Manufacturing process additive

Abstract

The use of queuine, queuosine or a salt or solvate thereof either: in cell-based or non-cell-based protein synthesis; as an additive to cell fermentation media; or as an additive to serum-free media for cell-based protein synthesis. The queuine, queuosine or salt or solvate thereof may be present in a concentration range from 0.01-200 micromolar. The cells may be selected from bacteria, yeast, baculovirus, insect, mammal or filamentous fungi cells. The cells may be proliferating. The use of queuine, queuosine or a salt or solvate thereof may increase the yield or quality of protein produced. The protein to be produced may be poorly expressed from cells in serum-free media. The protein to be produced may have a high AT content. The process may take 1-7 days. The cells may have been optimised to have elevated levels of the enzyme tRNA guanine transglycosylase (TGT).

Description

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), 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-[[((1S,4S,5R)-4,5-dihydroxycyclopent-2-en-1-y!]amino]methyl]-3,7-dihydropyrrolo[2,3-dJpyrimidin-4-one) is a substrate for the TGT enzyme (tRNA guanine transglycosylase) a complex made of two proteins known as queuine tRNA-ribosyltransferase 1, and the partner protein QTRTD1 (queuine tRNA transglycosylase domain containing 1), also referred to as QTRT2 or Qv1. Queuosine (7-({[(1S,4S,5R)-4,5-Dihydroxycyclopent-2-en-1-yl]amino}methyl)-7-carbaguanosine), systematic IUPAC name 2-Amino-5- ({[(1 S,4S, 5R)-4,5-dihydroxycyclopent-2-en-1 -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 tRNAasp, tRNAasn, tRNAhis and tRNA^, 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 and WO 2016 / 050806 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 re-optimised 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. Summary of the Invention The present invention provides for the use of queuine and queuosine as an additive in protein expression systems to facilitate protein production. Detailed Description of the Invention The present invention provides the use of queuine, queuosine or salts or solvates thereof in protein synthesis as set out in the appended claims. 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 or a salt or solvate thereof in cell expressed methods of protein synthesis The invention also provides for the use of queuine, queuosine or a salt or solvate thereof as an additive to cell fermentation media. The invention also provides queuine and queuosine or a salt or solvate thereof as an additive to serum free media for use in cell expressed method of protein synthesis The invention also provides for the use of queuine, queuosine or a salt or solvate thereof to optimise production of protein expression 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 to optimise production of protein expression. The invention provides for the use of queuine to increase protein expression. The invention provides for the use of queuine to increase the quality of protein expressed. The invention provides for the use of queuine in systems where protein is poorly expressed in serum free media. 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, multidomain eukaryotic proteins expressed in bacteria often are non-functional. 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 in vivo 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 are developed using unicellular eukaryotes (i.e. non-pathogenic ‘Leishmania’ cells). Suitable protein manufacture methods include those utilising bacterial cells. Escherichia coli[ E. coli is one of the most widely used 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. co / / 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. Coryn ebacterium 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. Pseudomonas fluorescens The non-pathogenic and gram-negative bacteria, Pseudomonas fluorescens, is used for high level production of recombinant proteins; commonly for the development of bio-therapeutics 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 C1 have been developed into expression platforms for screening and production of diverse industrial enzymes. The expression system C1 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. The present invention is suitable for use where the process takes from 1-7 days. The present invention is suitable where the process takes greater than 3 days. Suitable protein expression systems include cell free systems. Cell-free production of proteins is performed in vitro using relevant components such as enzymes, cofactors, ribosomes, tRNAs and amino acids. Queuine, queuosine or salts &solvates thereof can be incorporated into the serum free media in a concentration range from 0.01 to 200 micromolar. Suitable concentration range includes: 0.1 to 10 micromolar Other suitable ranges include: Queuine can also be present in a concentration range of 0.01 to 0.1 micromolar in the protein synthesis vessel. Queuine can also be present in a concentration range of 0.1 to 1 micromolar in the protein synthesis vessel. Queuine can also be present in a concentration range of 1 to 10 micromolar in the protein synthesis vessel. Queuine can also be present in a concentration range of 10 to 100 micromolar in the protein synthesis vessel. Queuine can also be present in a concentration range of 100 to 200 micromolar in the protein synthesis vessel. Queuine 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 to ensure sufficient incorporation of queuine into vacant tRNA in cells. Queuosine can also be present in a concentration range of 0.01 to 0.1 micromolar in the protein synthesis vessel. Queuosine can also be present in a concentration range of 0.1 to 1 micromolar in the protein synthesis vessel. Queuosine can also be present in a concentration range of 1 to 10 micromolar in the protein synthesis vessel. Queuosine can also be present in a concentration range of 10 to 100 micromolar in the protein synthesis vessel. Queuosine can also be present in a concentration range of 100 to 200 micromolar in the protein synthesis vessel. Queuosine 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 queuosine to ensure sufficient incorporation of queuine into vacant tRNA in cells. The molecule queuine or queuosine can be administered as a free base or as a salt or solvate. A suitable pharmaceutically acceptable salt of queuine or queuosine is for example an acidaddition salt, such as an acid-additional 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 of formula (I) 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 and queuosine 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 and queuosine. It is to be understood that the invention relates to all isomeric forms of queuine and queuosine. It is to be understood that queuine and queuosine 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), 11C, 13C, 14C, 15O, 17O, 180,18F 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 and queuosine may exhibit polymorphism and the invention encompasses all such forms. There are many commercially available media, suitable for protein synthesis , to which queuine and queuosine 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 optimises the rate at which the ribosome reads and assembles the tRNA 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 protein to be produced has a high AT 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 of material and solvents; 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; All leading to optimized manufacturing 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. 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 and queuosine were obtained from WuXi Apptec (Hong Kong) Ltd., (Unit C, 20 / F., OfficePlus @ Mong Kok, No. 998 Canton Road, Kowloon, Hong Kong). The serum free media was analysed by Mass spec to confirm the absence of queuine. This was further confirmed by comparison with a sample of queuine. Experimental method • Plasmid DNAs with his-tag in secreted system were prepared by Midiprep DNA kit. The target proteins (see table below) were selected based on size and expression levels. • The plasmid DNA encoding the protein sequence with Histidine tag were transiently transfected in EXPI293 cells in 4ml suspension culture. • After 30 minutes, stock solution Queuine and Queuosine were prepared in DMSO solvent and added into the culture in different concentrations (0.1, 1, 10,100, 200 micromolar plus an untreated control). After 72 hours, spin down the transfected cells at 200xg for 3 minutes. The supernatants were tested for protein expression using Western blot by anti-His antibody and small-scale purification using Nickel-NTA magnetic beads. Results showed an increased yield of quality protein with the addition of queuine or queuosine. 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 queuine, queuosine or a salt or solvate thereof in protein synthesis.2) The use of queuine, queuosine or a salt or solvate thereof in cell expressed method of protein synthesis.3) The use of queuine, queuosine or a salt or solvate thereof as an additive to cell fermentation media.4) Queuine and queuosine or a salt or solvate thereof as an additive to serum free media for use in cell expressed method of protein synthesis.5) Use of queuine, queuosine or a salt or solvate thereof to optimise production of protein expression.6) The use according to claims 1-5 where the compound is queuine or a salt or solvate thereof.7) The use according to claims 1-5 where the compound is queuosine or a salt or solvate thereof.8) The use according to claims 1-5 wherein Queuine, queuosine or a salt or solvate thereof is present in a concentration of range 0.01-200 micromolar.9) The use on claims 1-5 where cells are selected from bacteria, yeast, baculovirus / insect, mammalian cells or filamentous fungi.10) Use in claim 1 in Non cell based protein synthesis.11) Use in claims 1-5 where cells are proliferating as part of the process.12) Use according to any preceding claim to increase the yield of protein expression.13) Use according to any preceding claim to increase the quality of protein expressed.14) Use according to any preceding claim where protein to be produced is poorly expressed from cells in serum free media.15) Use according to any preceding claim where protein to be produced has a high AT content.16) Use according to any preceding claim where the process takes from 1-7 days.17) Use according to any preceding claim where the cells have been optimised to have elevated levels of the enzyme TGT.13