Recombinant human lactoferrin and methods and uses thereof

Abstract

The present invention relates to a recombinant human lactoferrin; a composition comprising the recombinant human lactoferrin; methods of producing the recombinant human lactoferrin; nucleic acids, vectors and host cells for producing a recombinant human lactoferrin; and methods and uses of the recombinant human lactoferrin.

Description

[0001] RECOMBINANT HUMAN LACTOFERRIN AND METHODS AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a recombinant human lactoferrin; a composition comprising the recombinant human lactoferrin; methods of producing the recombinant human lactoferrin; nucleic acids, vectors and host cells for producing a recombinant human lactoferrin; and methods and uses of the recombinant human lactoferrin.

[0004] BACKGROUND OF THE INVENTION

[0005] Lactoferrin was first isolated and identified simultaneously from human and bovine milks in the 1960’s (Groves, 1960; Johansson, 1960). Industrial scale production of bovine lactoferrin involving extraction and fractionation from skim milk and whey started in 1985 (Tomita et al., 2009). Currently, bovine lactoferrin is produced by several companies globally including Milei GmbH (the world largest manufacturer, acquired by Morinaga) in Germany, Tatua Nutritionals and Fonterra in New Zealand, as well as Murray Goulboum Nutritionals (now acquired by Saputo) and Bega Bionutrients, in Australia. Bovine lactoferrin is the only commercially available form in the market for use in food and nutraceutical applications.

[0006] The extraction and fractionation of bovine lactoferrin is an expensive process since lactoferrin is a minor component of bovine milk (0.03 to 0.5g / L) (Cheng et al., 2008). The amino acid sequence of bovine lactoferrin has some homology with the human equivalent however, up to 31% of the sequence is different which may result in functional differences. Compared to human lactoferrin, the number and location of N- glycosylation sites and glycan types vary significantly resulting in changes to functional attributes such as receptor binding, digestibility and bioavailability (Zlatina and Galuska 2021; Conesa et al., 2009). Commercial extraction and fractionation of human lactoferrin is not feasible due to supply issues, social licence and safety risks. Thus, there have been numerous attempts since the 1990s to produce recombinant human lactoferrin in microbial, transgenic animal and plant expression systems (Conesa et al., 2010) with limited success.

[0007] Accordingly, there remains a need for improved forms of lactoferrin for supply to the market, improved lactoferrin compositions and improved methods for producing lactoferrin. SUMMARY OF THE INVENTION

[0008] The present inventors have developed recombinant human lactoferrin proteins and methods and uses thereof.

[0009] In an aspect, the present invention provides a method of treating or preventing inflammation in a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0010] In an aspect, the present invention provides a use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing inflammation in a subject.

[0011] In an aspect, the present invention provides a method of treating or preventing iron deficiency in a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0012] In an aspect, the present invention provides a use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing iron deficiency in a subject.

[0013] In an aspect, the present invention provides a method of increasing iron absorption in the gastrointestinal tract of a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0014] In an aspect, the present invention provides a use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto for increasing iron absorption in the gastrointestinal tract of a subject.

[0015] In an aspect, the present invention provides a method of decreasing the activity of a pathogenic microbe in the microbiome of a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0016] In an aspect, the present invention provides a use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for decreasing the activity of a pathogenic microbe in the microbiome of a subject.

[0017] In an aspect, the present invention provides a method of decreasing the activity of a pathogenic bacteria in the microbiome of a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical to the subject.

[0018] In an aspect, the present invention provides a use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for decreasing the activity of a pathogenic bacteria in the microbiome of a subject.

[0019] In an aspect, the present invention provides a method of treating or preventing a microbial infection in a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0020] In an aspect, the present invention provides a use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing a microbial infection in a subject.

[0021] In an aspect, the present invention provides a method of treating or preventing a bacterial infection in a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0022] In an aspect, the present invention provides a use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing a bacterial infection in a subject.

[0023] In an aspect, the present invention provides recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto.

[0024] In an aspect, the present invention provides a composition comprising a recombinant human lactoferrin as described herein.

[0025] In an aspect, the present invention provides an isolated nucleic acid encoding a recombinant human lactoferrin.

[0026] In an aspect, the present invention provides a nucleic acid comprising a sequence selected from SEQ ID NO:4.

[0027] In an aspect, the present invention provides a vector comprising the nucleic acid as described herein.

[0028] In an aspect, the present invention provides a host cell comprising the nucleic acid or vector as described herein.

[0029] In an aspect, the present invention provides a method of producing a recombinant human lactoferrin as described herein, comprising culturing the host cell as described herein in cell culture medium and expressing the recombinant human lactoferrin.

[0030] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise. For instance, as the skilled person would understand the recombinant human lactoferrin outlined above for the method and uses of the invention equally apply to composition of the invention.

[0031] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0032] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0033] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0034] BRIEF DESCRIPTION OF THE ACCOMPANING DRAWINGS

[0035] Figure 1. Shows SDS-PAGE analysis of protein fractions collected during anion exchange chromatography purification process stained with Bulldog Aquastain. Protein gel bands boxed on the right were confirmed to contain predominantly recombinant target protein. M - NEB Broad Host Range molecular weight markers SDS-PAGE analysis of anion exchange chromatography fractionation. Lanes 1-6 column flow- through fractions 5A1-5B2; Lanes 7-18: fractions collected from the anion exchange column chromatography: fraction 1B1 to fraction 1C6; Std: Sigma rhLF standard 5pL (2 mg / ml).

[0036] Figure 2. Shows an SDS-PAGE gel of process samples for recombinant human lactoferrin fermentations T-008 and T-009, both expressing rhLF3. Lane 1 : T008-1 preinduction (before methanol feeding commenced); Lane 2: T008-2 15 hours postinduction (hpi); Lane 3: T008-3 19 hpi; Lane 4: T008-4 37 hpi; Lane 5: T008-5 42 hpi; Lane 6: T008-7 Culture prepared for harvest; Lane 7: T008-6 76 hpi; Lane 8: T008-8 post-chill to 12°C; Lane 9: T008 adjusted to pH 8.0; Lane 10: T008 Bulk supernatant post-centrifugation; Lane 11 : rhLF standard (Sigma); Lane 12: T009-1 pre-induction; Lane 13: T009-2 15 hpi; Lane 14: T009-3 19 hpi; Lane 15: T009-4 37 hpi; Lane 16: T009-5 42 hpi; Lane 17: T009-7 Culture prepared for harvest; Lane 18: T009-6 76 hpi; Lane 20: T009-8 Bulk supernatant post-centrifugation; Lane 21 : Sigma rhLF standard (2 mg / ml).

[0037] Figure 3. Shows tryptic digest LCMS proteomics analysis of rhLF3 strain flask scale expression - total relative abundance of peptides and distribution of identified peptides. Figure 4. Shows SDS PAGE analysis of samples taken from a recombinant human lactoferrin production batch T-011. The batch was completed in a bioreactor with a 10 L working volume. Samples were taken periodically before and after induction with methanol, and the cells were removed by centrifugation, after which the supernatant samples were analysed. Top and bottom boxes show native Pichia protein bands and the middle box shows rhLF protein bands. Lane 1 : T011-1 pre-induction; Lane 2: T011-2 12.5 hpi; Lane 3: T011-3 19 hpi; Lane 4: T011-4 26 hpi; Lane 5: T011-5 43 hpi; Lane 6: T011-6 50 hpi; Lane 7: T011-7 Culture prepared for harvest; Lane 8: T011-8 post-chill to 12°C; Lane 9: T011-9 Bulk supernatant post-centrifugation; Lane 10: T011-10 Final filtered (0.2 pm) supernatant before transfer to cold storage; Std: Sigma rhLF standard (2 mg / ml).

[0038] Figure 5. Comparison of protein production during the fermentation processes for batches T-008 (2 L) and T-011 (10 L) expressing rhLF3.

[0039] Figure 6. Comparison of growth of Pichia pastoris - optical density - in 2 L (T-008) and 10 L (T-011) bioreactors.

[0040] Figure 7. Shows in vitro anti-inflammatory activity, measured via nitrite production in lipopolysaccharide treated RAW264.7 cells and expressed as quercetin equivalents, in response to rhLF3 and commercial lactoferrin samples.

[0041] Figure 8. Representation of Caco-2 and HT29-MTX-E12 cells grown on transwell membranes to mimic the upper part of the small intestine and how human ferritin is detected in this model.

[0042] Figure 9. Shows in vitro intestinal iron absorption as measured by cellular ferritin production in response to lactoferrin samples without and with ferric chloride.

[0043] Figure 10. A) Shows A-glycan pattern of Ezc / zzrz-deri ved rhLF3 proteins as identified by LCMS analysis of excised SDS-PAGE gels slices. B) Typical A-glycosylation patterns of native human and bovine lactoferrin proteins (adapted from Zlatina and Galuska, 2021). C) Average mass spectra for rhLF3. Figure 11. Shows a summary of the amino acid sequences corresponding to the proteins and the nucleotide sequences encoding the recombinant lactoferrin proteins as described herein. Secretion signal sequence indicated in bold.

[0044] Figure 12. Shows SDS PAGE analysis of samples taken from a recombinant human lactoferrin production batch. The batch was completed in a bioreactor with a 10 L working volume. Samples were taken periodically before and after induction with methanol, and the cells were removed by centrifugation, after which the supernatant samples were analysed. Arrow A: lactoferrin (presence in bioreactor samples confirmed using mass spectroscopy of excised bands with the same mobility as the lactoferrin standard); Arrow B: Induced host-cell protein; Arrow C: Low molecular weight material which also contains lactoferrin peptides. Lane 1 : Molecular weight markers; Lane 2: Preinduction (before methanol feeding commenced); Lane 3: 12.5 hours post-induction (hpi); Lane 4: 19 hpi; Lane 5: 26 hpi; Lane 6: 43 hpi; Lane 7: 50 hpi; Lane 8: 67 hpi; Lane 9: Culture prepared for harvest; Lane 10: Bulk supernatant post-centrifugation; Lane 11 : Final filtered (0.2 pm) supernatant before transfer to cold storage.

[0045] Figure 13. Shows a time course of recombinant human lactoferrin production in a 10 L bioreactor. Lactoferrin titre was determined by HPLC. Samples were taken periodically during induction, the cells were then removed, and the supernatant retained for analysis.

[0046] Figure 14. Shows the recovery of recombinant human lactoferrin during the harvest procedure. Lactoferrin titre was determined by HPLC.

[0047] KEY TO SEQUENCE LISTING

[0048] DETAILED DESCRIPTION

[0049] General techniques and definitions

[0050] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., enzyme, fermentation, inoculation).

[0051] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0052] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0053] As used herein, the term “about”, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, even more preferably + / - 1%, of the designated value.

[0054] As used herein, the term “subject” is any animal. In an embodiment, the subject is a mammal. In an embodiment, the subject is a human. In an embodiment, the subject is select from a / an: neonate, infant, child, adolescent, adult and an elderly adult. In an embodiment, the subject is a neonate. In an embodiment, the subject is an infant. In an embodiment, the subject is a child. In an embodiment, the subject is an adolescent. In an embodiment, the subject is an adult. In an embodiment, the subject is an elderly adult (an adult 65 years or older). In an embodiment, the subject is pregnant. In an embodiment, the subject is postpartum. In an embodiment, the subject is iron deficient. In an embodiment, the subject has an iron deficiency condition as described herein. In an embodiment, the subject has an inflammatory condition as described herein. In an embodiment, the subject has a bacterial infection as described herein.

[0055] As used herein, “microorganism” or “microorganisms” or “microbe” or “microbial” refers to microscopic organisms including bacterial, viral, fungal or eukaryotic organisms. In an embodiment, the microorganism is a bacteria. In an embodiment, the microorganism is a virus. In an embodiment, the microorganism is a fungus. In an embodiment, the microorganism is a eukaryotic microorganism.

[0056] As used herein, “pre-biotic” refers to a substrate that is selectively utilized by host microorganisms conferring a health benefit. As used herein, “pro-biotic” refers to live microorganism which when administered in an adequate amount confers a health benefit to a subject as described herein.

[0057] As used herein, “recombinant” refers to DNA, proteins, cells, or organisms that are generated from the combination of genetic material from two or more different sources.

[0058] As used herein, the term “increase” or “increases” or “increased” or “increasing” refers to having a higher or greater level of a given parameter after application of the methods, uses or compositions as described herein compared to the level of a given parameter at baseline or compared to a control. In an embodiment, the increase of the given parameter is by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% compared to a given parameter at baseline or compared to a control.

[0059] As used herein, the term “reduce” or “reduces” or “reduced” or “reducing” refers to abolishing, decreasing or having a lower level of a given parameter after application of the methods, uses or compositions as described herein compared to the level of a given parameter at baseline or compared to a control. In an embodiment, the reduction of the given parameter is by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% compared to a given parameter at baseline or compared to a control.

[0060] In some embodiments, the control is a lactoferrin from a species other than human (e.g. bovine). In some embodiments, the control is endogenously produced human lactoferrin. In some embodiments, the control is human lactoferrin expressed in rice plant.

[0061] As used herein, the term “higher” means great or greater than a comparative e.g. value, for example size, quantity, or intensity, or greater than normal.

[0062] As used herein, the terms "treating" or "treatment" refers to at least partially obtaining a desired therapeutic outcome. In an embodiment, treatment comprises reducing or eliminating at least one symptom of a specified condition.

[0063] As used herein, the term "prevention" or “prophylaxis” refers to stopping or hindering the development of at least one symptom of a specified condition. Prevention need not be complete and does not imply that a subject will not eventually develop the symptom of the specified condition. As used herein, the term “anti-inflammatory” refers to treating or preventing inflammation as described herein.

[0064] A used herein, the term “anti-microbial” refers to an agent that is active against a microbe (kills, inhibits, inactivates or slows the growth of a microbe).

[0065] As used herein, the term “anti-bacterial” refers to an agent that is active against a bacteria (kills, inhibits, inactivates or slows the growth of a bacteria).

[0066] As used herein, “microbiome” refers to the microorganisms in a particular environment which can include the body or a part of the body of a subject. For example, the gastrointestinal microbiome refers to the community of microorganisms in the gastrointestinal tract of the body of a subject. For example, the dermal microbiome refers to the community of microorganisms of the skin.

[0067] As used herein, “relative abundance” refers to the percentage of a microbiome made up of a specific microorganism. A person skilled in the art will appreciate the relative abundance can be assessed via 16s ribosomal sequencing.

[0068] As used herein, “endogenous” refers to growing or being produced from within an organism. For example, endogenously produced human lactoferrin is produced within a human or human cell in culture.

[0069] Lactoferrin

[0070] Lactoferrin is a multifunctional glycoprotein found in most biological fluids such as milk, saliva, tears, and nasal secretions. It serves numerous functions including iron binding and transport, anti-microbial, antiviral, antioxidant, immunomodulatory, antiinflammatory, cell proliferation and differentiation functions (Ohradanova-Repic et al., 2023; Sienkiewicz et al., 2022). Notably, it is present in high concentrations in colostrum, the first form of milk produced immediately following the delivery of a newborn.

[0071] Lactoferrin plays a crucial role in the immune response due to its anti-microbial and anti-inflammatory properties, and may bind to bacterial and viral pathogens, preventing them from entering the body's cells, thus contributing to the body's first line of defence against infection. Lactoferrin also imparts anti-microbial activity by reducing essential iron (Fe) bioavailability for bacterial pathogens. Moreover, lactoferrin also has a significant role in iron metabolism in humans, having a very high affinity for iron enables lactoferrin to carry and deliver iron to cells while also preventing the accumulation of excess free iron in the body, which can lead to the production of harmful free radicals.

[0072] Lactoferrin exists in several variants and isoforms with the main secreted isoforms being the a, P, and y forms, whereas the 5 isoform is intracellularly produced. The functional properties of these isoforms are not well characterised. Limited data on bovine lactoferrin indicates that the isoforms are functionally different and their relative proportion in milk changes with lactation period. For instance, the a isoform of bovine lactoferrin was found to be ten times more resistant to tryptic proteolysis compared to the P isoform and its proportion in bovine milk decreases from 30% in colostrum to 15% in mature milk (van Veen et al., 2004), which indicates changes in the proportion of the isoforms depending on the need of the infant.

[0073] In an aspect, the present invention provides a recombinant human lactoferrin variant.

[0074] In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 70% identical thereto, or a sequence at least 80% identical thereto, or a sequence at least 85% identical thereto, or a sequence at least 88% identical thereto, or a sequence at least 90% identical thereto, or a sequence at least 92% identical thereto, or a sequence at least 94% identical thereto, or a sequence at least 96% identical thereto, or a sequence at least 98% identical thereto, or a sequence at least 98% identical thereto.

[0075] In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 70% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 80% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 85% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 88% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 90% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 92% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 94% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 96% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 98% identical thereto. In an aspect, the present invention provides a recombinant human lactoferrin rhLF3 comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 99% identical thereto.

[0076] In an embodiment, the recombinant human lactoferrin comprises a signal sequence. In an embodiment, the signal sequence comprises the amino acid sequence SEQ ID NO:3

[0077] In an embodiment, the recombinant human lactoferrin is produced in a yeast cell.

[0078] In an embodiment, the recombinant human lactoferrin comprises a yeast glycosylation pattern.

[0079] In an embodiment, the recombinant human lactoferrin has higher anti-microbial activity against a pathogenic microorganism or a spoilage microorganism as described herein compared to endogenously produced bovine lactoferrin.

[0080] In an embodiment, the recombinant human lactoferrin has higher anti-microbial activity against a pathogenic microorganism or a spoilage microorganism as described herein compared to a recombinant human lactoferrin expressed in rice plants.

[0081] In an embodiment, the recombinant human lactoferrin has higher antiinflammatory activity compared to a recombinant human lactoferrin expressed in rice plants.

[0082] In an embodiment, the recombinant human lactoferrin has higher antiinflammatory activity compared to endogenously produced bovine lactoferrin.

[0083] In an embodiment, the recombinant human lactoferrin has higher antiinflammatory activity compared to human lactoferrin expressed in rice plants and endogenously produced bovine lactoferrin.

[0084] In an embodiment, the recombinant human lactoferrin has higher iron intestinal absorption compared to recombinant human lactoferrin expressed in rice plants.

[0085] In an embodiment, the recombinant human lactoferrin has higher iron intestinal absorption compared to endogenously produced human lactoferrin.

[0086] In an embodiment, he recombinant human lactoferrin has higher iron intestinal absorption compared to endogenously produced bovine lactoferrin.

[0087] In an embodiment, the recombinant human lactoferrin, wherein when the recombinant human lactoferrin is combined with an iron supplement, iron intestinal absorption is higher compared to an iron supplement alone.

[0088] Glycosylation

[0089] A review of carbohydrate chemistry which uses this nomenclature is found in Hubbard et al., (1981). This nomenclature includes, for instance, Man, which represents mannose; GlcNAc, which represents 2-N-acetylglucosamine; Gal which represents galactose; Fuc for fucose; and Glc, which represents glucose. Sialic acids are described by the shorthand notation NeuNAc, for 5-N-acetylneuraminic acid, and NeuNGc for 5- glycolylneuraminic acid.

[0090] The term "glycosylation" means the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together e.g. from two to about twelve simple sugars linked together) to a glycoprotein. The oligosaccharide side chains are typically linked to the backbone of the glycoprotein through either N- or Cilinkages .

[0091] "N-linked glycosylation" refers to the attachment of the carbohydrate moiety to an asparagine residue in a glycoprotein chain. A summary of typical A-glycosylation patterns of native human and bovine lactoferrin proteins is provided in Figure 10. The skilled artisan will appreciate that human lactoferrin has three sites for N-linked glycosylation at amino acid residues Asnl38, Asn479 and Asn624 (described in van Veen et al., 2004). The skilled artisan will appreciate that bovine lactoferrin has 5 sites for N-linked glycosylation at amino acid residues Asn233, Asn281, Asn368, Asn476 and Asn545 of SEQ ID NO:2 (described in van Veen et al., 2004). In an embodiment, rhLF3 comprises a site for N-linked glycosylation at amino acid residue Asnl38. In an embodiment, rhLF3 comprises a site for N-linked glycosylation at amino acid residue Asn479. In an embodiment, rhLF3 comprises a site for N-linked glycosylation at amino acid residue Asn624. In an embodiment, rhLF3 comprises sites for N-linked glycosylation at amino acid residues Asnl38, Asn479 and Asn624.

[0092] "Glycoproteins" are polypeptides having one or more oligosaccharide side chains attached thereto.

[0093] In an embodiment, the recombinant human lactoferrin is a glycoprotein.

[0094] In an embodiment, the recombinant human lactoferrin comprises a yeast glycosylation pattern. In an embodiment, the yeast glycosylation pattern is a high mannose N-glycosylation. In an embodiment, the yeast glycosylation pattern does not comprise fucose.

[0095] In an embodiment, the recombinant human lactoferrin as described herein has an increased level of one or more of: H8N2, H9N2, H10N2, Hl 1N2 and H12N2 compared to endogenously produced human lactoferrin. In an embodiment, the recombinant human lactoferrin has an increased level of H8N2 compared to endogenously produced human lactoferrin. In an embodiment, the recombinant human lactoferrin has an increased level of H9N2 compared to endogenously produced human lactoferrin. In an embodiment, the recombinant human lactoferrin has an increased level of H10N2 compared to endogenously produced human lactoferrin. In an embodiment, the recombinant human lactoferrin has an increased level of Hl 1N2 compared to endogenously produced human lactoferrin. In an embodiment, the recombinant human lactoferrin has an increased level of H12N2 compared to endogenously produced human lactoferrin.

[0096] As used herein, “N” refers to N-acetylhexosamine. As used herein, “H” refers to hexose. In some, embodiment, the hexose is a mannose.

[0097] In an embodiment, the recombinant human lactoferrin has an increased level of mannose compared to endogenously produced human lactoferrin.

[0098] In an embodiment, the recombinant human lactoferrin has a reduced level of fucose compared to endogenously produced human lactoferrin.

[0099] In an embodiment, the recombinant human lactoferrin has a reduced abundance of N-GlcNac, Neu5Ac and / or fucose compared to endogenously produced human lactoferrin. In an embodiment, the recombinant human lactoferrin has a reduced abundance of N-GlcNac compared to endogenously produced human lactoferrin. In an embodiment, the recombinant human lactoferrin has a reduced abundance of N-Neu5Ac compared to endogenously produced human lactoferrin. In an embodiment, the recombinant human lactoferrin has a reduced abundance of N- fucose compared to endogenously produced human lactoferrin.

[0100] In an embodiment, the recombinant human lactoferrin has an increased level of one or more of: H8N2, H9N2, H10N2, H11N2 and H12N2 compared to recombinant human lactoferrin protein expressed in rice plants. In an embodiment, the recombinant human lactoferrin has an increased level of H8N2 compared to recombinant human lactoferrin protein expressed in rice plants. In an embodiment, the recombinant human lactoferrin has an increased level of H9N2 compared to recombinant human lactoferrin protein expressed in rice plants. In an embodiment, the recombinant human lactoferrin has an increased level of H10N2 compared to recombinant human lactoferrin protein expressed in rice plants. In an embodiment, the recombinant human lactoferrin has an increased level of Hl 1N2 compared to recombinant human lactoferrin protein expressed in rice plants. In an embodiment, the recombinant human lactoferrin has an increased level of H12N2 compared to recombinant human lactoferrin protein expressed in rice plants.

[0101] In an embodiment, the recombinant human lactoferrin has an increased level of mannose compared to recombinant human lactoferrin protein expressed in rice plants.

[0102] In an embodiment, the recombinant human lactoferrin has a reduced abundance of N-GlcNac, Neu5 Ac and / or fucose compared to recombinant human lactoferrin protein expressed in rice plants. In an embodiment, the recombinant human lactoferrin has a reduced abundance of N-GlcNac compared to recombinant human lactoferrin protein expressed in rice plants. In an embodiment, the recombinant human lactoferrin has a reduced abundance of N-Neu5Ac compared to human lactoferrin protein expressed from rice plants. In an embodiment, the recombinant human lactoferrin has a reduced abundance of N-fucose compared to recombinant human lactoferrin protein expressed in rice plants.

[0103] Methods and uses

[0104] Inflammation

[0105] In an aspect, the present invention provides a method of treating or preventing inflammation in a subject, the method comprising administering a recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0106] In an aspect, the present invention provides use of a recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing inflammation in a subject.

[0107] As used herein, “inflammation” refers to a local response to injury or infection characterised by one or more: vasodilation, increased vascular permeability, cellular infiltration, changes to the biosynthetic, metabolic and catabolic profiles of affected organs, and activation of the cells of the immune system. Without limiting the present invention to any one theory or mode of action, the inflammatory response is a complex response characterised by a series of physiological and / or immunological events which are induced to occur by the release of a cytokine cascade in response to any one of a variety of stimuli including, but not limited to, tissue injury, infection, an immune response (such as to a pathogen or an innocuous agent - as occurs with allergies) or disease (such as tumour formation or an autoimmune response). Inflammation serves as a biological mechanism initiating the elimination of noxious agents and / or damaged cells.

[0108] In an embodiment, the inflammation is selected from one or more of: skin inflammation; gastrointestinal inflammation; inflammatory bowel disease; urogenital inflammation; ulcerative colitis; response to infection anaemia of inflammation (Al) or chronic disease. In an embodiment, the inflammation is skin (dermal) inflammation. In an embodiment, the inflammation is gastrointestinal inflammation. In an embodiment, the inflammation is inflammatory bowel disease. In an embodiment, the inflammation is urogenital inflammation. In an embodiment, the inflammation is ulcerative colitis. In an embodiment, the inflammation is a response to infection. In an embodiment, the inflammation is anaemia of inflammation (Al) or chronic disease. In an embodiment, the skin inflammation is selected from one or more of: eczema, acne, seborrheic dermatitis, and psoriasis. In an embodiment, the skin inflammation is eczema. In an embodiment, the skin inflammation is acne. In an embodiment, the skin inflammation is seborrheic dermatitis. In an embodiment, the skin inflammation is psoriasis.

[0109] In an embodiment, the inflammation is associated with nitric oxide production. In an embodiment, inflammation is assessed by measuring nitric oxide production.

[0110] In an embodiment, inflammation is assessed by measuring nitric oxide production.

[0111] In an embodiment, the inflammation is associated with an increase in the level of one or more inflammatory cytokine / s. In an embodiment, the inflammatory cytokine is selected from one or more of: C-reactive protein (CRP), interleukin-6 (IL-6), interleukin- 8 (IL-8), tumor necrosis factor alpha (TNF-a), soluble tumor necrosis factor receptor I (sTNFR I) and soluble tumor necrosis factor receptor II (sTNFR II). In an embodiment, the inflammatory cytokine is CRP. In an embodiment, the inflammatory cytokine is IL- 6. In an embodiment, the inflammatory cytokine is IL-8. In an embodiment, the inflammatory cytokine is TNF-a. In an embodiment, the inflammatory cytokine is sTNFR I. In an embodiment, the inflammatory cytokine is sTNFR II.

[0112] In an embodiment, the inflammation is associated with a reduction in the level of one or more anti-inflammatory cytokines. In an embodiment, the anti-inflammatory cytokine is selected from one or more of: interleukin-4 (IL-4), interleukin- 10 (IL-10) or interleukin- 13 (IL-13). In an embodiment, the anti-inflammatory cytokine is IL-4. In an embodiment, the cytokine is IL- 10. In an embodiment, the cytokine is IL-13.

[0113] Iron deficiencies / iron absorption

[0114] In an aspect, the present invention provides a method of treating or preventing iron deficiency in a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0115] In an aspect, the present invention provides use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing iron deficiency in a subject.

[0116] A person skilled in the art will appreciate that iron deficiency can be determined by one or more of a: serum iron test, transferrin test, total iron-binding capacity test, haemoglobin test, haematocrit test, complete blood count, zinc protoporphyrin and a ferritin blood test. In an embodiment, the subject with iron deficiency is not anaemic. In an embodiment, the subject with iron deficiency is anaemic.

[0117] In an embodiment, the iron deficiency is caused by iron dietary insufficiency. In an embodiment, the iron deficiency is caused be insufficient iron absorption (e.g. coeliac disease, autoimmune gastritis, inflammatory bowel disease, ulcerative colitis, or Crohn’s disease, Helicobacter pylori infection, gastrointestinal surgery (e.g. gastric bypass surgery or gastrectomy)). In an embodiment, iron deficiency is caused by blood loss (e.g. from heavy menstrual cycle, giving blood too often, loss during surgery, inflammatory bowel disease, parasite infection (hookworms)) or any other inflammation.

[0118] Iron deficiency can cause one or more of the following symptoms: extreme fatigue, weakness, pale skin, chest pain, fast heartbeat, shortness of breath, headache, dizziness, light-headedness, cold hands and feet, bruises, chills, inflammation, tongue soreness, brittle nails and poor appetite.

[0119] In an aspect, the present invention provides a method of increasing iron absorption in the gastrointestinal tract of a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

[0120] In an aspect, the present invention provides a use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto for increasing iron absorption in the gastrointestinal tract of a subject.

[0121] As used herein, the “gastrointestinal tract” refers to the pathway by which food enters the body and solid wastes are expelled. In an embodiment, the gastrointestinal tract comprises one or more of the small intestine, large intestine and stomach.

[0122] In an embodiment, iron absorption is increased in one or more of small intestine, large intestine and the stomach. In an embodiment, iron absorption is increased in the small intestine. In an absorption is increased in one or more of the duodenum, jejunum and the ileum. In an embodiment, absorption is increased in duodenum. In an embodiment, absorption is increased in the jejunum. In an embodiment, absorption is increased in the upper jejunum. In an embodiment, absorption is increased in the ileum.

[0123] Pathogenic microorganism

[0124] In an aspect, the present invention provides a method of decreasing the activity of a pathogenic microorganism in the microbiome of a subject, the method comprising administering a recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject. In an aspect, the present invention provides use of a recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for decreasing the activity of a pathogenic microorganism in the microbiome of a subject.

[0125] As used herein, a “pathogenic microorganism” is an organism which is capable of causing disease in a host.

[0126] As used herein, “decreasing the activity” in the context of a pathogenic microorganisms comprises one or more of: decreasing the activity of the microorganism, inhibiting the activity of the microorganism, inactivation of the microorganism, decreasing the level / number of a microorganism, and decreasing the abundance of the microorganism in a subjects microbiome.

[0127] In an embodiment, the pathogenic microorganism is a pathogenic bacteria.

[0128] In an embodiment, the pathogenic microorganism is a pathogenic fungi.

[0129] In an embodiment, the pathogenic bacteria is a bacteria in a family selected from one or more of: Enter obacteriaceae, Staphylococcaceae, Bacillaceae, Campylobacter spp., Pseudomonadaceae an Mor axellaceae. In an embodiment, the pathogenic bacteria is a bacteria in a family selected from one or more of: Enter obacteriaceae, Staphylococcaceae and Bacillaceae. In an embodiment, the pathogenic bacteria is a Campylobacter spp. In an embodiment, the pathogenic bacteria is a Enter obacteriaceae. In an embodiment, the pathogenic bacteria is a Staphylococcaceae. In an embodiment, the pathogenic bacteria is a. Bacillaceae. In an embodiment, the pathogenic bacteria is a Pseudomonadaceae. In an embodiment, the pathogenic bacteria is aMoraxellaceae.

[0130] In an embodiment, the pathogenic bacteria is selected from one or more of: Escherichia coli, Staphylococcus aureus, Bacillus cereus, Cronobacter sakazakii, Salmonella spp., Yersinia spp., Listeria spp., Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas spp., Shigella spp., Shigella sonnei, Cutibacterium acnes and Acinetobacter baumanni. In an embodiment, the pathogenic bacteria is selected from one or more of: Escherichia coli, Staphylococcus aureus and Bacillus cereus.

[0131] In an embodiment, the bacteria is Escherichia coli. In an embodiment, the bacteria is Staphylococcus aureus. In an embodiment, the bacteria is Bacillus cereus. In an embodiment, the bacteria is Cronobacter sakazakii. In an embodiment, the bacteria is Salmonella spp. In an embodiment, the bacteria is Salmonella typhimurium. In an embodiment, the bacteria is Klebsiella pneumoniae. In an embodiment, the bacteria is Pseudomonas aeruginosa. In an embodiment, the bacteria is Pseudomonas spp. In an embodiment, the bacteria is Shigella spp. In an embodiment, the bacteria is Shigella sonnei. In an embodiment, the bacteria is Cutibacterium acnes. In an embodiment, the bacteria is Acinetobacter baumanni.

[0132] In an embodiment, the pathogenic fungi is selected from one or more of: Candida spp., Cryptococcus spp. and Aspergillus spp. In an embodiment, the pathogenic fungi is Candida spp. In an embodiment, the pathogenic fungi is Cryptococcus spp. In an embodiment, the pathogenic fungi is Aspergillus spp.

[0133] Spoilage microorganism

[0134] As used herein, a “spoilage microorganism” is an organism which causes food to deteriorate and an develop unpleasant odour. In an embodiment, the spoilage microorganism is a spoilage bacteria, yeast or fungi. In an embodiment, the spoilage microorganism is a spoilage bacteria. In an embodiment, the spoilage microorganism is a spoilage yeast. In an embodiment, the spoilage microorganism is a spoilage fungi.

[0135] In an embodiment, the spoilage bacteria is a bacteria selected from one or more of: Brochothrix spp., Carnobacterium spp., Kurthia spp. and Clostridium spp. In an embodiment, the spoilage bacteria is Brochothrix spp. In an embodiment, the spoilage bacteria is Carnobacterium spp. In an embodiment, the spoilage bacteria is Kurthia spp. In an embodiment, the spoilage bacteria is Clostridium spp.

[0136] In an embodiment, the spoilage yeast is a yeast selected from one or more of: Dekkera bruxellensis, Zygosaccharomyces bisporus, Schizosaccharomyces pombe, Issatchenkia orientalis, Debaryomyces hansenii, Candida holmii, Pichia membranifaciens, Zygosaccharomyces bailii, Saccharomyces cerevisiae, Zygosaccharomyces rouxii and Kloeckera apiculate. In an embodiment, the spoilage yeast is Dekkera bruxellensis. In an embodiment, the spoilage yeast is Zygosaccharomyces bisporus. In an embodiment, the spoilage yeast is Schizosaccharomyces pombe. In an embodiment, the spoilage yeast is Issatchenkia orientalis. In an embodiment, the spoilage yeast is Debaryomyces hansenii. In an embodiment, the spoilage yeast is Candida holmii. In an embodiment, the spoilage yeast is Pichia membranifaciens. In an embodiment, the spoilage yeast is Zygosaccharomyces bailii. In an embodiment, the spoilage yeast is Saccharomyces cerevisiae. In an embodiment, the spoilage yeast is Zygosaccharomyces rouxii. In an embodiment, the spoilage yeast is Kloeckera apiculate.

[0137] In an embodiment, the spoilage fungi is a mould selected from one or more of: Aspergillus spp., Rhizopus spp., Penicillium spp. and Botrytis spp. In an embodiment, the spoilage mould is Aspergillus spp. In an embodiment, the spoilage fungi is Rhizopus spp. In an embodiment, the spoilage mould is Penicillium spp. In an embodiment, the spoilage fungi is Botrytis spp.

[0138] Infection

[0139] In aspect, the present invention provides use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing a microbial infection in a subject.

[0140] In an embodiment, the microbial infection is a bacterial infection.

[0141] In an embodiment, the infection is selected from one or more of a / an: i) skin infection; ii) urinary tract infection; iii) Enterobacteriaceae infection; iv) Staphylococcaceae infection; v) Bacillaceae infection; vi) Pseudomonadaceae infection; vii) Moraxellaceae infection; viii) Escherichia coli infection; ix) Staphylococcus aureus infection; x) Bacillus cereus infection; xi) Cronobacter sakazakii infection; xii) Salmonella spp. infection; xiii) Salmonella typhimurium infection; xiv) Klebsiella pneumoniae infection; xv) Pseudomonas aeruginosa infection; xvi) Pseudomonas spp., infection; xvii) Shigella spp., infection; xviii) Shigella sonnei infection; xix) Acinetobacter baumanni infection; xx) respiratory infection; xxi) periodontitis; and xxii) peri-implantitis. In an embodiment, the bacterial infection is a skin infection. In an embodiment, the bacterial infection is a urinary tract infection. In an embodiment, the bacterial infection is a Enterobacteriaceae infection. In an embodiment, the bacterial infection is a Staphylococcaceae infection. In an embodiment, the bacterial infection is a Bacillaceae infection. In an embodiment, the bacterial infection is a Pseudomonadaceae infection. In an embodiment, the bacterial infection is a Moraxellaceae infection. In an embodiment, the bacterial infection is a Escherichia coli infection. In an embodiment, the bacterial infection is a Staphylococcus aureus infection. In an embodiment, the bacterial infection is a Bacillus cereus infection. In an embodiment, the bacterial infection is a Cronobacter sakazakii infection. In an embodiment, the bacterial infection is a Salmonella spp. infection. In an embodiment, the bacterial infection is a Salmonella typhimurium infection. In an embodiment, the bacterial infection is a Klebsiella pneumoniae infection. In an embodiment, the bacterial infection is a Pseudomonas aeruginosa infection. In an embodiment, the bacterial infection is a Pseudomonas spp., infection. In an embodiment, the bacterial infection is a Shigella spp., infection. In an embodiment, the bacterial infection is a Shigella sonnei infection. In an embodiment, the bacterial infection is a Acinetobacter baumanni infection. In an embodiment, the bacterial infection is a respiratory infection. In an embodiment, the bacterial infection is periodontitis. In an embodiment, the bacterial infection is peri-implantitis.

[0142] In an embodiment, the recombinant human lactoferrin as described herein is not used as adjuvant e.g. as an adjuvant for cancer therapy or a vaccine.

[0143] Production methods

[0144] Upstream production methods

[0145] A person skilled in the art will appreciate that the recombinant human lactoferrin as described herein can be produced using a cell based expression system. In one example, a recombinant human lactoferrin of the disclosure is produced by culturing a cell under conditions sufficient to produce the recombinant human lactoferrin as described herein.

[0146] In some embodiments, a nucleic acid encoding a recombinant human lactoferrin as described herein is placed into one or more expression construct / s, e.g., expression vector(s), which is / are then transfected into a host cell as described herein.

[0147] In an aspect, the present invention provides an isolated nucleic acid encoding a recombinant human lactoferrin as described herein. In an embodiment, the nucleic acid sequence encodes a signal sequence. In an embodiment, the signal sequence comprises the nucleic acid sequence (SEQ ID NO:5). In an embodiment, the nucleic acid sequence encodes a purification tag. In an embodiment, the tag is e.g. a strep-tagll.

[0148] In an aspect, the present invention provides a nucleic acid comprising the nucleotide sequence SEQ ID NO:4 or a sequence at least 70% identical thereto. In an aspect, the present invention provides a nucleic acid comprising the nucleotide sequence SEQ ID NO:4 or a sequence at least 80% identical thereto. In an aspect, the present invention provides a nucleic acid comprising the nucleotide sequence SEQ ID NO:4 or a sequence at least 85% identical thereto. In an aspect, the present invention provides a nucleic acid comprising the nucleotide sequence SEQ ID NO:4 or a sequence at least 90% identical thereto. In an aspect, the present invention provides a nucleic acid comprising the nucleotide sequence SEQ ID NO:4 or a sequence at least 95% identical thereto.

[0149] In an aspect, the present invention provides a vector comprising the nucleic acid as described herein.

[0150] In some embodiments, the nucleic acid is operably linked to a promoter. As used herein, the term “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and / or external stimulus, or in a tissue specific manner. In the present context, the term “promoter” is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter the spatial expression and / or temporal expression of said nucleic acid. As used herein, the term “operably linked to” means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0151] It will be understood that it is possible to improve the expression of a nucleic acid in a host organism or host cell by replacing the nucleotide sequences coding for a particular amino acid (i.e., a codon) with another codon which is better expressed in the host organism (i.e., codon optimization). One reason that this effect arises is due to the fact that different organisms show preferences for different codons. In some embodiments, a nucleic acid as disclosed herein is modified or optimized such that the nucleotide sequence reflects the codon preference for the particular host cell. Method of codon optimization will be apparent to the skilled person. For example, tools for codon optimization include, for example, GeneArt GeneOptimizer (Thermofisher®) or GenSmart® (GeneScript®).

[0152] In an aspect, the present invention provides a host cell comprising the nucleic acid or vector as described herein. In an embodiment, the host cell is a yeast cell. In an embodiment, the yeast cell is Pichia pastoris.

[0153] A skilled person would understand that the cells of invention can be a yeast cell or a yeast like cell which can be cultured in vitro and in which the recombinant lactoferrin can be expressed. In an embodiment, the yeast cell is selected from one or more of: Pichia pastoris (Komagaetella phaffii), Pichia hangzhouana, Candida utilis, Pichia jadinnii and Aspergillus spp. In an embodiment, the yeast cell is Pichia pastoris (Komagaetella phaffii). In an embodiment, the yeast cell is Pichia hangzhouana. In an embodiment, the yeast cell is Candida utilis. In an embodiment, the yeast cell is Pichia jadinnii. In an embodiment, the yeast cell is Aspergillus spp In an embodiment, the yeast like cell is a Hypocreaceae. In an embodiment, the yeast like cell is Trichoderma spp.

[0154] The cells of the present invention can be cultured in any cell culture medium that allows the expansion of the cells as described herein in vitro and allows for expression of recombinant lactoferrin by the cells. 1

[0155] In an embodiment, the cell culture medium is selected from one or more of: Yeast Extract Petone Dextrose (YDP) Medium, Buffered Glycerol-complex Medium (BMGY), Buffered Minimal Methanol (BMM) and Buffered Minimal Methanol with Yeast Extract (BMMY). In an embodiment, the cell culture medium is YDP Medium. In an embodiment, the cell culture medium is BMGY. In an embodiment, the cell culture medium is BMM. In an embodiment, the cell culture medium is BMMY. It will be appreciated by persons skilled in the art that such mediums may be supplemented with additional growth factors, for example, but not limited to; amino acids, hormones sugars, glycerol or methanol.

[0156] In an embodiment, the cell culture is batch cell culture. In an embodiment, the cell culture is fed-batch cell culture. In an embodiment, the cell culture is perfusion cell culture.

[0157] In an embodiment, the cell culture process comprises glycerol feeding. In an embodiment, the cell culture process comprises methanol feeding to induce recombinant lactoferrin production.

[0158] In an embodiment, the cell culture volume of the bioreactor is in the range of about WOOL to about 375,000L. In an embodiment, the cell culture volume of the bioreactor is in the range of from about WOOL to about 50,000L. In an embodiment, the cell culture volume of the bioreactor is in the range of from about 50,000L to about 150,000L. In an embodiment, the cell culture volume of the bioreactor is in the range of from about 150,000L to about 300,000L. In an embodiment, the cell culture volume of the bioreactor is in the range of from about 300,000L to about 375,000L

[0159] In an aspect, the present invention provides a method of producing a recombinant human lactoferrin as described herein, comprising culturing the host cell as described herein in cell culture medium and expressing the recombinant human lactoferrin.

[0160] In an embodiment, the recombinant human lactoferrin is produced at a concentration of about 0.2g / L to about 50g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of about 0.2g / L to about 40g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of about 0.2g / L to about 30g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of about 0.2g / L to about 25g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of about 0.8g / L to about 2.5g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of about 0.5 g / L to about 2.2g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least Ig / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least 1.5g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least about 1.8g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least about 2g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least about 2.3g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least about 5g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least about lOg / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least about 15g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least about 20g / L. In an embodiment, the recombinant human lactoferrin is produced at a concentration of at least about 25g / L.

[0161] Downstream production methods

[0162] The recombinant human lactoferrin of the present disclosure can be isolated or purified using any method known to a person skilled in the art, including, for example a method or combination of steps as described in Krolitzki et al., (2022). In an embodiment, the recombinant human lactoferrin is isolated from the media.

[0163] When using recombinant techniques, the recombinant human lactoferrin of the disclosure can be present intracellularly, or directly secreted into the medium. If the protein is present intracellularly, as a first step, the cell is separated from the media, lysed and the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation, filtration or ultrafiltration. Where the protein is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, a tangential flow filtration (TFF) system with flat, spiral wound or hollow fiber membranes. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. Supernatants can also be used directly for purification.

[0164] The recombinant human lactoferrin prepared from the cells or supernatant can be isolated using, for example, filtration, clarification, ultrafiltration-diafiltration, chromatography, ion exchange chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography) or any combination thereof. These methods are known in the art and described, for example in WO99 / 57134 or Zola (1997). In an embodiment, the recombinant human lactoferrin is isolated using chromatography. In an embodiment, the recombinant human lactoferrin is isolated using ion exchange chromatography. In an embodiment, the recombinant human lactoferrin is isolated using anion exchange chromatography. In an embodiment, the recombinant human lactoferrin is isolated using cation exchange chromatography.

[0165] In an embodiment, the recombinant human lactoferrin is isolated using one or more of the following steps: filtration / clarification, buffer exchange ultrafiltration- diafdtration, purification chromatography, buffer exchange ultrafiltration-diafiltration, sterile filtration followed by spray drying or freeze-drying for production in a powder form.

[0166] The skilled artisan will also be aware that a the recombinant human lactoferrin of the disclosure can be modified to include a tag to facilitate purification or detection. The tag may be any suitable tag known to a person skilled in the art. In some embodiments, the tag is strep-tagll. The resulting protein is then purified using methods known in the art, such as affinity purification. For example, a protein comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-His-tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. This may be followed by e.g. an enzymatic cleaving step to remove the tag, ultrafiltration-diafiltration to remove buffer salts and spray drying or freeze drying.

[0167] Compositions

[0168] In an aspect, the present invention provides a composition comprising a recombinant human lactoferrin as described herein.

[0169] In an embodiment, the composition comprises rhLF3 or a sequence at least 70% identical thereto. In an embodiment, the composition does not comprise a further lactoferrin. In an embodiment, the composition does not comprise a further recombinant lactoferrin. In an embodiment, the composition does not comprise a further recombinant human lactoferrin. In an embodiment, the composition does not comprise bovine lactoferrin. In an embodiment, the composition does not comprises a human endogenously produced lactoferrin. In an embodiment, the composition does not comprise recombinant human lactoferrin expressed in a rice plant.

[0170] In an embodiment, the composition comprising a recombinant human lactoferrin consists of the recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto. In an embodiment, the composition is an isolated or purified composition comprising rhLF3 or a sequence at least 70% identical thereto. In an embodiment, the composition is an enteral composition.

[0171] In an embodiment, the composition is a topical composition. In an embodiment, the composition is a food composition.

[0172] In an embodiment, the composition is not an adjuvant composition.

[0173] In an embodiment, the food composition is selected from one or more of: infant formula, premature infant formula, low birth weight infant formula, a human milk fortifier, toddler milk formula, functional food and a powdered food. In an embodiment, the infant formula is administered at least 5 times a day. In an embodiment, the infant formula is administered at least 6 times per day.

[0174] In an embodiment, the food composition is infant formula. In an embodiment, the food composition is premature infant formula. In an embodiment, the food composition is low birth weight infant formula. In an embodiment, the food composition is a human milk fortifier. In an embodiment, the food composition is toddler milk formula.

[0175] In an embodiment, the food composition is a functional food. In an embodiment, the food composition is a powdered food.

[0176] In an embodiment, the food composition is a food composition for the elderly.

[0177] In an embodiment, the food composition is selected from one or more of: an animal feed, animal feed supplement, pre-starter feed, starter feed, milk replacer, aquaculture feed, pet food and pet treat. In an embodiment, the food composition is an animal feed. In an embodiment, the food composition is an animal feed supplement. In an embodiment, the food composition is pre-starter feed. In an embodiment, the food composition is starter feed. In an embodiment, the food composition is milk replacer. In an embodiment, the food composition is aquaculture feed. In an embodiment, the food composition is pet food. In an embodiment, the food composition is a pet treat.

[0178] In an embodiment, the composition is a beverage composition.

[0179] In an embodiment, the beverage composition is selected from one or more of: a dairy product, a dairy product substitute, a functional beverage, a powdered beverage, a sports drink and an energy drink. In an embodiment, the beverage composition is a dairy product. In an embodiment, the beverage composition is a dairy product substitute. In an embodiment, the beverage composition is a functional beverage. In an embodiment, the beverage composition is a powdered beverage. In an embodiment, the beverage composition is a sports drink. In an embodiment, the beverage composition is an energy drink.

[0180] In an embodiment, the composition is an oral health composition.

[0181] In an embodiment, the composition is a bone health composition.

[0182] In an embodiment, the composition is a nutraceutical composition.

[0183] In an embodiment, the composition is an anti-microbial composition. In an embodiment, the anti-microbial composition comprises one or more of: a bacteriocin, an organic acid, an enzyme, monolaurin and a polyphenolic extract. In an embodiment, the anti-microbial composition comprises a bacteriocin. In an embodiment, the anti-microbial composition comprises an organic acid. In an embodiment, the antimicrobial composition comprises an enzyme. In an embodiment, the anti-microbial composition comprises monolaurin. In an embodiment, the anti-microbial composition comprises a polyphenolic extract.

[0184] In an embodiment, the bacteriocin is selected from one or more of: nisin, pediocin and enterocin. In an embodiment, the bacteriocin is nisin. In an embodiment, the bacteriocin is pediocin. In an embodiment, the bacteriocin is enterocin.

[0185] In an embodiment, the organic acid is selected from one or more of: lactic acid, acetic acid, citric acid and malic acid. In an embodiment, the organic acid is lactic acid. In an embodiment, the organic acid is acetic acid. In an embodiment, the organic acid is citric acid. In an embodiment, the organic acid is malic acid.

[0186] In an embodiment, the enzyme is lysozyme. In an embodiment, the enzyme is lactoperoxidase.

[0187] In an embodiment, the composition is an anti-bacterial composition.

[0188] In an embodiment, the composition is a pre-biotic composition.

[0189] In an embodiment, the composition is a pro-biotic composition.

[0190] In an embodiment, the composition is an anti-inflammatory composition.

[0191] In an embodiment, the composition is an immune enhancing composition.

[0192] In an embodiment, the composition is a skin care composition. In an embodiment, the composition is a cosmetic composition. In an embodiment, the skin care composition or cosmetic composition is selected from one or more of: a facial moisturiser, a body moisturiser, a facial cream a body cream, a serum, an eye cream, a face mask, a sunscreen, a sun protection factor (SPF) product, an acne treatment, a body lotion, a hair care product, a lip balm, a lip treatment, wound dressing and burn treatment. In an embodiment, the skin care composition or cosmetic composition is a facial moisturiser. In an embodiment, the skin care composition or cosmetic composition is a body moisturiser. In an embodiment, the skin care composition or cosmetic composition a facial cream a body cream. In an embodiment, the skin care composition or cosmetic composition is a serum. In an embodiment, the skin care composition or cosmetic composition is an eye cream. In an embodiment, the skin care composition or cosmetic composition is a face mask. In an embodiment, the skin care composition or cosmetic composition is a sunscreen. In an embodiment, the skin care composition or cosmetic composition is a sun protection factor (SPF) product. In an embodiment, the skin care composition or cosmetic composition is an acne treatment. In an embodiment, the skin care composition or cosmetic composition is a body lotion. In an embodiment, the skin care composition or cosmetic composition is a hair care product. In an embodiment, the skin care composition or cosmetic composition is a lip balm. In an embodiment, the skin care composition or cosmetic composition is a wound dressing. In an embodiment, the skin care composition or cosmetic composition is a burn treatment.

[0193] In an embodiment, the composition is in a form selected from one or more of a: powder, tablet, capsule, liquid, emulsion, syrup, gummy, gel and a sachet. In an embodiment, the composition is a powder. In an embodiment, the composition is a tablet. In an embodiment, the composition is a capsule. In an embodiment, the composition is a liquid. In an embodiment, the composition is a emulsion. In an embodiment, the composition is a syrup. In an embodiment, the composition is a gummy. In an embodiment, the composition is a gel. In an embodiment, the composition is a sachet.

[0194] In an embodiment, the composition is formulated for enteral administration. In an embodiment, the enteral composition comprises one or more of the following: protein, peptide, carbohydrate, lipid, water, vitamin, mineral, fibre, pre-biotic, pro-biotic, antioxidant, polyphenol, flavouring agent and milk protein / s. In an embodiment, the enteral composition comprises a protein. In an embodiment, the enteral composition comprises a peptide. In an embodiment, the enteral composition comprises a carbohydrate. In an embodiment, the enteral composition comprises a lipid. In an embodiment, the enteral composition comprises water. In an embodiment, the enteral composition comprises a vitamin. In an embodiment, the enteral composition comprises a mineral. In an embodiment, the enteral composition comprises a fibre. In an embodiment, the enteral composition comprises a pre-biotic. In an embodiment, the enteral composition comprises a pro-biotic. In an embodiment, the enteral composition comprises an antioxidant. In an embodiment, the enteral composition comprises a flavouring agent. In an embodiment, the enteral compositions comprise one or more milk protein / s.

[0195] In an embodiment, the vitamin is selected from one or more of: vitamin Bl, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D and vitamin E. In an embodiment, the vitamin is vitamin B 1. In an embodiment, the vitamin is vitamin B2. In an embodiment, the vitamin is vitamin B3. In an embodiment, the vitamin is vitamin B5. In an embodiment, the vitamin is vitamin B6. In an embodiment, the vitamin is vitamin B7. In an embodiment, the vitamin is vitamin B9. In an embodiment, the vitamin is vitamin B12. In an embodiment, the vitamin is vitamin C. In an embodiment, the vitamin is vitamin D. In an embodiment, the vitamin is vitamin E.

[0196] In an embodiment, the mineral is selected from one or more of: zinc, selenium, iron as FeCh and iron as FeSCh. In an embodiment, the mineral is zinc. In an embodiment, the mineral is selenium. In an embodiment, the mineral is iron as FeCh. In an embodiment, the mineral is iron as FeSC>4.

[0197] In an embodiment, the polyphenol is selected from one or more of: ferulic acid, coumaric acid and tyrosol. In an embodiment, the polyphenol is ferulic acid. In an embodiment, the polyphenol is coumaric acid. In an embodiment, the polyphenol is tyrosol. In an embodiment, the milk protein is selected from one or more of: an immunoglobulin G, osteopontin and transferrin. In an embodiment, the milk protein is an immunoglobulin G. In an embodiment, the milk protein is osteopontin. In an embodiment, the milk protein is transferrin.

[0198] In an embodiment, the composition is formulated for topical administration. In an embodiment, the topical composition comprises one or more of the following: active pharmaceutical ingredient (API), active cosmetic ingredient, micronutrient, emollient, humectant, occlusive, antioxidant, preservative, fragrance, penetration enhancer and stabiliser. In an embodiment, the topical composition comprises an active pharmaceutical ingredient (API). In an embodiment, the topical composition comprises an active cosmetic ingredient. In an embodiment, the active cosmetic ingredient comprises one or both of the following: hyaluronic acid and retinol. In an embodiment, the active cosmetic ingredient is hyaluronic acid. In an embodiment, the active cosmetic ingredient is retinol. In an embodiment, the topical composition comprises a micronutrient. In an embodiment, the micronutrient comprises one or more of: vitamin A, vitamin C and vitamin E. In an embodiment, the micronutrient is vitamin A. In an embodiment, the micronutrient is vitamin C. In an embodiment, the micronutrient is vitamin E. In an embodiment, the topical composition comprises an emollient. In an embodiment, the topical composition comprises a humectant. In an embodiment, the topical composition comprises an occlusive. In an embodiment, the topical composition comprises an antioxidant. In an embodiment, the topical composition comprises a preservative. In an embodiment, the topical composition comprises a fragrance. In an embodiment, the topical composition comprises a penetration enhancer. In an embodiment, the topical composition comprises a stabiliser.

[0199] Other active agents A person skilled in the art will appreciate that the compositions, methods and uses described herein comprise one or more other active agents.

[0200] In an embodiment, the active agent is selected from one or more of: an iron supplement, pre-biotic, pro-biotic, post-biotic, vitamin and mineral, vitamin B12, transferrin, immunoglobulin and osteopontin.

[0201] In an embodiment, the active agent is an iron supplement. In an embodiment, the iron supplement is selected from one or more of: ferrous gluconate, ferric citrate, ferric chloride (FeCh), ferric sulfate (Fe2(SO4)s) and ferrous sulfate (FeSCh) In an embodiment, the iron supplement is ferrous gluconate. In an embodiment, the iron supplement is ferric citrate. In an embodiment, the iron supplement is ferric chloride (FeCh). In an embodiment, the iron supplement is ferric sulfate (Fe2(SC>4)3). In an embodiment, the iron supplement is ferrous sulfate (FeSCh).

[0202] In an embodiment, the active agent is a pre-biotic. In an embodiment, the active agent is a pro-biotic. In an embodiment, the active agent is a post-biotic. In an embodiment, the active agent is a vitamin. In an embodiment, the active agent is a mineral. In an embodiment, the active agent is a vitamin B12. In an embodiment, the active agent is a transferrin. In an embodiment, the active agent is an immunoglobulin. In an embodiment, the active agent is an osteopontin.

[0203] In an embodiment, the active agent is not a vaccine. In an embodiment, the active agent is not a tuberculosis vaccine. In an embodiment, the active agent is not a bovine vaccine.

[0204] Administration

[0205] A person skilled in the art will appreciate that the compositions, methods, uses as described herein are administered enterally or topically to the subject.

[0206] In an embodiment, the composition is for daily administration. In an embodiment, the composition is for twice daily administration. In an embodiment, the composition is administered three times per day. In an embodiment, the composition is administered 4 times per day. In an embodiment, the composition is administered three times per day. In an embodiment, the composition is administered 5 times per day. In an embodiment, the composition is administered 6 times or more per day.

[0207] In an embodiment, administration is for about 7 days. In an embodiment, administration is for about 7 days to about 70 days. In an embodiment, administration is for about 20 days to about 65 days. In an embodiment, administration is for about 25 days to about 65 days. In an embodiment, administration is for about 30 days to about 60 days. In an embodiment, administration is for at least about 30 days. In an embodiment, administration is for at least about 45 days. In an embodiment, administration is for at least about 60 days.

[0208] In an embodiment, the composition is administered before, after or at the same time as one or more other active agents as described herein. In an embodiment, the composition is administered before the one or more other active agents as described herein. In an embodiment, the composition is administered after the one or more other active agents as described herein. In an embodiment, the composition is administered at the same time as one or more other active agents as described herein.

[0209] EXAMPLES

[0210] Example 1 - Methods

[0211] Background strains

[0212] Background strains for strain engineering: the P. pastoris host strains used in this project were sourced from ATUM Bio (USA) or Biogrammatics Inc. USA : PPS-9010 BG10 (wildtype), PPS-9011 BG11 (aoxl A; MutS), and PPS-9016 BG16 (pep4A, prbl A, protease deficient). All background strains were routinely maintained on YPD agar (1% yeast extract, 2% peptone, 2% glucose; 2% bacteriological agar) plates containing suitable antibiotic selection as required. Master stocks were made by culturing a single colony of each strain overnight in YPD broth (1% yeast extract, 2% peptone, 2% glucose), and suspending cell pellet in YPD Broth containing 30% glycerol. Cells were frozen on a dry ice / ethanol bath and stored at - 80°C.

[0213] Media

[0214] Media used for strain engineering and flask cultivations were as described in the ATUM Pichia Culture and Induction protocol and the Invitrogen Pichia manual.

[0215] Yeast Extract Peptone Dextrose (YPD) Medium: 1% yeast extract, 2% peptone, 2% dextrose (glucose), supplemented with 20 g / L for solid medium.

[0216] Yeast Extract Peptone Glycerol (YP glycerol) Medium: 1% yeast extract, 2% peptone, 2% glycerol, supplemented with 20 g / L for solid medium. YPD + Zeocin or YP glycerol + Zeocin: supplemented with lOOpg / mL Zeocin when required for selection.

[0217] Buffered Glycerol-complex Medium (BMGY): 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base (YNB), 4 x 10-5 % biotin, 1% glycerol, buffered with 0.1 M potassium phosphate buffer, pH 6.0; supplemented with lOOpg / mL Zeocin when required for selection. Buffered Minimal Methanol (BMM): 300 mM potassium phosphate pH 6.0, 1.34% YNB (yeast nitrogen base), 4 x 10-5 % biotin, 1% methanol. BMM + Zeocin: BMM supplemented with lOOpg / mL Zeocin when required for selection.

[0218] Buffered Minimal Methanol with Yeast Extract (BMMY): 1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB (yeast nitrogen base), 4 x 10-5 % biotin, 2% methanol. BMMY + Zeocin: BMMY supplemented with lOOpg / mL Zeocin when required for selection.

[0219] DNA manipulation

[0220] Gene fragments used for construction of vectors were designed using SnapGene (Dotmatics, USA) or Geneious Prime 2021 v2.2 (Dotmatics, USA) and then codon- optimised using Geneart codon optimisation tool (Invitrogen, Thermofisher). Gene fragments were synthesised as G Blocks (IDT, USA) or as priority gene plasmid inserts (Genewhiz, Azenta, USA). Plasmid DNA was routinely prepared for analysis using Bioline Isolate II Plasmid DNA Miniprep Kit (Bioline, USA) and larger scale preparations were prepared using CompactPrep Midi plasmid kit (Qiagen, USA).

[0221] Recombinant protein expression constructs were prepared using three different backbones and a variety of promotor, terminator, antibiotic selection, secretion signal and amino acid sequence arrangements (Table 1). The constructs were designed and synthesised for expression of nature-equivalent human lactoferrin fused with the Saccharomyces cerevisiae alpha-mating factor secretion signal and assessed in at least two of four selected plasmid backbones (pCHPIC, pAK902, pGAP and pCHG2)

[0222] Plasmid vector backbones and inserts were prepared by restriction enzyme digestion (New England Biolabs, USA), gel-purified using Isolate II PCR and Gel Extraction Kit (Bioline, USA) and ligated using T4 DNA ligase (New England Biolabs, USA), following manufacturer’s instructions with incubation at 16°C overnight. Ligations were transformed into chemically competent Escherichia coli NEB 5-alpha (NEB, USA) or Escherichia coli TOPP 10 (Invitrogen, Thermo, USA) according to manufacturer’s instructions and selected on Luria agar plates containing the appropriate antibiotic selection reagent (Zeocin, Invitrogen, Thermo, USA; Geneticin, Gold Biotechnology, USA; Hygromycin, Sigma-Aldrich, USA). All constructs were confirmed by DNA Sanger sequencing (Macrogen, S. Korea).

[0223] Human genetic variant rhLF3 was chosen for recombinant expression. Genetic constructs for each of these isoforms were codon-optimised (Geneart, Invitrogen, USA) and fused with the coding sequence for alpha-mating factor from Saccharomyces cerevisiae for expression and secretion into the supernatant. Table 1. Recombinant protein expression plasmid vector design.

[0224] Plasmid DNA preparation

[0225] Plasmid DNA was routinely prepared for transformations using CompactPrep Midi plasmid kit (Qiagen) following the manufacturer's instructions. DNA was quantified using a Nanodrop 2000 Spectrophotometer (Thermofisher Scientific), following the manufacturer’s instructions.

[0226] Preparation of electrocompetent P. pastoris cells

[0227] P. pastoris cultures (50 mL) were prepared in YPD + Zeocin in 250 mL baffled flasks from overnight cultures at 0.2-0.3 OD600nm. At 0.8-1.0 OD cells were centrifuged at 500 *g for 15 min at RT in 50 ml tubes. Supernatant was discarded and the pellet resuspended with gentle swirling in 9 ml ice-cold BEDS solution (lOmM Bicine-NaOH, pH8.3, 3% (v / v) ethylene glycol, 5% (v / v) DMSO, IM sorbitol) each. lOOmM DTT (1 mL of IM DTT) was added to each tube and the solution mixed then incubated at 30°C, 200 rpm for 5 minutes. Cells were spun down again then pellets suspended in 1ml ice- cold BEDS solution. Tubes were kept at room temp while dividing into 100ml aliquots. Aliquots were placed in a pre-chilled (4°C) freezer box lined with polystyrene then transferred to a -80°C freezer for storage.

[0228] Transformation of electrocompetent P. pastoris cells

[0229] Required plasmids were linearised in 10 pg aliquots with suitable single-site restriction enzymes (Pmel for integration into AOX1 locus on genome or Swal for nonspecific site integration) by incubation at 37°C for >lhr. Agarose gel electrophoresis was used to check linearisation, after which the linear DNA was precipitated with 1 / 10 volume 3M NaOAc pH 5.3, and 2.5 volumes absolute ethanol at -20°C for at least 1 hr. DNA was pelleted 6,000 x g and room temperature (RT) for 15 mins. Pellets were washed with 500 mL 70% ethanol with a 2-minute re-spin as above. Pellets were allowed to air-dry then resuspended in 30 pl milliQ water. DNA was quantified using a Nanodrop 2000 Spectrophotometer following the manufacturer’s instructions. Linearised plasmid DNA in < lOpl volume was used for each transformation with 50 pL competent cells, by mixing with freshly thawed competent cells, incubating on ice for 5 min and then electroporating in chilled 2mm electroporation cuvettes (Cell Projects, USA) using a Biorad Gene Pulser, following manufacturer’s instructions for electroporation of P. pastoris cells (1.5kV, 25F, 200 pM). After electroporation, 500pL chilled IM sorbitol was immediately added to the electroporation cuvette, mixed gently, and the solution transferred to a 15mL tube containing 500pL YPD medium. After 2-4 hr outgrowth at 30°C with shaking, transformation reactions were diluted appropriately, plated onto suitable selection media (e.g. YPD plus antibiotic) and incubated at 28°C for 48-72 hrs. The concentration of antibiotic was increased above standard selection concentrations in order to enhance selection for high copy number recombinant cassette genomic insertions for base recombinant expression strains, using e.g. 500-1000 pg / mL zeocin or geneticin. After incubation, colonies were patched onto similar YPD selection media for working stocks and short-term storage.

[0230] Genomic DNA extraction and determination of copy number

[0231] Cultured cells were pre-treated as follows and then genomic DNA was prepared using Isolate II Genomic DNA prep (Bioline, Meridian Biosciences, Australia), as per manufacturer’s instructions. An overnight culture of yeast was pelleted 500g, 10 mins, room temperature (RT) and resuspended in 1ml lOmM EDTA, pH8. After mixing well the suspension was pelleted by centrifugation 5000g, lOmin, RT, and resuspended in 600 pl sorbitol buffer (1.2M sorbitol, lOmM CaC12, 0.1M Tris-HCL, pH7.5, 35mM P- mercaptoethanol) + 20 pl lyticase (lOU / ml). After incubation at 30°C for 30 minutes, spheroplasts 7were pelleted 2,000xg for 10 min and resuspended in 180 pl Buffer GL + 25 pl Proteinase K (50mg / ml) with vigorous vortexing, before incubation 56°C overnight and subsequent addition of RNase A (lOpg / ml) with mixing and incubation at 37°C for 60 min. Thereafter genomic DNA was prepared as per manufacturer’s instructions from step 3. qPCR relative copy number in Pichia genome vs PGK-1 single copy gene

[0232] Primers specific to the gene of interest were designed using Primer Quest and synthesised (IDT, USA). Primer sequences are shown in Table 2. For PCR, RNaseA- treated Pichia genomic DNA was diluted to ~ O.Olng / pl in sterile, distilled water and 1 pL mixed with 9 pL master mix (iTag Universal SYBR Mix (Biorad), 200pM forward primer, 200pM reverse primer). Amplification was performed using Biorad CFX96 (Biorad, Australia). The conditions were optimised for each primer set, but standard amplification conditions involved denaturation at 95 °C for 5 minutes, 39 cycles of denaturation (95°C, 5 sec) and annealing / extension (60°C, 20 sec), followed by a final extension (65°C, 5 sec; 95°C, 30 sec). Primers specific to PGK were co-amplified in each dataset and comparative amplification used to calculate the relative copy number using Biorad CFX96 software (Biorad).

[0233] Table 2: Primers used for determining qPCR relative copy number

[0234] Culture conditions: small scale (growth block) and shake flask culture and expression

[0235] For both 48-well growth block culture (1 mL culture) and flask culture (50-400 mL cultures), the culture and protein expression conditions followed a standard two-step procedure of biomass production followed by induction of the gene of interest for recombinant milk protein expression, as outlined below.

[0236] Basic overnight biomass production: The required volume of BMGY or YP glycerol broth (+ selection marker e.g. zeocin) in a 5-fold volume culture vessel (e.g. 50 mL in a 250 mL flask) was inoculated with a large fresh colony and grown overnight at 28-30°C, 230 rpm. The OD600 was measured using a 1 / 10-1 / 40 dilution. Cells were pelleted by centrifugation at 3000 rpm, 10 min, 25°C or at room temperature (not cold).

[0237] Basic gene of interest expression protocol: Basic induction protocol for GOI expressed from AOX promotor: 50mL BMGY or YP glycerol broth (+ selection marker e.g. zeocin) in a 250mL culture vessel was inoculated with a large fresh colony and grown at 28-30°C, 230 rpm overnight. Cells were harvested as per biomass production protocol above and resuspended in either BMM medium (when the GOI was expressed from the AOX promoter) or MM medium (when the GOI was expressed from the GAP promoter) of desired volume to an OD600nm of ~5.0 - 8.0. Cells were cultured at 28-30°C, 230 rpm for 72 - 108 hours (time varied according to the protein being expressed; for caseins between 72-96 hpi is generally best). When the GOI was expressed from the AOX promoter, the culture was supplemented with 1% methanol every 24hrs, or 1% methanol + 0.5% glucose, every 24 hrs (for BG11 or Mxrl overexpression strains) using direct addition of neat methanol. Samples of cultures were taken at t=0 (resuspension in induction medium), and every 24 hrs thereafter usually at ~ 24, 48, 72 and 96 hours post induction (hpi). Samples were analysed for growth by measuring OD600nm (1-10 to 1- 40 dilution in water) using a platereader (SpectraMax M3, Molecular Devices, USA) then centrifuged at 3000g, 10 min, 4°C and the supernatant analysed for total protein (Biorad Protein Dye, Biorad) recombinant lactoferrin titre (HPLC and proteomics) and SDS-PAGE protein profile.

[0238] SDS-PAGE

[0239] Culture supernatants were routinely analysed for total protein and recombinant protein content using SDS-polyacrylamide gel electrophoresis (PAGE). Whole cell or cell pellet samples were analysed where considered appropriate.

[0240] Sample preparation: 30pL of neat or concentrated supernatant samples were mixed with 16pL of blue protein loading dye (NEB, Australia) containing reductant (DTT). Additional 5mM EDTA was added to the loading dye for increased denaturation when required. For whole cell or cell pellet samples, 20pL of sample was mixed with 20pL loading dye. Samples were then heated to 96°C for 6 minutes, cooled briefly on ice and 30pL loaded into wells of pre-made Invitrogen Bolt 4-12% Bis-Tris Plus gel (1.0mm * 12 well or 1.0mm * 17 well), electrophoresed at 120-130V for ~ 1 hour. After electrophoresis gels were stained with AcquaStain protein staining reagent (Bulldog Bio), usually overnight, then destained using Milli-Q deionized water for 20-30 minutes before visualisation and photography with Vu GelDoc digital imaging system (PopBio Systems, UK). For concentration of samples, 500pL of sample was applied to either 3kDa or lOkDa cut-off column (Merck Millipore, USA) and centrifuged for 10 minutes, 14000 rpm at room temp or 5°C, as appropriate. This resulted in ~ 5-fold concentration of total protein.

[0241] High performance liquid chromatography (HPLC)

[0242] Accurate quantification of secreted, recombinant, non-catalytic proteins such as lactoferrin is complex and difficult. An HPLC method was developed to provide a rapid, reproducible, quantitative method of analysing recombinant protein titre in supernatants, and thus of assessing strain and ferment performance. The HPLC method used was adapted from two literature reports (Bordin et al., 2001; Bonfatti et al. 2008) and a methodology obtained from Dr. Roderick Williams (pers comms). A BioRad column (Hi-Pore RP-318 Column 250 x 4.6 C18 5 pm 300A 3 mg 2-7 125-0551) or Zorbax column (300SB-C18 4.6*250*5u column PN 880995-902) was used. HPLC equipment: The HPLC equipment consisted of an Agilent 1200 Series chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump (Agilent 1200 Series, G1312A). A variable wavelength ultraviolet detector (Agilent 1200 Series, G1314B) was also used. The equipment was controlled by the Agilent Chem-Station for LC Systems software which sets solvent gradient, data acquisition and data processing. Separations were performed on a reversed-phase analytical column C18 (Zorbax 300SB-C18 RP, Agilent Technologies Part No. 880995- 902) with a silica-based packing (5pm, 300A°,250><4.61.D.). A Security Guard Cartridge System was used as pre-column (Eclipse C18 4mm * 3.0 mm, Agilent Technologies Ltd.). The sample vial was kept at constant low temperature (10°C) by a liquid refrigerator (Agilent 1200 series, G1330B) and injected via an auto-sampler (Agilent 1200 series, G1329 A).

[0243] Chromatographic conditions: Gradient elution was carried out with a mixture of two solvents. Solvent A consisted of 0.1% trifluoroacetic acid (TFA) in water and solvent B was acetonitrile. Separations were performed with the following program: linear gradient from 27 to 32% B in 2 min from 32 to 39% B in 15 min (0.5% Bmin-1), from 39 to 60 % B in 13 min, followed by an isocratic elution at 60 % B for 3 minutes and return linearly to the starting condition in 2 min (total gradient time 38 minutes). Before the following sample, the column was re-equilibrated under the starting conditions for 3 min. Therefore, the total analysis time per sample was 40min. The flowrate was 0.7 mL / min, the column temperature was kept at 40°C and the detection was made at wavelength of 280 & 214 nm. The injection volume consisted of 10-20 pL.

[0244] Quantitation: Samples containing recombinant lactoferrin proteins were quantified by peak area for the target protein (mg / mL) by comparison with a standard curve prepared using recombinant human lactoferrin protein expressed in rice plants (L1294, Sigma-Aldrich, now Merck Millipore, Australia). Samples were diluted 1 : 1 (150 pL: 150 pL) with a guanidinium hydrochloride solubilization buffer (0.1 M Tris buffer pH 6.8, 6 M GdnHCl, 5.37 mM sodium citrate, and 19.5 mM DTT pH 7) if required, i.e. when aggregation of proteins was present or likely.

[0245] Standard tryptic digest proteomic analysis by LC-MS

[0246] Chemicals including acetone, ammonium bicarbonate, DTT, formic acid, urea were purchased from Sigma. Acrylamide and Braford assay reagent were purchased from BioRad. Acetonitrile, FlexMix Calibration Solution and Retention Time Calibration Mixture were purchased from Thermo Scientific. Protein extraction and quantification: Proteins from culture supernatant were precipitated by cold acetone at-20°C overnight. After being air dried the protein pellets were dissolved in 8 M Urea. Protein concentration was determined using Bradford assay in comparison with a BSA standard curve.

[0247] Reduction, alkylation and tryptic digestion: 10 pL (5 pg) of protein was reduced with 1.5 pL of 15% (w / v) DTT for 30 min at room temperature and alkylated with 1.5 pL of 40% (w / v) acrylamide for 30 min at room temperature. Proteins were digested by adding 47 pL of trypsin solution (0.1 pg of trypsin in 25 mM ammonium bicarbonate) and incubated at 37°C for 3 hours to overnight. The digestion was stopped with 1 pL of 10% (v / v) formic acid and filtered with a 0.22pm filter. 100 ng of the tryptic digested peptides was injected to LC-MS analysis.

[0248] LC-MS analysis: Tryptic peptides (100 ng) were desalted and concentrated with a trap column (PepMaplOO C18 5 mm x 300 pm, 5 pm, Thermo Scientific) and separated on a nano column (PepMaplOO C18 150 mm x 75 pm, 2 pm, Thermo Scientific) using an UltimateTM 3000 RSLC nano LC system (Thermo Scientific). Mobile phase A consisted of water and 0.1% (v / v) formic acid and mobile phase B consisted of 80% (v / v) acetonitrile and 0.08% (v / v) formic acid. Tryptic peptides were eluted using a gradient of 5% to 40% solvent B for 60 minutes and 40% to 99% solvent B for 10 minutes. The eluted peptides were ionized with a Nanospray Flex Ion Source (Thermo Scientific). The spray voltage was set to 2.3 kV and the temperature of the heated capillary was set at 300 °C. After ionization, mass spectra (MSI) and tandem mass spectra (MS / MS) analysis was performed using an Orbitrap Fusion MS (Thermo Scientific). MS survey scans of peptide precursors were performed in the Orbitrap detector, and the scan range was 400 to 1500 m / z at resolution of 120 K (at 200 m / z). The target value of automatic gain control (AGC) was set as 4 x 105. The maximum injection time for the MS was 50 ms. MS / MS was performed on the most abundant precursors of charge states 2+ to 7+ with intensity greater than 1 x 105. They were isolated by the quadrupole with a window of 1.6 m / z. Fragmentation was achieved by high-energy collisional dissociation (HCD) with collision energy of 28%. Fragments were detected in the ion trap detector in rapid scan rate mode. The AGC target was 4 x 103, maximum injection time was 300 ms and the dynamic exclusion was 15 seconds. The instrument was set to run in top speed mode with a three second cycle for both the MS and MS / MS scans.

[0249] Data analysis: Protein Discoverer 2.2 (Thermos Scientific) and Sequest HT search engine were used to identify peptides / proteins and quantify relative abundance of proteins. The spectrum data was searched against the Uniprot Pichia pastoris and the expected recombinant lactoferrin protein sequences. Precursor mass tolerance was set to lOppm and product ions were searched at 0.6 Da. Three missed tryptic cleavages were allowed. Modification included Oxidation (+ 15.995Da), Deamidation (+ 0.984 Da), Amidation (- 0.984Da), and Propionamidation (+ 71.037 Da). Peptide spectral matches were validated using the Percolar algorithm, based on q-values and 1% False Discovery Rate (FDR). Relative abundance is calculated from precursor abundance intensity and is normalized from total peptide amount.

[0250] Detailed post-translational modification (PTM) tryptic digest proteomic analysis

[0251] Protein quantitation and extraction: The protein concentration of the supplied samples was determined using a Bradford colorimetric assay (read at 595nm). Protein (40-50pg) was extracted using 200pl of urea-based buffer (UA buffer: 8M urea in 0. IMTris-HCl, pH8). All samples were vortexed, sonicated for 5 minutes in a sonic water bath, and shaken at 1000 rpm for 30 minutes at RT in a thermomixer.

[0252] Tryptic digestion: Protein was subjected to FASP (filter-assisted sample preparation) wherein the protein extract was applied to a 10 kDa molecular weight cutoff (MWCO) filter (Millipore, Australia) and centrifuged (20,800 x g, 15 min). The protein on the filter was washed with two 200 pL volumes of UA buffer with centrifugation (20,800 x g, 15 min). Protein was reduced on-filter with 200 pL of 50mM DTT followed by two washes with 200 pL of UA buffer with centrifugation (20,800 x g, 15 min). To alkylate the cysteine residues, iodoacetamide (50 mM, 100 mL) was applied to the protein on the filter with incubation for 30 min at room temperature in the dark. The filter was again washed with 200 pL of UA buffer with centrifugation (20,800 x g, 15 min). The buffer was exchanged using 50 mM ammonium bicarbonate (pH 8.0) by two consecutive wash / centrifugation steps. Sequencing grade porcine trypsin (Promega, Alexandria, Australia) was added (0.005 pg / pL, i.e. 1 pg in 200 pL of 50 mM ammonium bicarbonate with 1 mM CaC12) to the samples and incubated for 16 h at 37°C in a thermomixer at 300rpm. The filters were transferred to fresh centrifuge tubes and the filtrate (digested peptides) was collected following centrifugation (20,800 x g, 10 min). The filters were washed twice with 200 pL of 50 mM ammonium bicarbonate and the filtrates were combined and lyophilised. The resultant peptides were re-suspended in 50 pL of 0.1% formic acid and 2 pL (equivalent to ~2 pg of total protein) was analysed by LC-MS / MS

[0253] LC-MS analysis: Proteolytically digested proteins were analysed with chromatographic separation (1 pL) on an Ekspert nanoLC415 (Eksigent, Dublin, CA, U.S.A.) directly coupled to the OptiFlow ion source of a TripleTOF 6600 LC-MS / MS (SCIEX, Redwood City, CA, USA). The peptides were desalted for 3 min on a Trajan ProteCol C18 (3 m, 120 A, 10 mm x 0.3 mm) trap column at a flow rate of 10 pL / min 0.1% FA, and separated on a ChromXP C18 (3 pm, 120 A, 150 mm x 0.3 mm) column at a flow rate of 5 pL / min at 30°C. A linear gradient from 3-25% solvent B over 68 min was employed followed by: 5 min from 25% B to 35% B; 2 min 35% B to 80% B; 3 min at 80% B, 80-3% B, 1 min; and 8 min re-equilibration. The solvents were: (A) 5% DMSO, 0.1% FA, 94.9% water; (B) 5% DMSO, 0.1% FA, 90% acetonitrile, 4.9% water. The instrument parameters were: ion spray voltage 4500 V, curtain gas 30 psi, GS1 30 psi and GS230 psi, heated interface 150°C. Data were acquired in information-dependent acquisition (IDA) mode comprising a time-of-flight (TOF)-MS survey scan followed by 30 MS / MS product ion scans. First stage MS analysis was performed in positive ion mode, mass range m / z 400-1250 and 0.25 s accumulation time. Tandem mass spectra were acquired on precursor ions >150 counts / s with charge state 2-5 and dynamic exclusion for 15 s with a 100 ppm mass tolerance.

[0254] Protein identification: Tandem mass spectrometry data was searched against in silico tryptic digests of a custom-built database. The database comprised the Uniprot sequences for the target proteins appended with Pichia pastoris (version 2019 / 11) and a database of contaminant proteins (known as the common repository of adventitious proteins, cRAP). The search parameters were defined as: thorough (to facilitate biological modifications); phosphorylation emphasis; iodoacetamide modified for cysteine alkylation (producing carbamidomethyl cysteine); and trypsin as the digestion enzyme. Additional modifications and cleavages were defined previously (Michelle L. Colgrave, 2014). The database search results were manually curated to yield the protein identifications using a 1% global false discovery rate (FDR) determined by the ProteinPilot companion FDR tool (Tang et al., 2008).

[0255] Detection of phosphosite occupancy: In addition to IDA analysis, sample digests were subjected to LC-MRM acquisition using a panel of transitions spanning the variable occupancy of all potential phosphosites. Reduced and alkylated tryptic peptides (5 pL) were chromatographically separated on a Kinetex C18 column (2.1 mm x 100 mm, Phenomenex) using a linear gradient of 5-45% acetonitrile in 0.1% formic acid, over 10 min at a flow rate of 400 pL / min. The eluent from the Shimadzu Nexera UHPLC was directed to a QTRAP 6500 mass spectrometer (SCIEX) equipped with a TurboV ion source operated in positive ion mode for data acquisition and analysis. The MS parameters were as follows: ion spray voltage, 5500 V; curtain gas, 35; GS1, 35; GS2, 40; source temperature, 500°C; declustering potential, 70 V; and entrance potential, 10 V. Peptides were fragmented in the collision cell with nitrogen gas based on rolling collision energy equations that are dependent on the mass and charge of the precursor ion. Peptide detection was confirmed using unscheduled MRM scanning experiments with a 0.6 second cycle time. Data were acquired using Analyst vl.7 software. Peak groups were examined using Skyline software (MacLean, Bioinformatics 2010) wherein quality peak groups encompassing multiple peaks with s:n >3 were considered detectable peptides.

[0256] Detection of disulphide bond arrangements: The samples were digested as per above without the addition of reducing or alkylating reagents to preserve disulphide bond arrangement. LC-MSMS was carried out as per section 2.3 with the MSI mass range extended to 2000 Th, and IDA criteria for MSMS acquisition also extended to 2000 Th. Concomitantly, disulphide cross-link database entries were generated using the X- COMB perl programs: protein2digest.ppl and digest2cxdb.pl. Data were searched against the new database using the Paragon algorithm within ProteinPilot software with special conditions attributed to the occurrence of ~8 mass units to cysteine residues (with high occurrence probability) for inter-peptide crosslinking and a loss of ~1 mass units to cysteine residues (with high occurrence probability) for intra-peptide cross-linking. Spectra were manually validated before reporting cross links.

[0257] N-glycan analysis of gel slices

[0258] Samples were prepared according to Kolarich et al., 2012, with modification.

[0259] Gels were cut into 1 - 1.5 mm pieces, and samples reduced and alkylated by treatment with dithiothreitol and iodoacetamide. N-glycans were released by addition of 10U PNGase F (Promega) and incubated at 37°C overnight, and the supernatant collected.

[0260] N-glycans were extracted by incubating gel pieces in acetonitrile and incubation for 15 mins, and supernatant collected. Gel pieces were washed twice more with 5 % formic acid with 15 min incubations and supernatant from washes combined with all collected supernatant, and dried to completion. Glycans were reduced by treatment with sodium borohydride and cleaned with graphite carbon packed tips.

[0261] For each sample (30%) was injected onto the LC-MS and analysed in negative mode with MS 1 scanning between m / z 400 - 2000. Top 5 most intense ions were selected for fragmentation. Glycan were separated on a Hypercarb column (1 mm ID x 30 mm, 3 pm particle size; ThermoFisher) using a gradient elution profile. Buffer A consisted of 10 mM ammonium bicarbonate in water, and Buffer B consisted of 10 mM ammonium bicarbonate in 70 % ACN. Gradient elution occurred as follows time (min) / Eluent B%: 0 / 0.0 (time min / Eleunt B%); 3 / 0.0; 4 / 14.0; 40 / 40.0; 48 / 56.0; 50 / 100; 54 / 100; 56 / 0.0 and 60 / 0.0. N-glycans were detected, and composition assigned and relative amounts of each glycan were quantified in each sample. Glycan composition shorthand, moetity and exact mass are as follows: N, HexNAc, 203.0794; H, Hexose, 162.0528; X, Pentose, 132.0423; N / A, Phosphate, 79.9663; N / A, Reduced end, 20.0262; N / A, Hydrogen, 1.0078. N / A = nonapplicable.

[0262] Protein purification

[0263] A variety of different methods were explored for the partial purification of the recombinant lactoferrin proteins and assessed for suitability regarding the functional requirements of the desired application. For nature equivalence, functional assessment and post-translational modification assessment, proteins were purified using ion exchange chromatography to provide a relatively pure sample for analysis, but with considerable loss of protein during the process. Somewhat surprisingly, anion exchange chromatography at high pH proved more successful in purification of recombinant lactoferrin away from the similarly sized alcohol oxidase enzyme protein present in P. pastoris supernatants. For functional assays, the partially purified eluted protein solutions were partially dialysed against Milli-Q water at 5°C (5 fold volume ratio, 2 buffer exchanges) to remove excess salt, frozen at -80°C and freeze-dried to a powder overnight (-40°C).

[0264] Strep-tagll-lactoferrin protein purification

[0265] The lactoferrins as described herein can also be purified using step-tag II protein purification. Vectors incorporating a strep-tag II fusion to the N-terminus of the lactoferrin protein coding sequence, followed immediately by a SUMO or enterokinase protease cleavage site for cleavage of the tag post-purification were designed and cloned in the pAK back bone used before to create pAK-rhLF3 -SSI or pAK-rhLF3-SEKl (SEQ ID NO: 11 and SEQ ID NO: 13). Pichia strains expressing these fusion proteins (SEQ ID NO: 12 and SEQ ID NO: 14) were produced as described. Supernatants containing Strep-tag II-lactoferrin fusion proteins were purified following the methodology of Schmidt et al (2007). Briefly supernatants were dialysed into buffer W (100 mM Tris- HC1 pH 8.0, 150 mM NaCl and 1 mM EDTA), loaded onto a StrepTactin Super Flow 5mL column (IBA Lifesciences, catalogue no. 2-1258-001) at 0.7mL / min flow rate, washed, and then eluted from the column using Buffer E (Buffer W containing 2.5 mM D-desthiobiotin) at 0.7ml / min with a 0 to 100% Buffer E gradient over 20 column volumes. Fractions (7mL per fraction) containing Strep-rhLF were cooled, dialysed into buffer W and assessed for Strep-rhLF content and functionality as described Western blot

[0266] Transfer and antibody incubation: Protein samples were separated by SDS-PAGE as above. Briefly 30pL of neat or concentrated supernatant samples were mixed with 16pL of blue protein loading dye (NEB, Australia) containing reductant (DTT). Additional 5mM EDTA was added to the loading dye for increased denaturation when required. For whole cell or cell pellet samples, 20pL of sample was mixed with 20pL loading dye. Samples were then heated to 96°C for 6 minutes, cooled briefly on ice and 30pL loaded into wells of pre-made Invitrogen Bolt 4-12% Bis-Tris Plus gel (1.0mm * 12 well or 1.0mm * 17 well), electrophoresed at 120-130V for ~ 1 hour.

[0267] After electrophoresis gels were transferred to a nitrocellulose membrane (iBlot NC Regular Stack, Invitrogen, USA) using iBlot 2.0 transfer device (Invitrogen, USA) following the recommended iBlot Gel Transfer Method P0 (20V 1 min, 23V 4 min, 25V 3 min). After transfer, membranes were washed briefly with sterile deionised water and then blocked by incubating in 5% bovine serum albumin (BSA, Sigma-Aldrich) in trisbuffered saline (TBS; 20mM Tris-HCl pH 7.6, 150mMNaCl), 4°C, overnight with gentle shaking. The block solution was replaced with primary antibody diluted 1 :300 in the blocking solution above and incubated for 3-24 hours as required. For B-lactoglobulin detection, a directly labelled primary antibody was used, proceeding straight from the 3 washes to detection and visualisation. For all other Western Blots, the primary antibody solution was removed, the blot was washed and then incubated with a suitable labelled secondary antibody diluted 1 :300 in TBS.

[0268] Washes: For all wash steps, blots were washed 4 times with excess volume of trisbuffered saline (TBS) plus Tween (TEST, 20mM Tris-HCl pH 7.6, 150mM NaCl, 0.1% Tween-20), for 5 minutes at room temperature with gentle shaking (40 rpm).

[0269] Detection and visualisation: Bound conjugated (horse radish peroxidase, HRP) antibody on membranes was detected by incubation of the membranes in freshly prepared 4-chloro-l-napthol solution (1 tablet 4-chl oro-1 -napthol [Sigma-Aldrich] was dissolved in 3 mL methanol), followed by addition of 10 mL TBS), and then documented using standard light photography or using GelDoc PopBio Image Analyser (PopBio, UK) after an appropriate incubation time at room temperature (23°C), approximately 5-20 minutes depending on signal intensity.

[0270] Example 2 - Strain engineering for expression of human lactoferrin variants

[0271] Pichia pastoris recombinant human lactoferrin protein production strains were developed for human lactoferrin protein variants, in a variety of P. pastoris background strains (including both Mut+ and Mut-). Over 200 different production strains with copy numbers ranging from 1 to 30 were assessed for rhLactoferrin expression and secretion in 48-well growth block and flask expression assays, using HPLC quantitation, LCMS proteomics and SDS-PAGE analysis (Figures 2 and 3). Titre data from shake flask fermentation experiments using select strains are presented in Table 3. Titres are within similar ranges reported in literature. Based on the flask titre data, an optimal initial rhLactoferrin production strain was chosen for variants and underwent bioreactor fermentation. In all cases, Mut+ strains produced superior rhLactoferrin titres under these expression conditions. The results are shown in comparison to control human Lactoferrin A (rCLFA), control human Lactoferrin B (rCLFB), control human Lactoferrin C (rCLFC) and control human Lactoferrin D (rCLFD).

[0272] Table 3: Recombinant lactoferrin production strains.

[0273] The patterns of N-glycan post-translational modifications for rhLactoferrin proteins derived from bioreactor fermentation were assessed and compared with typical native human and bovine lactoferrin N-glycan post-translational modifications. All the rhLactoferrin proteins assessed predominantly demonstrated the expected high hexose (likely mannose) HxN2 pattern, with a variety of hexose modifications from 7-15 but predominantly H9 or H10. As anticipated, no complex or hybrid N-glycan post- translational modifications were detected.

[0274] Example 3 - Small-scale production of recombinant human lactoferrin

[0275] Laboratory trials in Erlenmeyer flasks demonstrated production of recombinant human lactoferrin derivatives using engineered P. pastoris strains. As production in flasks is not suitable for large-scale / commercial manufacture, protein expression was initially tested in 2 L bench top bioreactors. Expression was tested using a high cell density fed-batch process configuration with glycerol used as the carbon source to increase cell density, and methanol was used to induce production of recombinant protein. High cell density processes were used to maximise process efficiency and protein titre. The growth medium used for all trials was a fully defined medium to minimise cost of goods (ingredients for the growth medium) and reduce batch-to-batch variation which can arise when using complex medium components such as yeast extract and partially digested protein. The use of bioreactor systems also enables greater process control (for parameters such as pH and dissolved oxygen) which can improve product titre and consistency.

[0276] Media

[0277] Seed medium: The seed medium used for this experiment comprised BMGY base medium (10 g of yeast extract, 20 g peptone), 1 M potassium phosphate buffer pH 6, 13.4% yeast nitrogen base with ammonium sulphate without amino acids, 0.02% (w / v) biotin solution and 10% (v / v) glycerol.

[0278] Base medium for bioreactors: The base medium used for bioreactors comprised 26.7 mL phosphoric acid, 0.93 g calcium sulphate dihydrate, 18.2 g potassium sulphate, 14.9 g magnesium sulphate 7H2O, 4.13 g potassium hydroxide and 40.0 g glycerol. Components were dissolved in the order listed above and made up to volume with reverse osmosis filtered water.

[0279] Trace metals solution (PTMi): The trace metals solution comprised: 6.0 g cupric sulphate 5H2O, 0.08 g Sodium iodide, 3.0 g manganese sulphate H2O, 0.2 g sodium molybdate 2H2O, 0.02 g boric acid, 0.5 g cobalt chloride, 20.0 g zinc chloride, 65.0 g ferrous sulphate 7H2O, 0.2 g biotin and 5.0 mL sulphuric acid.

[0280] Seed culture

[0281] Seed medium 200 ml was placed in a each of 2 x 1000 mL baffled flasks and inoculated with 400 pL of a glycerol stock of the Pichia pastoris Apep strain expressing rhLF3 or Mutsstrain expressing rhLF3. The flasks were incubated at 30°C with shaking at 200 rpm (25 mm orbit). The optical density of the seed culture after incubation for 23 hours was for the Apep strain was 23.64 and 23.30 for the Mutsstrain at OD 600 nm. Under microscopic analysis, the seed culture consisted of healthy yeast cells with no contamination evident.

[0282] Bioreactor operating conditions

[0283] Two Sartorius glass autoclavable bioreactors with a maximum working volume of 2 L connected to a Sartorius Biostat B controller were used for cultivation. The starting medium volume used was 1.0 L. The base medium was very acidic (~pH 1.8) and so was adjusted to pH 6 using the bioreactor pH controller and 28% ammonia solution (medium formed precipitate while adjusting the pH). The starting temperature setpoint was 30°C. The target optical density at inoculation was 0.30. Dissolved oxygen control (multiple parameter cascade) setpoint was 30% of saturation. The agitator was set to a minimum speed of 500 rpm and a maximum speed of 1200 rpm. The airflow was set to a minimum rate of 0.3 L / min and a maximum rate of 0.9 L / min. The oxygen flow was set to a minimum rate of 0 L / min and a maximum rate of 8 L / min. The cascade control strategy is summarised in Table 4. The pH control setpoint was pH 6.0. The acid used for automated pH control was 10% (v / v) H3PO4 and the base used for automated pH control was 28% NH3 solution. For foam control 5 mL of 10% Sigma Antifoam 204 was manually added before inoculation.

[0284] Table 4. Summary of cascade contro strategy.

[0285] Process

[0286] Batch mode: The bioreactors were operated in batch mode until the glycerol became depleted.

[0287] Glycerol fed-batch mode: The glycerol feed composition used was 200 mL of 25% (w / v) glycerol with 1.2 mL of PTMi added. Once the residual glycerol in the medium had been depleted the glycerol feed was started at a flow rate of 24 mL / h / L. Glycerol was fed at this rate for five hours.

[0288] Methanol fed-batch mode: For induction of protein expression the methanol feed composition used was 1000 mL of 50% methanol with 6 mL of PTMi added. Temperature was decreased to 27°C thirty minutes before methanol induction. At 27°C, the glycerol feed was stopped and the methanol feed tubing was primed. Once signs of glycerol depletion were observed the methanol induction feed was started as summarised in Table 5.

[0289] Table 5. Methanol feeding strategy.

[0290] Harvest

[0291] Both batches T-008 and T-009 were harvested 94.3 hours after inoculation and T- 010 was harvest 93.9 hours after inoculation. The temperature was decreased to 12°C over sixty minutes at the end of the process. The culture was drained from the bioreactor and the cells removed by centrifugation at 5,000 x g for 10 minutes. Samples were taken for measurement of optical density throughout the process. Samples were taken prior to induction (i.e. before methanol feed commenced) and at several time points postinduction for HPLC analysis. Supernatants were filtered through 1 x 500 mL 0.2pm bottle top filters, while the remaining supernatants were unfiltered. All the supernatants were stored at -80°C. Pellets were discarded.

[0292] For batch T-008 the final optical density was 333. The harvest volume was 1.8 L and the supernatant volume was 1.5 L. The filtered volume was 450 mL and the pellet weight was 373 g.

[0293] For batch T-009 the final optical density was 131. The harvest volume was 1.1 L and the supernatant volume was 0.97 L. The filtered volume was 430 mL and the pellet weight was 146 g.

[0294] For batch T-010 the final optical density was 160. The harvest volume was 0.9 L and the supernatant volume was 0.8 L. The filtered volume was 800 mL and the pellet weight was 138 g.

[0295] Biomass production

[0296] Biomass concentration increased then slowed when the glycerol feed rate was decreased. No methanol accumulation was seen.

[0297] Protein production The in-process fermentation samples for batches T-008 and T-009, expressing rhLF3 were analysed for protein production by HPLC at various time points and the analyses summarised in Table 6 and 7. Batch T-008 produced more than 1 g / L of target protein. The titre was still increasing at the time of harvest, as was the biomass concentration. In contrast, batch T-009 produced less than 0.5 g / L of the target protein. In T-009, the protein production did not increase after 46 hours (I + 18) and the biomass was also static. T-009 accumulated methanol, and did not recover. The methanol concentration for T-009 at harvest was 11.8 g / L (1.18%). The methanol concentration for T-008 at harvest was 0.322 g / L (0.03%), which is at the low end, but enough to keep the culture going. SDS-PAGE gel analysis of process samples from T-008 and T-009 are shown in Figure 2. Table 7. Process data for batch T-009.

[0298] The production of recombinant human lactoferrin was tested in 2 L bioreactors. To provide larger amounts of material for testing purposes and initial development of downstream processing options, the process was scaled 5-fold using bioreactors with 10 L working volumes. Recombinant human lactoferrin variant titres from selected batches are shown in Table 8. The titres were relatively high for rhLF3 and higher than the highest titre (1.2 g / L) reported for recombinant human lactoferrin production using Pichia pastoris until recently. Only the latest titre of 5.4 g / L reported by Lv et al., 2023 for recombinant human lactoferrin production in a bioreactor system is higher than the titres achieved for two of the lactoferrin variants indicated in Table 8 below.

[0299] Table 8. Recombinant human Lactoferrin titres from selected production batches completed in bioreactors. Lactoferrin titres were measured using HPLC with recombinant human lactoferrin as the standard.

[0300] The titres shown in Table 8 demonstrate the difference in titre achieved with different lactoferrins, the various derivatives. In some instances, the increase in scale resulted in an increase in titre (again dependent on the human lactoferrin variant). At the larger operating scale, the highest titre attained was 2.3 g / L (measured by HPLC). SDS PAGE analysis of a selected example (10 L) batch is shown in Figure 12.

[0301] The data shown in Figure 12 provides evidence of an induced protein being produced with the same mobility as a recombinant human lactoferrin. The protein band in Figure 12 that is proximate to Arrow A was excised from the gel and analysed by mass spectroscopy of tryptic digests, and the data confirmed the presence of peptides from human lactoferrin. Human lactoferrin peptides were also detected in bands with smaller molecular weights which may indicate some proteolytic cleavage of the target protein. Proteolytic cleavage is not uncommon in Pichia processes and, if deemed to be having a significant impact on titre, can be reduced through changes to process parameters and further strain engineering. SDS-PAGE analyses of samples taken during recovery of the spent culture supernatant also show a minimal decrease in intensity, suggesting that the protein is largely retained during this part of the process. The same samples were analysed by HPLC to determine the titre (Figure 13).

[0302] HPLC data indicates some lactoferrin expression prior to induction with an increasing titre during the induction period. The decrease in titre at 26 hours may be process or sample related but production does recover and lactoferrin accumulates in the culture during the remainder of the process. The titre does appear to be increasing at the end of the process therefore, a longer induction time may result in a further improvement in titre.

[0303] HPLC analysis of samples taken during the recovery of the spent culture supernatant does indicate some losses during the process (Figure 14).

[0304] The lactoferrin titre during the processing of the spent culture did show some variation with an increase in titre during preparation step and subsequent losses during centrifugation and filtration. The recoveries were greater than 66% which is encouraging from a process that has not been optimised. With greater characterisation of each process step higher recoveries are expected to be achieved.

[0305] Example 4 - Large-scale production of recombinant human lactoferrin rhLF3

[0306] Batch T-011 provides an example of scaled up growth and protein expression of a Pichia pastoris Apep strain expressing recombinant human lactoferrin rhLF3 using a fed-batch process with a slow increase in the methanol feed rate. The temperature was decreased to 12°C before harvesting the supernatant. The process was completed in a fully defined medium.

[0307] Cell line

[0308] A Pichia pastoris Apep strain expressing rhLF3 was used for scaled up growth and expression.

[0309] Media

[0310] Seed medium: The seed medium used for this experiment comprised BMGY base medium (10 g of yeast extract, 20 g peptone), 1 M potassium phosphate buffer pH 6, 13.4% yeast nitrogen base with ammonium sulphate without amino acids, 0.02% (w / v) biotin solution and 10% (v / v) glycerol. Base medium for bioreactors: The base medium used for bioreactors comprised 26.7 mL phosphoric acid, 0.93 g calcium sulphate dihydrate, 18.2 g potassium sulphate, 14.9 g magnesium sulphate 7H2O, 4.13 g potassium hydroxide and 40.0 g glycerol. Components were dissolved in the order listed above and made up to volume with reverse osmosis filtered water.

[0311] Trace metals solution (PTMi): The trace metals solution comprised: 6.0 g cupric sulphate 5H2O, 0.08 g Sodium iodide, 3.0 g manganese sulphate H2O, 0.2 g sodium molybdate 2H2O, 0.02 g boric acid, 0.5 g cobalt chloride, 20.0 g zinc chloride, 65.0 g ferrous sulphate 7H2O, 0.2 g biotin and 5.0 mL sulphuric acid.

[0312] The optical density of the seed culture after incubation for 28 hours was 21.6 at OD 600 nm. Under microscopic analysis, the seed culture consisted of healthy yeast cells with no contamination evident. This experiment is referred to as Batch T-011.

[0313] Bioreactor operating conditions

[0314] A New Brunswick BioFlo 310 bioreactor with a maximum working volume of 10 L which had been sterilised at 121°C for 30 minutes was used for cultivation. The starting medium volume used was 500 mL. The base medium was very acidic (~pH 1.8) and so was adjusted to pH 6 using the bioreactor pH controller and 28% ammonia solution (medium formed precipitate while adjusting the pH). The starting temperature setpoint was 30°C. The target optical density at inoculation was 0.30. Dissolved oxygen control (multiple parameter cascade) setpoint was 30% of saturation. The agitator was set to a minimum speed of 500 rpm and a maximum speed of 800 rpm. The airflow was set to a minimum rate of 1.5 L / min and a maximum rate of 4.5 L / min. The pH control setpoint was pH 6.0. No acid was used for automated pH control. The base used for automated pH control was 28% NH3 solution. For foam control 25 mL of 10% Sigma Antifoam 204 was manually added before inoculation.

[0315] Process

[0316] Batch mode: The bioreactor was operated in batch mode until the glycerol became depleted.

[0317] Glycerol fed-batch mode: The glycerol feed composition used was 1000 mL of 25% (w / v) glycerol with 6.0 mL of PTMi added. Once the residual glycerol in the medium had been depleted the glycerol feed was started at a flow rate of 24 mL / h / L. Glycerol was fed at this rate for five hours.

[0318] Methanol fed-batch mode: For induction of protein expression the methanol feed composition used was 4500 mL of 50% methanol with 27 mL of PTMi added. Temperature was decreased to 27°C thirty minutes before methanol induction. At 27°C, the glycerol feed was stopped and the methanol feed tubing was primed. Once signs of glycerol depletion were observed the methanol induction feed was started as summarised in Table 9.

[0319] Table 9. Methanol

[0320] Harvest

[0321] The ferment was harvested 92.3 hours after inoculation. The temperature was decreased to 12°C at the end of the process. This is usually done over 30 minutes for the 2 L ferments, but after 90 minutes, the temperature had only cooled to 18°C in the larger bioreactor. The culture harvest was drained into a 10 L carboy that was placed in an ice bath and the cells removed by centrifugation at 10,000 x g for 10 minutes. Samples were taken for measurement of optical density throughout the process. Samples were taken prior to induction (i.e. before methanol feed commenced) and at several time points postinduction for HPLC analysis. The supernatant was filtered through 1 x 500 mL 0.2pm bottle top filter, while the remaining supernatant was unfiltered. All the supernatant was stored at -80°C. Pellets were discarded. The final optical density was 312. The harvest volume was 9.2 L and the supernatant volume was 7.2 L. The filtered volume was 1250 mL and the pellet weight was 1945 g.

[0322] Biomass production

[0323] Biomass concentration increased then slowed when the glycerol feed rate was decreased. No methanol accumulation was seen.

[0324] Protein production

[0325] The in-process fermentation samples for batch T-011, expressing rhLF3 were analysed for protein production by HPLC at various time points and is summarised in Table 10. Batch T-011 produced approximately 1.1 g / L of target protein just after induction, with a maximum of 2.7 g / L detected in the 12°C harvest samples. The titre dropped considerably (25%) after centrifugation. This experiment shows that the process can be successfully scaled to 10 L. SDS-PAGE analysis of the process samples of batch T-011 is shown in Figure 4.

[0326] Table 10. Process data for batch T-011.

[0327] Conclusions

[0328] Comparing data from ferments T-008 and T-011 (Figures 5 and 6) shows that the current process for the production of rhLF3 can be successfully scaled to 10 L. T-011, the 10 L ferment had similar biomass production to T-008, the 2 L process, although the target protein titre was much higher. Losses that occurred between the harvest and subsequent processing need to be addressed. The 10 L process produced a higher titre of target protein than the 2 L (Batch T-008) process.

[0329] Example 5 - Downstream processing

[0330] The bioreactor fermentation supernatants served as the starting material for the partial purification of the rhLF3. This process was followed by subsequent dialysis to remove salts and freeze-drying to generate a powder form suitable for functionality assessment.

[0331] The downstream processing involved the following steps:

[0332] 1. Dialysis: The protein solution was first dialysed into an appropriate buffer for anion exchange chromatography, to ensure efficient separation.

[0333] 2. Ion Exchange Chromatography: The dialysed solution was then subjected to ion exchange chromatography, separating the target protein based on charge. Elution was performed using a gradient of sodium chloride (NaCl), to facilitate the effective separation of rhLF3 from host cell proteins, other protein species and contaminants.

[0334] 3. Post-chromatography dialysis: Following elution, the resultant protein fractions underwent a second dialysis treatment to remove excess salt to ensure the stability and functionality of the dried product.

[0335] 4. Freeze drying: The dialysed solutions were chilled to -80°C and freeze-dried overnight at -70°C under a vacuum of 150mTorr. This process effectively removed moisture, converting the protein into a stable powdered form.

[0336] Resultant partially purified powdered protein samples were analysed by HPLC, SDS-PAGE and tryptic digest proteomics to confirm rhLF3 content and integrity (Figure 1). The powders were then stored at -20°C for functionality assessments.

[0337] The rhLf3 variant has been assessed for anti-microbial, anti-inflammatory, pre- biotic and intestinal iron absorption functionality. The results of the functionality assays are presented in the following sections. Current efforts are focused on refining and improving the preliminary downstream processing (DSP) steps. This ongoing development aims to enhance the efficiency, yield, and quality of the final rhLF3 product.

[0338] Example 6 - Anti-microbial activities of recombinant human lactoferrin variants

[0339] The in vitro anti-microbial activities of recombinant human lactoferrin variants were investigated against Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), and Bacillus cereus (ATCC 10876). The organisms were selected based on their association with infections related to breast-feeding, donated human milk, breast milk substitutes and other food-borne disease. The efficiency of the recombinant human lactoferrin variants for inhibiting these organisms was compared with human and bovine lactoferrin standards purchased from Sigma Aldrich. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) methods were used for assessing the anti-microbial activities of the lactoferrin samples in accordance with the methods described by Wiegand et al., 2008 and the guidelines of the Clinical and Laboratory Standard Institute (Cockerill, 2012) The MIC experiment was conducted using 96 well microtiter plate, whereby the target microorganisms were inoculated into Mueller- Hinton broth (MHB) with lactoferrin added at concentration ranging from 3 mg / mL to 0.023 mg / mL. Samples with added ciprofloxacin (an anti-microbial agent) at concentrations ranging from 2 pg / mL to 0.0156 pg / mL were used as negative controls, whereas samples with the growth medium (MHB) and the inoculum were used as growth controls and the sterile MHB broth without inoculum was used as a sterile control. The microtiter plates were wrapped with parafilm and were incubated at 37°C in the case of Escherichia coli and Staphylococcus aureus and at 30°C in the case of Bacillus cereus. The growth of the microorganisms was monitored spectrophotometrically (Thermo Scientific Varioskan® Flash) by measuring optical density at 600 nm (OD600) at time zero and at the end of incubation. Experiments were conducted in triplicates. Microbial growth was calculated from the mean absolute change in OD600 compared to time zero. MIC was defined as the lowest lactoferrin concentration at which reduction in growth relative to control was observed. The percentage growth inhibition (I, %) of microorganisms by each lactoferrin variant was calculated in accordance with the formula: I (%) = (ODC -ODT / ODC) * 100, where ODC is the optical density of the growth control and ODT is the optical density of the tested microbial growth in each well containing lactoferrin. The minimum bactericidal concentration (MBC) was determined by subculturing the wells (with no OD value change) from the broth dilution of MIC tests onto fresh agar plates without the lactoferrin. After the incubation, the lowest concentration that did not show any visible growth was taken as the MBC. The MIC and MBC experiments were performed once with rhLF3 samples.

[0340] The percentage growth inhibition of the three microorganisms compared to growth control by rhLF3 and bovine lactoferrin and human lactoferrin standards are presented in Table 11. As can be seen, all the lactoferrin samples tested had antimicrobial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Bacillus cereus (B. cereus) at 3 mg / L concentration.

[0341] Table 11. Anti-microbial effect (percentage growth inhibition relative to control) of recombinant lactoferrin variant (rhLF3) and human and bovine lactoferrin standards against Escherichia coli, Staphylococcus aureus, and Bacillus cereus at 3 mg / L concentration. Example 7 - Pre-biotic (growth stimulation) effects of recombinant human lactoferrin variants

[0342] The growth stimulating effects of lactoferrin was investigated using a similar method as the MIC method described in Example 5 using Lactobacillus rhamnosus GG (LGG) as a model pro-biotic organism (recently renamed as Lactocaseibacillus rhamnosus as described in Zheng et al., 2020). The experiment was conducted using 96 well microtiter plate, whereby LGG was inoculated into MRS broth with lactoferrin added at concentration ranging from 3 mg / mL to 0.023 mg / mL. Samples with added ciprofloxacin (an anti -microbial agent) at concentrations ranging from 2 pg / mL to 0.0156 pg / mL were used as negative controls, whereas samples with the growth medium (MRS) and the inoculum were used as growth controls while the sterile MRS broth without inoculum was used as a sterile control. The experiments were performed in triplicates and were repeated twice. The results are presented in Table 12.

[0343] With respect to growth promotion and potential pre-biotic effect, bovine lactoferrin showed the highest growth stimulation effect of 31%. The human lactoferrin standard did not show growth stimulation effect whereas rhLF3 had the opposite effect of inhibiting the growth of LGG. The assessment of the pre-biotic properties of the other recombinant human lactoferrin will be assessed in further experiments in order to select the best targets for a pre-biotic lactoferrin formulation.

[0344] Table 12. Growth stimulation effects (relative to control) of Eclipse recombinant lactoferrin variant (rhLF3) and human and bovine lactoferrin standards on Lactobacillus rhamnosus GG (LGG).

[0345] Example 8 - Anti-inflammatory property of recombinant human lactoferrin variants

[0346] The in vitro anti-inflammatory activities of the lactoferrin variants were evaluated and compared with that of human and bovine lactoferrin standards using a cell-based assay as described in Cho et al., 2000 and Kim et al., 2005, with modifications. Bacterial lipopolysaccharide (LPS) was used to induce an inflammatory cellular state in the murine macrophage cell line, RAW 264.7 (Sosroseno et al., 2002). Inflammation was indicated by the cellular production of nitric oxide (NO) as measured using 2,3- diaminonaphthalene (DAN). A decrease in the production of NO by the samples indicated anti-inflammatory activity. Accordingly, 300 ng / mL LPS in culture media was added to the cells with 600 - 250 pg / mL of different lactoferrin samples. Cells were cultured in 96 micro titre plates for 48 hours, and on day 3, NO and cytokines (see method below) secreted by the RAW 264.7 cells were measured in the cell media. A standard curve was also prepared with sodium nitrite and used to interpolate the NO production in the cell media. A quercetin response curve was also generated to determine pg quercetin activity / mg lactoferrin protein assuming quercetin as the ideal antiinflammatory compound. All cell treatments were performed in triplicate and were assayed in duplicates (n=6). Results were analysed for significant differences using unpaired t tests. Differences were deemed significant when P<0.05. All statistical analyses were performed using GraphPad Prism 9 software.

[0347] The anti-inflammatory activities of the lactoferrin samples are presented in Figure 7 and Table 13.

[0348] Table 13. In vitro anti-inflammatory activity, measured via nitrite production in lipopolysaccharide treated RAW264.7 cells and expressed as quercetin equivalents, in response to commercial and Eclipse lactoferrin samples

[0349] Note: Different letters (a, b, c) denote significant differences as determined by unpaired t tests

[0350] As can be seen, the highest in vitro anti-inflammatory activity, measured through inhibition of NO production, was observed in response to rhLF3 (Figure 7 and Table 13). All commercial lactoferrin samples inhibited NO production significantly less than rhLF3 (as determined by unpaired t tests, Table 13). The result indicates that rhLF3 has superior anti-inflammatory properties. Example 9 - Intestinal iron absorption property of recombinant human lactoferrin isoforms

[0351] The impact of the recombinant human lactoferrin variants on in vitro intestinal iron absorption were assessed together with human and bovine lactoferrin standards through cellular ferritin production by human Caco-2 and HT29-MTX-E12 cells cocultured on semi-permeable membranes to form an in vitro polarised intestinal barrier in accordance with the methods described in Mahler et al., 2009. Lactoferrin preferentially binds ferric iron, so intestinal iron absorption was measured in response to all samples with and without FeCh. Human Caco-2 (enterocytes) and HT29-MTX-E12 (goblet-like) cells grown on a semi-permeable membrane was used as intestinal barrier model to mimic the upper part of the small intestine. To ensure that any treatment effect on the intestinal barrier model is not related to cytotoxicity, Caco-2 and HT29-MTX-E12 coculture cell viability was measured in response to all lactoferrin samples. To mimic intestinal iron absorption in vivo, 80 pM ascorbic acid was added to all experimental treatments applied to the intestinal barrier model (Badu-Boateng and Naftalin, 2019) to reflect the presence of dietary ascorbic acid that is required to help reduce ferric iron to ferrous iron for intestinal absorption by divalent metal transporter 1 (i.e., ferric iron is not directly transported by absorptive enterocytes, it must be reduced by dietary components or cytochrome b reductase). The cell co-cultures were grown on transwells for 21 days with media changed every 2 - 3 days. At 21 days, transepithelial electric resistance (or TEER) was measured using a Millicell voltohmeter from the apical to basolateral chamber to determine the integrity of the cell layer and ensure cells are polarised and an intact barrier is ready for experimentation. Following preparation of an intact intestinal cell barrier, impact of lactoferrin samples on intestinal iron absorption was observed by measuring ferritin formation as depicted in Figure 8. The cell cultures were incubated with lactoferrin samples (with and without FeCh) dissolved in Hank’s balanced salt solution (HBSS) buffer for 2 hrs, followed by replacement with HBSS buffer, incubation overnight and harvest after 16-18 hrs. The cells were then lysed, and ferritin production was assayed using Abeam human ferritin ELISA kit (ab200018) according to manufacturer’s instructions. All experiments were conducted in triplicates and assayed in duplicates and results were expressed in ng / mL ferritin as mean ± standard error (n=6).

[0352] The in vitro ferritin production data are presented in Figure 9. The results show that all lactoferrin samples significantly improved in vitro intestinal absorption of FeC13 as can be seen in the higher ferritin production. The highest ferritin production was stimulated by 250 pg / mL Sigma rice human recombinant lactoferrin and 250 pg / mL Sigma bovine milk lactoferrin. The next highest ferritin production was stimulated by 250 pg / mL rhLF3 (that was not significantly different to the Sigma human recombinant lactoferrin), followed by 250 pg / mL of Sigma’s human lactoferrin standard.

[0353] Example 10 - Glycosylation patterns of yeast derived rhLactoferrin proteins

[0354] N-glycan analysis of excised SDS-PAGE gel slices was used to assess the overall glycan composition for each of the rhLactoferrin isoforms.

[0355] Gel slices examined showed high hexose(H)-HexNac (N) N-glycosylation post- translational modifications with the composition HxN2 glycans and a distribution of hexose sugars from hexose H7 to Hl 5, predominantly H9-H10. Based on known post- translational modification biochemistry of Pichia pastoris, the hexoses are likely mannose. The HxN2 distribution of rhLF3 and average mass spectra is shown in Figure 10A and is summarised below in Table 14.

[0356] Table 14. HxN2 distribution of rhLF3.

[0357] The yeast-derived rhLactoferrin that were examined from these gel slices have similar core and high hexose N-glycosylation modifications to native human lactoferrin, but lack the complex and hybrid glycosylation patterns that occur on native human and bovine lactoferrin, such as N-acetylneuraminic acid (Neu5Ac) glycan branches (comparison between Figure 10A and 10B) (Zlatina and Galuska 2021; van Veen et al., 2004).

[0358] Although the analysis herein cannot assign the glycans detected to specific amino acid residues, it is probable that at least some of the N-glycosylations occur on Asn 138, 249 and 624, similar to native human lactoferrin. However, the wider distribution of N- glycan patterns detected (up to 11 different N-glycan structures detected per gel slice) suggest there is either considerable heterogeneity of N-glycan patterns on the isolated proteins, or that other Asn residues are also N-glycosylated. Although the protein gel slices assessed contained predominantly rhLactoferrin, other Pichia pastoris proteins were also detected at low abundance in the gel slices and may have contributed a small portion of the N-glycans detected.

[0359] Example 11 - Discussion

[0360] Pichia pastoris was engineered to enable recombinant production of rhLF3. rhLF3 performed well in bioreactor fermentation trials at 2L and 10L scales. Overall titre was improved with scale up. The level of improvement was significant for rhLF3 whereby the titres almost doubled from 1.08 g / L and 1.25 g / L for rhLF3 respectively. Culture supernatants were partially purified using ion exchange chromatography to produce partially purified samples for functional characterisation. So far, rhLF3 has been characterised for antimicrobial, pre-biotic, anti-inflammatory and iron absorption properties. rhLF3 stimulated in vitro intestinal iron absorption. rhLF3, had an effect equivalent to bovine and human lactoferrin standards on in vitro intestinal iron absorption.

[0361] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0362] This application claims priority from Australian Provisional Application No. 2024900624 entitled “Recombinant human lactoferrin and methods and uses thereof’ filed on 8 March 2024, the entire contents of which are hereby incorporated by reference.

[0363] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0364] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. REFERENCES

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Claims

CLAIMS1. A method of treating or preventing inflammation in a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto.

2. Use of a recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing inflammation in a subject.

3. The method of claim 1, or the use of claim 2, wherein the inflammation is selected from one or more of: skin inflammation; gastrointestinal inflammation; inflammatory bowel disease; urogenital inflammation; ulcerative colitis; response to infection, anaemia of inflammation (Al) and chronic disease.

4. The method or use of claim 3, wherein the inflammation is skin inflammation.

5. The method or use of claim 4, wherein the skin inflammation is selected from one or more of: eczema, acne, seborrheic dermatitis and psoriasis.

6. A method of treating or preventing iron deficiency in a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

7. Use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing iron deficiency in a subject.

8. A method of increasing iron absorption in the gastrointestinal tract of a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

9. Use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto for increasing iron absorption in the gastrointestinal tract of a subject.

10. A method of decreasing the activity of a pathogenic bacteria in the microbiome of a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

11. Use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for decreasing the activity of a pathogenic bacteria in the microbiome of a subject.

12. The method of claim 10 or the use of claim 11, wherein the pathogenic bacteria is a bacteria in a family selected from one or more of: Enter obacteriaceae, Staphylococcaceae, Bacillaceae, Pseudomonadaceae and Moraxellaceae.

13. The method or use of claim 12, wherein the pathogenic bacteria is a bacteria in a family selected from one or more of: Enter obacteriaceae, Staphylococcaceae and Bacillaceae.

14. The method or claim 12 or use of 13, wherein the bacteria is selected from one or more of: Escherichia coli, Staphylococcus aureus, Cutibacterium acnes, Bacillus cereus, Cronobacter sakazakii, Salmonella spp. Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas spp., Shigella spp., Shigella sonnei and Acinetobacter baumanni.

15. The method or use of claim 14, wherein the bacteria is selected from one or more of: Escherichia coli, Staphylococcus aureus and Bacillus cereus.

16. A method of treating or preventing a bacterial infection in a subject, the method comprising administering recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto to the subject.

17. Use of recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto in the manufacture of a medicament for treating or preventing a bacterial infection in a subject.

18. The method or use of claim 16 or claim 17, wherein the bacterial infection is selected from one or more of a / an: i) skin infection; ii) urinary tract infection; iii) Enter obacteriaceae infection; iv) Staphylococcaceae infection; v) Bacillaceae infection; vi) Pseudomonadaceae infection; vii) Moraxellaceae infection; viii) Escherichia coli infection; ix) Staphylococcus aureus infection; x) Bacillus cereus infection; xi) Cronobacter sakazakii infection; xii) Salmonella spp. infection; xiii) Salmonella typhimurium infection; xiv) Klebsiella pneumoniae infection; xv) Pseudomonas aeruginosa infection; xvi) Pseudomonas spp., infection; xvii) Shigella spp., infection; xviii) Shigella sonnei infection; xix) Acinetobacter baumanni infection; xx) respiratory infection; xxi) periodontitis; and xxii) peri-implantitis.

19. The method or use of claim 18, wherein the bacterial infection is a skin infection.

20. The method or use of any one of claims 1 to 19, wherein the subject is a human.

21. The method or use of claim 20, wherein the subject is selected from one or more of: a neonate, infant, child, adolescent, adult and an elderly adult.

22. The method or use of any one of claims 1 to 21, wherein recombinant human lactoferrin rhLF3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:

123. A recombinant human lactoferrin rhLF3 or a sequence at least 70% identical thereto.

24. The recombinant human lactoferrin of claim 23, wherein recombinant human lactoferrin rhLF3 comprises or consists of the amino acid sequence set forth in SEQ IDNO:

125. The recombinant human lactoferrin of claim 24, wherein the recombinant human lactoferrin is produced in a yeast cell.

26. The recombinant human lactoferrin of claim 25, wherein the yeast cell is selected from one or more of: Pichia pastor is (Komagaetella phaffii), Pichia hangzhouana, Candida utilis, Pichia jadinnii, Aspergillus spp. and Trichoderma spp.

27. The recombinant human lactoferrin of any one of claims 23 to 26, wherein the recombinant human lactoferrin has higher iron intestinal absorption compared to recombinant human lactoferrin expressed in rice plants.

28. The recombinant human lactoferrin of any one of claims 23 to 27, wherein the recombinant human lactoferrin has higher iron intestinal absorption compared to endogenously produced human lactoferrin.

29. The recombinant human lactoferrin of any one of claims 23 to 28, wherein the recombinant human lactoferrin has higher iron intestinal absorption compared to endogenously produced bovine lactoferrin.

30. The recombinant human lactoferrin of any one of claims 23 to 29, wherein when the recombinant human lactoferrin is combined with an iron supplement, iron intestinal absorption is higher compared to an iron supplement alone.

31. The recombinant human lactoferrin of any one of claims 23 to 30, wherein when the recombinant human lactoferrin is combined with an iron supplement, iron intestinal absorption is higher compared to endogenously produced human lactoferrin combined with iron supplement.

32. The recombinant human lactoferrin of claim 30 or 31, wherein the iron supplement is selected from one or more of: ferrous gluconate, ferric citrate, ferric chloride (FeCh), ferric sulfate (F 62(804)3) and ferrous sulfate (FeSO4).

33. The recombinant human lactoferrin of any one of claims 23 to 32, wherein the recombinant human lactoferrin has higher anti-microbial activity against a pathogenic microbe compared to endogenously produced bovine lactoferrin.

34. The recombinant human lactoferrin of claim 33, wherein the pathogenic microbe is a bacteria in a family selected from one or more of: Enter obacteriaceae, Staphylococcaceae and Bacillaceae.

35. The recombinant human lactoferrin of claim 33, wherein the pathogenic microbe is a bacteria selected from one or more of: Escherichia coli, Staphylococcus aureus and Bacillus.

36. The recombinant human lactoferrin of any one of claims 23 to 33, wherein the recombinant human lactoferrin has higher anti-microbial activity against a pathogenic microbe compared to recombinant human lactoferrin expressed in rice plants.

37. The recombinant human lactoferrin of any one of claims 23 to 36, wherein the recombinant human lactoferrin has higher anti-inflammatory activity compared to endogenously produced bovine lactoferrin.

38. The recombinant human lactoferrin of claim 36 or claim 37, wherein the pathogenic microbe is a bacteria in the family Enter obacteriaceae.

39. The recombinant human lactoferrin of claim 36 or claim 37, wherein the pathogenic microbe is the bacteria Escherichia coli.

40. The recombinant human lactoferrin of claim 39, wherein the anti-inflammation activity is assessed by nitric oxide assay.

41. The recombinant human lactoferrin of any one of claims 23 to 40, wherein the recombinant human lactoferrin has an increased level of one or more of: H8N2, H9N2, H10N2, Hl 1N2 and H12N2 compared to endogenously produced human lactoferrin.

42. The recombinant human lactoferrin of any one of claims 23 to 41, wherein the recombinant human lactoferrin has an increased level of H10N2 compared to endogenously produced human lactoferrin.

43. The recombinant human lactoferrin of any one of claims 23 to 42, wherein the recombinant human lactoferrin has an increased level of mannose compared to endogenously produced human lactoferrin.

44. The recombinant human lactoferrin of any one of claims 23 to 43, wherein the recombinant human lactoferrin has a decreased abundance of N-GlcNac, Neu5Ac and / or fucose compared to endogenously produced human lactoferrin.

45. A composition comprising the recombinant human lactoferrin of any one of claims 23 to 44.

46. The composition of claim 45, wherein the composition is an enteral composition.

47. The composition of claim 45, wherein the composition is a parenteral composition.

48. The composition of claim 45 or claim 46, wherein the composition is a food composition.

49. The composition of claim 48, wherein the food composition is selected from one or more of: infant formula, premature infant formula, low birth weight infant formula, a human milk fortifier, toddler milk formula, functional food and a powdered food.

50. The composition of claim 49, wherein the food composition is selected from one or more of: an animal feed, animal feed supplement, pre-starter feed, starter feed, milk replacer, aquaculture feed, pet food and pet treat.

51. The composition of any one of claims 45, 46 and 49 to 50, wherein the composition is a beverage composition.

52. The composition of claim 51, wherein the beverage composition is selected from one or more of: a dairy product, a dairy product substitute, a functional beverage, a powdered beverage, a sports drink and an energy drink.

53. The composition of any one of claims 45, 46 and 48 to 52, wherein the composition is an oral health composition or a bone health composition.

54. The composition of any one of claims 45 to 53, wherein the composition is a nutraceutical composition.

55. The composition of any one of claims 45 to 54, wherein the composition is an anti-microbial composition.

56. The composition of any one of claims 45 to 55, wherein the composition is an anti-inflammatory composition.

57. The composition of any one of claims 45 and 54 to 56, wherein the composition is a skin care composition or a cosmetic composition.

58. The composition of claim 57, wherein the skin care composition or cosmetic composition is selected from one or more of: a facial moisturiser, a body moisturiser, a facial cream a body cream, a serum, an eye cream, a face mask, a sunscreen, a sun protection factor (SPF) product, an acne treatment, a body lotion, a hair care product, a lip balm, a lip treatment, wound dressing and burn treatment.

59. The composition of any one of claims 45 to 58, wherein the composition comprises one or more of: an iron supplement and a vitamin.

60. The composition of claim 59, wherein the iron supplement is selected from one or more of: ferrous gluconate, ferric citrate, ferric chloride (FeCh), ferric sulfate (Fe2(SO4)s) and ferrous sulfate (FeSCh).

61. The composition of claim 59 or claim 60, wherein the vitamin is selected from one or more of: vitamin Bl, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin Bl 2, vitamin C and vitamin D.

62. The composition of any one of claims 45 to 61, wherein the composition is in a form selected from one or more of a: powder, tablet, capsule, liquid, emulsion, syrup, gummy, gel and a sachet.

63. The composition of claim 62, wherein the composition does not comprise another human lactoferrin variant.

64. An isolated nucleic acid encoding a recombinant human lactoferrin of any one of claims 1 to 44.

65. An isolated nucleic acid of claim 64, wherein the nucleic acid comprises a sequence selected from SEQ ID NO:4 or a sequence at least 70% identical thereto.

66. A vector comprising the nucleic acid of claim 64 or claim 65.

67. A host cell comprising the nucleic acid of claim 64 or claim 65, or the vector of claim 66.

68. The host cell of claim 67, wherein the host cell is a yeast cell.

69. The host cell of claim 67, wherein the yeast cell is a Pichia pastoris.

70. A method of producing a recombinant human lactoferrin of any one of claims 23 to 26, comprising culturing the host cell of any one of claims 67 to 69 in cell culture medium and expressing the recombinant human lactoferrin.

71. The method of claim 70, wherein the recombinant human lactoferrin is produced at a concentration of at least Ig / L.

72. The method of claim 70 or claim 71, wherein the recombinant human lactoferrin is produced at a concentration of at least 2g / L.

73. The steps, features, integers, compositions and / or compounds disclosed herein or indicated in the specification of this application individually or collectively, and any and all combinations of two or more of said steps or features