture, enabling the use of complex growth and production media.

Suitable protein manufacture methods include those utilising baculovirus/insect cells. Baculovirus-infected insect cells (Sf9, Sf21 , High five strains) allow production of glycosylated or membrane proteins that cannot be produced using fungal or bacterial systems. Genes are not expressed continuously because infected host cells eventually lyse and die during each infection cycle.

Non-lytic insect cell expression is an alternative to the lytic baculovirus expression system. In non-lytic expression, vectors are transiently or stably transfected into the chromosomal DNA of insect cells for subsequent gene expression. This is followed by selection and screening of recombinant clones. The non-lytic system has been used to give higher protein yield and quicker expression of recombinant genes compared to baculovirus-infected cell expression. Cell lines used for this system include:Sf9, Sf21 from Spodoptera frugiperda cells, Hi-5 from Trichoplusia ni and Schneider 2 cells and Schneider 3 cells from Drosophila melanogaster cells. With this system, cells do not lyse and several cultivation modes can be used. Additionally, protein production runs are reproducible. This system gives a homogeneous product. A drawback of this system is the requirement of an additional screening step for selecting viable clones.

Suitable protein manufacture methods include those utilising unicellular eukaryote cells.

Leishmania tarentolae (cannot infect mammals) expression systems allow stable and lasting production of proteins at high yield, in chemically defined media. Produced proteins exhibit fully eukaryotic post-translational modifications, including glycosylation and disulfide bond formation.

Suitable protein manufacture methods include those utilising mammalian cells.

• Chinese Hamster ovary cells

• Mouse myeloma lymphoblastoid (e.g. NSO cell)

• Fully Human o Human embryonic kidney cells (HEK-293) o Human embryonic retinal cells (Crucell's Per.C6) o Human amniocyte cells (Glycotope and CEVEC) The most common mammalian expression systems are Chinese Hamster Ovary (CHO) and Human embryonic kidney (HEK) cells.

The present invention is suitable for use in an embodiment where the cells which express protein have been optimised to have elevated levels of the enzyme TGT.

In one embodiment the present invention is suitable for use where the process takes from 1-7 days. In another embodiment the present invention is suitable for use where the process takes greater than 3 days.

In one embodiment the present invention is suitable for use where the process takes >7 days.

In one embodiment the present invention is suitable for use where the process takes >10 days.

In one embodiment the present invention is suitable for use where the process takes >12 days.

In another embodiment, the present invention can be used to extend the amount of days the process is conducted for, with enhanced viability of cells and quality of the protein expressed allowing greater protein yield over the manufacturing process time span.

Suitable protein expression systems include cell free systems.

Cell-free production of proteins is performed in vitro using purified RNA polymerase, ribosomes, tRNA, amino acids, enzymes, cofactors and ribonucleotides. These reagents may be produced by extraction from cells or from a cell-based expression system.

Queuine, queuosine, and queuine analogues or salts & solvates thereof can be incorporated into the serum free media in a concentration range from 0.01 to 500 micromolar.

Queuine, queuosine, and queuine analogues or salts & solvates thereof can be incorporated into the serum free media in a concentration range from 0.01 to 300 micromolar.

Suitable concentration range includes: 0.1 to 10 micromolar

Other suitable ranges include:

Queuine, queuosine, and queuine analogues can also be present in a concentration range of 0.01 to 0.1 micromolar in the protein synthesis vessel.

Queuine analogues can also be present in a concentration range of 0.01 to 0.1 micromolar in the protein synthesis vessel.

Queuine, queuosine, and queuine analogues can also be present in a concentration range of 0.1 to 1 micromolar in the protein synthesis vessel.

Queuine analogues can also be present in a concentration range of 0.1 to 1 micromolar in the protein synthesis vessel.

Queuine, queuosine, and queuine analogues can also be present in a concentration range of 1 to 10 micromolar in the protein synthesis vessel.

Queuine analogues can also be present in a concentration range of 1 to 10 micromolar in the protein synthesis vessel. Queuine, queuosine, and queuine analogues can also be present in a concentration range of 10 to 100 micromolar in the protein synthesis vessel.

Queuine analogues can also be present in a concentration range of 10 to 100 micromolar in the protein synthesis vessel.

Queuine, queuosine, and queuine analogues can also be present in a concentration range of 100 to 300 micromolar in the protein synthesis vessel.

Queuine analogues can also be present in a concentration range of 100 to 300 micromolar in the protein synthesis vessel.

Queuine, queuosine, and queuine analogues can also be present in a concentration range of 200 to 300 micromolar in the protein synthesis vessel.

Queuine analogues can also be present in a concentration range of 200 to 300 micromolar in the protein synthesis vessel.

Queuine, queuosine, and queuine analogues can also be present in a concentration range of 10 to 500 micromolar in the protein synthesis vessel.

Queuine analogues can also be present in a concentration range of 10 to 500 micromolar in the protein synthesis vessel.

The skilled person will understand the necessary amount of queuine, queuosine, and queuine analogue to ensure sufficient incorporation of queuine, queuosine, and queuine analogue into vacant tRNA in cells.

The queuine, queuosine, and queuine analogue can be administered as a free base or as a salt or solvate.

A suitable pharmaceutically acceptable salt of queuine, queuosine, and queuine analogue is for example an acid-addition salt, such as an acid-addition salt with hydrochloric acid, citric acid, tartaric acid and fumaric acid (particularly hydrochloric acid). An acid-addition salt may be obtained, for example, by reaction of a compound with a suitable acid (such as hydrochloric acid, citric acid, tartaric acid and fumaric acid) using a conventional procedure.

A pharmaceutically acceptable salt may alternatively be formed by converting one salt of a compound of the invention to another by reaction with an appropriate acid or base, or by means of a suitable ion exchange column.

The preparation of a pharmaceutically acceptable salt is typically conducted in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent.

It is to be understood that queuine may exist in solvated or unsolvated forms, such as for example hydrated forms. The invention encompasses all pharmaceutically acceptable solvated forms. It is to be understood that the invention relates to all tautomeric forms of queuine analogue.

It is to be understood that the invention relates to all isomeric forms of queuine analogue.

It is to be understood that queuine analogue includes forms that are isotopically-labelled (i.e. radio-labelled). In such compounds, one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of radionucleotides that can be included in the compounds of the invention include ²H (also written as “D” for deuterium), ³H (also written as “T” for tritium), ^UC, ¹³C, ¹⁴C, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F and the like. The particular radionucleotide used will depend on the specific application of the radio-labelled compound.

It is to be understood that queuine may exhibit polymorphism and the invention encompasses all such forms.

There are many commercially available media, suitable for protein synthesis, to which queuine may be added.

The present invention is also suitable to increase the yield of protein expression in any process.

The present invention is also suitable to increase the quality of the protein expressed. Queuine, queuosine, and queuine analogue optimises the rate at which the ribosome reads and assembles the amino acid into protein. That rate is key to the folding of the protein and the ‘quality’ of the protein expressed.

The present invention is suitable for the production of protein, where the protein to be produced is poorly expressed from cells in serum free media.

The present invention is particularly suitable for use where the mRNA for the protein to be produced has a high AU content.

Advantages of the present invention include one or more of:

The ability to make commercially valuable proteins in greater quantity; in optimal folded state; at greater speed; in a shorter time; requiring smaller volumes and/or raw materials; the ability to make protein which cant be made in a commercially viable yield when made by queuine free media; the ability to increase cell proliferation in the manufacturing process; the ability to increase cell differentiation in the manufacturing process;

The ability to improve the viability of the cells used in the manufacturing process All leading to optimized manufacturing yields and costs.

Optimised proliferation of cells to support producing the protein of interest.

Optimised differentiation of the cells to support producing the protein of interest.

Optimised speed of protein production by the relevant cells.

Improved cell viability.

Production of increased levels of optimally folded protein.

Increased yields of optimal protein.

Increased speeds of manufacture.

Reduced manufacturing costs per unit protein.

Ability to produce viable levels of difficult to express proteins.

Examples

The invention will now be illustrated by the following non-limiting examples:

Queuine, queuosine, and queuine analogues were obtained from WuXi Apptec (Hong Kong) Ltd., (Unit C, 20/F., OfficePlus @ Mong Kok, No. 998 Canton Road, Kowloon, Hong Kong). Synthesis of queuine analogue compound 2 was made using the route described in example 4 of WO2022/023433.

The route is reproduced below:

2-Chloro-3-oxopropanenitrile

In a dry round bottomed flask under a positive pressure of argon, a suspension of NaOMe (7.14g, 0.13mol) in dry THF (90mL) was cooled to -5 °C. Methyl formate (9mL, 0.15 mol) was added dropwise over 1 min by syringe and stirring was continued at -5 °C for 20 min. Then chloroacetonitrile (8.33mL, 0.13 mol) was added dropwise via a dropping funnel over 45 min. The mixture turned from white to yellow and was stirred for a further 2 h at -5 °C at which point the reaction mixture was orange. The bath was removed and the reaction was allowed to warm up to room temperature. An aliquot of the reaction mixture was treated with a drop of concentrated HC1 and analysed by TLC which indicated the presence of the desired product with an Rf = 0.45, eluting with 100% EtOAc. The mixture was cooled to 0 °C and concentrated HC1 (12 mL) was added dropwise during which time the mixture reaction became cherry-red. The resultant suspension was filtered through a pad of celite, and the celite was washed with EtOAc until the filtrate became colourless. The collected filtrates were concentrated at reduced pressure with the water bath at a temperature no higher than 40 °C to afford chloro(formyl)acetonitrile¹ as a black oil, in quantitative yield, which was used without further purification.

2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-cf|pyrimidine-5-carbonitrile

2,4-Diamino-6-hydroxypyrimidine (3.00 g, 24 mmol) was added to a solution of sodium acetate (6.4g, 76 mmol) in millipore water (90 mL) and stirred at 50 °C for 1 hour. While still at 50 °C a solution of crude chloro(formyl)acetonitrile (S2) (3.00 g, 32 mmol) in mQ water (44 mL) was added dropwise with a dropping funnel, during which time the reaction turned beige and heating continued for 18 h at 50 °C, after which time the reaction was heated to 100 °C for 3 h. The reaction mixture was allowed to cool to room temperature and the solid removed by filtration. The solid was suspended in EtOH and 5M aqueous KOH solution was added until the solid dissolved. Charcoal was added to the solution and the mixture stirred for 30 minutes before removal of the solid by filtration. The pH of the filtrate was adjusted to pH=6 with concentrated aqueous HC1 solution during which time a precipitate formed and was collected by filtration. In order to remove the final traces of water from the solid it was dissolved in a mixture of toluene/methanol 1/1 and then concentrated at reduced pressure. The resultant solid was dried over P2O5 to afford the desired compound (1.68 g, 9.6 mmol, 40% yield) as beige solid. mp: > 250 °C (decomp.).

6_H (400 MHz, DMSO-de): 6.49 (2H, bs, NH2), 7.59 (1H, s), 10.78 (1H, bs), 11.90 (1H, bs)

HRMS m/z -ES): Found: 174.0420 ([M - H]' C7H4N5O; Requires: 174.0421)

4-Oxo-2-(tritylamino)-4,7-dihydro-3H-pyrrolo[2,3-d|pyrimidine-5- carbonitrile

In a dry round bottomed flask under an atmosphere of argon, trityl chloride (1.20 g, 4.28 mmol) was added to a solution of 2-amino-4,7-dihydro-4-oxo-3J/-pyrrolo[2,3- d]pyrimidine-5-carbonitrile (0.50 g, 2.85 mmol) in dry pyridine (29 mL). The mixture reaction was heated at 90 °C for 48 h. The reaction mixture was concentrated under reduced pressure then absorbed on silica gel and purified by flash chromatography on silica gel eluting with dichloromethane/MeOH with a gradient starting at 2% of MeOH and rising to 10%. The desired compound was obtained as a brown solid (0.63 g, 1.5 mmol, 53% yield). mp: 196-198 °C.

6_H (400 MHz, DMSO-d₆): 7.13-7.26 (15H, m), 7.37 (1H, s), 7.57 (1H, bs), 10.67 (1H, bs), 11.74 (1H, bs, NH)

HRMS m/z -ES): Found: 418.1665 ([M + H]⁺ C26H26N5O; Requires: 418.1662)

4-Oxo-2-(tritylamino)-4,7-dihydro-3H-pyrrolo[2,3-d|pyrimidine-5- carbaldehyde (S4)

Hexamethyldisilazane (HMDS) (6mmol, 1 ,3mL) was added to a mixture of 4,7-dihydro- 4-oxo-2-[(triphenylmethyl)amino]-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (1.30 g, 3 mmol) with ammonium sulphate (397 mg, 0.3mmol) in dry toluene (8mL) in a round bottomed flask. A reflux condenser was fitted, and the flask was heated at reflux temperature overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. Under a positive pressure of argon, the crude reaction mixture was solubilised in dry dichloromethane (8mL) and cooled to -78 °C. At this temperature, diisobutylaluminium hydride (DiBAL-H) (4.5 mL, 1 M in di chloromethane, 4.5 mmol) was added dropwise. After 2 hours, analysis by thin layer chromatography (TLC) (ethyl acetate (EtOAc) 100%) indicated that some starting material remained. So, a further 2mL diisobutylaluminium hydride (DiBAL-H) solution was added dropwise. After 1 hour, the reaction was complete and a mixture of water/acetic acid (9/1, 3.5 mL) was added at -78 °C. The reaction mixture was allowed to warm to room temperature slowly. A mixture of ethyl acetate/water (1/1, 300 mL) was added to the reaction mixture and stirring continued at room temperature for 2 hours. The layers were separated and the organic layer was washed with brine and the aqueous layers were extracted with ethyl acetate. The combined organic fractions were dried over anhydrous magnesium sulfate, filtered and concentrated at reduced pressure. The crude reaction product was filtered through a pad of silica gel eluting with ethyl acetate to afford a yellow solid (1.01 g, 2.38 mmol, 76 %). mp: > 250 °C (decomp.).

6H (400 MHz, DMSO-de): 7.15-7.29 (16H, m), 7.54 (1H, bs, NH), 9.99 (1H, s), 10.64 (1H, bs), 11.81 (1H, bs, NH)

HRMS (m/z -ES): Found: 443.1478 ([M + Na]⁺ C26H₂oN₄Na02; Requires:

443.1478) 2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-<flpyrimidine-5-carbaldehyde oxime

(Compound 2) was prepared by adding a spatula tip of Na2SO4 to a solution of S4 (300 mg, 0.72 mmol) and hydroxylamine (107 mg , 0.78 mmol) in MeOH (5 cm³). The resulting suspension was stirred at room temperature for 20 h before being concentrated in vacuo and purified by flash chromatography (7:3 Hex/EtOAc). The product was dissolved in IM HC1 in dioxane (2.4 mL, 9 eq.) and the solution stirred at room temperature overnight during which time a precipitate of product was seen to form. The product was isolated via vacuum filtration and washed with CH2Q2 to yield compound 2 as a white powder (34 mg, 39%) containing an inseparable mixture of E/Z isomers in a 92:8 ratio, mp >250 °C (decomp.).

5H (400 MHz, DMSO-d₆): major isomer - 7.54 (1H, d, J2.4), 7.84 (1H, s), 11.00 (1H, bs), 11.67 (lH, bs) minor isomer - 7.16 (0.08H, d, J2.2), 8.37 (0.08H, s), 11.81 (0.08H, bs)

5_C (100 MHz, DMSO-d₆): major isomer - 102.7, 1 14.6, 129.2, 142.9, 143.0, 156.9, 162.7 minor isomer - 102.5, 115.9, 127.9, 146.2, 146.3, 157.6, 163.5

HRMS (m/z -APCI): Found: 192.0525 ([M - H]‘ C7H6N5O2; Requires: 192.0527) Vmax (film)/cm^-1: 1578, 1671, 2625, 2971, 3088, 3676

Processes for the manufacture of suitable queuine analogues are further disclosed in WO2022/023438, WO2022/023433 & WO2016/050804.

Example 1

An example of serum free media was analysed by Mass spec to confirm the absence of queuine. This was further confirmed by comparison with a mass spec of a sample of queuine.

Example 2 Experimental method

Plasmid DNAs for Histidine(His)-tagged secreted proteins were prepared by Midiprep DNA kit. The target proteins (see table below) were selected based on size and expression levels.

Name of protein Mol. Wt Origin (kDa)

SARS-CoV-2 26 viral Omicron

BA.4 RBD (omicron) Thrombopoietin 36.4 cytokines (TPO) bone morphogenetic 43.4 cytokines protein 2a (BMP2a) Colony-Stimulating human Factor 1 Receptor (CSF1R)

Fab 186

C5 trimer 45 nanobody

SmCD human

Interferon beta 22.5 human

Sulfatase human

The plasmid DNAs encoding the protein sequence with His tag were transiently transfected into EXPI293 cells in 3 mL suspension cultures.

After 16 hrs, stock solutions of Quenine, queuosine and queuine analogues, including compound 2 were prepared in DMSO solvent and added into the culture in different concentrations (10, 100 & 300 micromolar plus an untreated control).

After the 3^rd, 4^th and 5^th days, the transfected cells were spun down at 200xg for 5 minutes. The supernatants were tested for protein expression using biolayer interferometry (BLI) using an anti-His sensor. The cell viability and density were studied on these days.

Biolayer interferometry (BLI) optimization:

BLI is label free technology to measure biomolecular interactions such as binding rate and dissociation constant. In this work, BLI was used to study the binding kinetics of His-tagged protein to an anti-His biosensor. A purified His-tag mNeongreen protein was used as a standard and used to calculate the concentration of proteins in the supernatant.

Results.

Results for the effect of a queuine analogue compound 2 and queuine after 5 days of culture are shown in Fig 1 & 2.

The cell density and viability of cells treated with different compounds were measured and it was found density and viability of queuine analogue compound 2 treated cells were 6-9 million cells/mL and 80%. Cell density and viability was much higher than the untreated conditions, which prompted further exploration of the effects on the cell viability.

Example 3

Untransfected EXPI293 cells and EXP 1293 cells transfected with different proteins were treated with different concentrations of queuine analogue compound 2 and cell density and viability of the cells was measured. The viability started to decrease from the 4^th day in untreated cells, whereas the viability of cells was improved in cells in the presence of 100-300micromolar compound 2.

Results are shown in Fig 3.

Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of the foregoing embodiment s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1) The use of a queuine, queuosine, a queuine analogue or a salt or solvate thereof in protein synthesis.

2) The use of queuine, queuosine, a queuine analogue or a salt or solvate thereof in cell expressed methods of protein synthesis.

3) The use of queuine, queuosine, a queuine analogue or a salt or solvate thereof as an additive to cell fermentation media.

4) Queuine, queuosine, a queuine analogue or a salt or solvate thereof for use as an additive to serum free media for use in cell expressed methods of protein synthesis.

5) Use of queuine, queuosine, a queuine analogue or a salt or solvate thereof to optimise protein expression.

6) The use of queuine, queuosine, and queuine analogues or a salt or solvate thereof for increasing cell viability.

7) The use of queuine, queuosine, and queuine analogues or a salt or solvate thereof for maintaining cell viability in a protein expression process that takes >7 days.

8) The use according to claims 1-7 where the queuine analogue or a salt or solvate thereof is a positive substrate for TGT in a TGT tRNA displacement assay.

9) The use according to claims 1-7 wherein queuine queuosine, and queuine analogues or salts & solvates thereof is present in a concentration of range from 0.01 to 500 micromolar.

10) The use on claims 1-7 where cells are selected from bacteria, yeast, baculovirus/insect, mammalian cells or filamentous fungi.

1 l)Use in claim 1 in Non cell based protein synthesis.

12) Use in claims 1-7 where cells are proliferating as part of the process.

13) Use according to any preceding claim to increase the yield of protein expression.

14) Use according to any preceding claim to increase the quality of protein expressed.

15) Use according to any preceding claim where protein to be produced is poorly expressed from cells in serum free media.

16) Use according to any preceding claim where the mRNA encoding the protein to be produced has a high AU content.

17) Use according to any preceding claim where the process takes takes >7 days.