High level production of GLP1 analogues

Abstract

The invention relates to multimers containing (i) a plurality of target peptide (TP) sequences, each of which is a GLP-1 analogue or a fragment thereof, (ii) Cleavage site (CS) between the target peptide (TP) sequences, and (iii) optionally one or more Tag amino acid sequences that modulate pI and hydrophobicity. The multimers permit high levels of expression of target peptide (TP) sequences and have desired solubility for ease of downstream processing and purification.

Description

[001] The present application claims the benefit of the Indian provisional application 202421006466, filed January 31, 2024, which is hereby incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[002] The instant application contains a Sequence Listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing was created on January 31, 2025, is named 159121 -06301_SL.xml and is 100,522 bytes in size. FIELD OF THE INVENTION

[003] The invention relates to multimers containing (i) a plurality of target peptide sequences, each of which is a GLP-1 analogue or a fragment thereof, (ii) cleavage sites between the target peptide sequences, and (iii) optionally one or more Tag amino acid sequences that modulate pl and hydrophobicity. The multimers permit high levels of expression of target peptide sequences and have improved solubility for ease of downstream processing and purification. BACKGROUND OF THE INVENTION

[004] Peptides are involved in various biological processes and thousands of different natural peptides have been identified (Barman etal., Int J Peptide Res. and Ther., 29:61, 2023). (Barman P. et al., Int J Pept Res Ther. 2023;29(4):61. doi: 10.1007 / sl0989-023-10524-3) Peptides are involved in important functions such as hormonal functions, neurotransmission and anti-microbial activity (Barman, supra). Due to this, peptides have gained immense commercial attention in the field of medicine with more than one hundred peptides approved for diagnostic and therapeutic use so far (Barman, supra). Recombinant bioactive peptides have been largely used as biotherapeutic agents. However, there is increasing demand in the fields of cosmetics and nutraceuticals. Bioactive peptides can serve as antioxidants, anti-aging and anti-inflammatory agents with the potential to enhance skin health and beauty (Le Thi Nhu Ngoc, Feature Papers in Cosmetics, 2023), (Le Thi Nhu Ngoc et. al, Cosmetics 2023;70(4). doi: 10.3390 / 10040111) Nutraceutical peptides can provide a daily dose of healthy nutrients offering preventive and treatment benefits (A Giriraj, Science direct, 2018; 157-181. doi:10.1016 / B978-0-08-100736-5.00006-5).

[005] Recombinant peptides are often expressed in a suitable host such as E.coli or yeast. However, there are challenges faced in the production of peptides. One of the major challenges of expressing peptides is due to their small size, they are highly prone to cleavage via the proteases of the host organism (Ajingi YS, Current Pharmaceutical Biotechnology, 23:645-663, 2022). This cleavage leads to lower yields and truncated versions of the peptide of interest (POI). To increase yield, fusion partners have been attached to peptides to increase their expression level and protect them from protease cleavage. For example, thioredoxine (TRX), small ubiquitin-like modifier (SUMO) and glutathione-S-transferase (GST) can be fused with the POI, and later separated by a protease cleavage site. The fusion partner protects the POI from intrinsic proteolytic enzymes of the host (S. Coasta 2014, SC Bernier 2018, LaVallie et al. (S. Coasta et.al., Front microbiol, 2014; 19:5:63 ; S CBernier et.al., ProteinExprPurif, 2018; 152:92-106 ; LaVallieeta / . BioTechnology, 1993; 11(2): 187-93 ). The POI is then obtained by cleaving off the fusion partner. These fusion partners, however, decrease the efficiency of peptide production by reducing the overall amount of the POI produced. The major challenge with expression of a recombinant peptide of interest as a multimer is the multimers often form highly insoluble compact inclusion bodies. Solubilization of the inclusion bodies is extremely challenging as it involves the use of a strong chaotropic agent such as guanidium hydrochloride (GuCl). GuCl is an expensive raw material, and its usage increases the cost of production, impacts enzymatic digestion and scalability of the process.

[006] There is a need to not only overcome the problem of cytosolic cleavage of peptides, but improve the efficiency of peptide production that is lost from using fusion proteins tags. One possibility is a method that includes adding repeats of the POI upon itself (Shen SH 1984 (Shen SH, Proc Natl Acad Sci U 5 A,1984; 81(15):4627-31). The intrinsic properties of a peptide such as its isoelectric point (pl) and hydrophobicity play a major role in its solubility (Ryan M. Kramer 2012 (RyanM. Kramer etaL, Biophys J, 2012; 18;102(8): 1907-15).

[007] U.S. Patent Publication No. 2022 / 0195004 discloses certain fusion proteins comprising a plurality of target protein sequences which are connected in series, wherein every two adjacent target protein sequences are connected by means of a linker sequence.

[008] Hou et al., Biosci Biotechnol Biochem., 71(6):1462-1469, 2007, discloses expression of a fusion protein containing 10 tandem repeated GLP-1 analogs in yeast Pichia pastoris.

[009] International Publication No. WO 2018 / 172921 discloses a method for preparation of a biologically active recombinant peptide by overexpressing it as a concatemer having specific intervening Kex2 protease and Carboxypeptidase B cleavage sites separating each monomer.

[0010] Chinese Patent Publication No. 101172996 discloses a connection peptide and polyfusion expression method.

[0011] There is a continuing need for improved processes for preparing polypeptides in high yield. SUMMARY OF THE INVENTION

[0012] The present invention provides a method for enhanced expression of a multimer (MU) containing a plurality of Target Peptide (TP) sequences, wherein each target peptide (TP) sequence is a GLP-1 analogue or a fragment thereof. Furthermore, the solubility of the multimer (MU) can be adjusted by using amino acid Tag (Tags) to either express the multimer (MU) as insoluble inclusion bodies or in the soluble form, to allows for convenient downstream processing, conversion of multimer (MU) to target peptide (TP) followed by the purification of the target peptide (TP), to achieve a final purity level of at least 95%.

[0013] The present invention is directed to the efficient production of a target peptide (TP) using a multimer (MU) comprising a plurality of target peptide sequences connected in series, wherein (i)     each target peptide sequence is a GLP-1 analogue or a fragment thereof, (ii)    every two adjacent target peptide (TP) sequences are connected by a separator sequence, which is referred as a Cleavage Site (CS) is capable of being cleaved by proteolytic enzymes (e.g., Kex2, EK, Carboxypeptidase B) or chemicals to form the target peptide upon complete digestion, (iii) optionally additional separator sequence referred to as Tag, may also be present between cleavage sites (CS), to modify the hydrophobicity and / or pl of the multimer (MU), (iv)   the choice of cleavage sites (CS) shall have no identical sequence within the target peptide (TP), (v) the N- and / or C-terminus of the multimer (MU) comprises of amino acid sequence which is capable of being cleaved by one or more of the proteolytic enzymes (Kex2, EK, Carboxypeptidase B) or chemicals to give target peptide (TP).

[0014] In one embodiment, each separator sequence independently comprises a cleavage site (also referred to as CS) and optionally a Tag amino acid sequence that alters the hydrophobicity, pl, or both of the multimer (MU).

[0015] In another embodiment a multimer (MU) having the formula MU = Met - (M0)n or MU = (M0)n, where n is an integer ranging from 2-50, preferably 2-20 and more precisely 10. Each MO has the formula - MO = (CS-TP-CS) and / or MO = (Tag-CS-TP-CS) and / or MO = (CS-TP-CS-Tag), where Tag is a sequence of amino acids to modulate pl and / or hydrophobicity of MO and MU. The Tag is not positioned between TP and CS,

[0016] each TP is independently a target peptide sequence selected from GLP-1 analogues and fragments thereof, selected from TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 1), EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 22), GTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 67), FTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 68), TSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 25), SDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 69), DVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 70), VSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 71), SSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 72), SYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 73), YLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 74), LEGQAAKEFIAWLVRGRGL (SEQ ID NO: 75), EGQAAKEFIAWLVRGRGL (SEQ ID NO: 76), GQAAKEFIAWLVRGRGL (SEQ ID NO: 77), QAAKEFIAWLVRGRGL (SEQ ID NO: 78), AAKEFIAWLVRGRGL (SEQ ID NO: 79), AKEFIAWLVRGRGL (SEQ ID NO: 80), KEFIAWLVRGRGL (SEQ ID NO: 81), Conjugating any of the TP sequences as shown above with a fatty acid moiety to give an intermediate product.

[0017] The number of Tag moieties to be included will depend on the degree of change in the pl and / or hydrophobicity desired for the MO and / or MU, i.   The choice of amino acids used in the Tags shall be based on whether the expression of the MU is desired in the soluble form or as insoluble inclusion bodies, ii. The choice of the cleavage site (CS) should be such that upon cleavage, by chemicals or one or more proteases, shall leave no additional amino acids at either the N or the C terminus of the TP. [

[0018] In some embodiments, the affinity tag is added either at the N- or C-terminus of the multimer (MU) separated by the cleavage site (CS).

[0019] In some embodiments, the amino acid at the N-terminus of the multimer is methionine.

[0020] In some embodiment and optionally poly-histidine is added either at N- or C-terminus of the multimer (MU), represented by the formula M(H)P, where p is 1 to 8 (such as MHHHHHH).

[0021] In some embodiments, one or more of the cleavage sites comprise the amino acid sequence KR.

[0022] In some embodiments, the R of the KR amino acid sequence is directly bound to the N-terminus of the target peptide (TP) sequences, and the K of the KR amino acid sequence is directly bound to the C-terminus of the target peptide sequences.

[0023] In some embodiments, one or more of the cleavage site (CS) comprises of amino acid sequences DDDDK (SEQ ID NO: 53).

[0024] In some embodiments, the K of the DDDDK sequence (SEQ ID NO: 53) is directly bound to the N-terminus of one of the target peptide (TP) sequences.

[0025] In some embodiments, the multimer includes one or more Tag amino acid sequences to alter the hydrophobicity, pl, or both of the multimer (MU).

[0026] In some embodiments, each Tag amino acid sequence comprises one or more amino acids selected from aspartic acid (D), glutamic acid (E), serine (S), threonine (T), arginine (R), glutamine (Q), lysine (K), histidine (H), leucine (L) and isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F), tyrosine (Y), cysteine (C), and methionine (M).

[0027] In some embodiments, the multimer has a pl of from about 3 to about 12.

[0028] In some embodiments, the multimer has a hydrophobicity of from 20% to 50%.

[0029] In some embodiments, the multimer (MU) has a hydrophobicity of 25% to about 40% and / or pl from 4 to 10.

[0030] In some embodiments, each Tag amino acid sequence between two target peptide (TP) sequences in the multimer is the same.

[0031] In some embodiments, each Tag amino acid sequence in the target peptide (TP) is the different.

[0032] In some embodiments, the multimer (MU) comprises at least two different Tag amino acid sequences.

[0033] In some embodiments, the multimer (MU) includes 2 to 50 copies of a target peptide (TP) sequence.

[0034] In some embodiments, the multimer (MU) includes 2 to 20 copies of a target peptide (TP) sequence.

[0035] In some embodiments, the multimer (MU) includes 10 copies of a target peptide (TP) sequence.

[0036] In some embodiments, the multimer (MU) has a molecular weight of from about 5 to about 200 kilodaltons.

[0037] In some embodiments, the multimer (MU) has a molecular weight of from about 30 to 60 kilodaltons.

[0038] In some embodiments, one or more of the Tag amino acid sequences are selected from SEQ ID NOs: 7-11, 14-19, and 28-41 and any combination of any of the foregoing.

[0039] In some embodiments, the present invention is directed towards a DNA construct comprising a nucleic acid sequence encoding a multimer.

[0040] In some instances, a host cell is transfected with the DNA construct.

[0041] In some embodiments, the present invention uses an expression vector, wherein the DNA sequence coding for the multimer (MU) is introduced under the T7 promoter.

[0042] In some instances, a host cell comprises is transformed with the expression vector.

[0043] In some embodiments, the method further comprises digesting the multimer (MU) with one or more proteolytic enzymes or chemicals to obtain a plurality of the target peptide (TP).

[0044] In some embodiments, the present invention is directed a method of preparing a peptide comprising: (a) obtaining a multimer; and (b) digesting the multimer with one or more digestive enzymes or chemicals to obtain a plurality of the target peptide sequence in free form.

[0045] In some embodiments, step (a) of the method comprises expressing the multimer in E. coli, and optionally isolating the multimer.

[0046] In some embodiments directed towards a method, the N-terminus includes methionine and a histidine tag, and the multimer is isolated using Nickel-NTA resin via the polyhistidine affinity tag.

[0047] In some embodiments directed towards a method, the E. coli is BL21, BL21(DE3), ToplO, or DH5a cells.

[0048] In some embodiments directed towards a method, the multimer (MU) is digested using one or more of the proteases selected from Kex2, carboxypeptidase B, and enterokinase.

[0049] In some embodiments when multiple proteases are used the cleavage is carried out sequentially or simultaneously using Kex2 and / or enterokinase followed by carboxypeptidase.

[0050] In some embodiments when multiple proteases are used the cleavage is carried out sequentially or simultaneously using Kex2 followed by carboxypeptidase.

[0051] In another embodiment the final peptide after complete digestion is target peptide (TP) which is further purified using one or more chromatography or precipitation techniques.

[0052] In some embodiments directed towards a method, the isolating step comprises one or more chromatography steps.

[0053] In some embodiments directed towards a method, wherein the method further comprises conjugating one or more chemical moieties to the target peptide sequence.

[0054] In some embodiments directed towards a method, wherein the method further comprising conjugating a second peptide to the target peptide sequence to form a third peptide and, optionally, conjugating one or more moieties to the third peptide.

[0055] In some embodiments, the method further comprises (c) isolating the target peptide sequence. In some embodiments, the isolating step comprises one or more chromatography steps.

[0056] In some embodiments, the method further comprises converting the target peptide sequence to a GLP-1 analogue. In some embodiments, the method comprises converting the target peptide sequence to semaglutide. In some embodiments, the method comprises converting the target peptide sequence to liraglutide.

[0057] In some embodiments, the method further comprises conjugating one or more chemical moieties to the target peptide sequence.

[0058] In some embodiments, the method further comprises conjugating a second peptide to the target peptide sequence to form a third peptide and, optionally, conjugating one or more moieties to the third peptide.

[0059] Yet another embodiment is a method for preparing a glucagon-like peptide-1 (GLP-1) agonist peptide comprising: (a) conjugating a protected activated amino acid fragment to a target peptide sequence prepared by any method described herein to form an intermediate product; and (b) conjugating a fatty acid moiety to the intermediate product.

[0060] In some embodiments, the activated amino acid fragment is conjugated to the target peptide sequence through a single amino acid coupling, direct fragment coupling, or a combination thereof.

[0061] In some embodiments, the fatty acid moiety is conjugated at the E of lysine.

[0062] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1).

[0063] In some embodiments, the target peptide sequence is selected from EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 22), GTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 67), FTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQID NO 68), TSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 25), SDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 69), DVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 70), VSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 71), SSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 72), SYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 73), YLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 74), LEGQAAKEFIAWLVRGRGL (SEQ ID NO: 75), EGQAAKEFIAWLVRGRGL (SEQ ID NO: 76), GQAAKEFIAWLVRGRGL (SEQ ID NO: 77), QAAKEFIAWLVRGRGL (SEQ ID NO: 78), AAKEFIAWLVRGRGL (SEQ ID NO: 79), AKEFIAWLVRGRGL (SEQ ID NO: 80), KEFIAWLVRGRGL (SEQ ID NO: 81),

[0064] In some embodiments, the fatty acid moiety is -^-U-W-Y-Z (I) wherein U represents -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-} wherein “}” is the point of attachment to W; W represents -C(O)-NH-(CH2)3-NH-], -C(O)-NH-(CH2)4-NH-], -C(O)-C(CH3)2-NH-], or -C(O)-N , wherein “]” is the point of attachment to Y; Y represents -C(O)-(CH2)2-CH(COOH)NH— and " is the point of attachment to Z; Z represents -C(O)-(CH2)n-COOH or -C(O)-(CH2)n-CH3, and n is an integer from 14 to 20.

[0065] In some embodiments, the U-W-Y-Z represents a group of the formula: wherein n is an integer from 14-20 and Ro is CO2H or CH3.

[0066] In some embodiments, the fatty acid moiety has the formula:

[0067] In some embodiments, the fatty acid moiety has the formula:

[0068] In some embodiments, the fatty acid moiety has the formula:

[0069] In some embodiments, the fatty acid moiety has the formula:

[0070] In some embodiments, the fatty acid moiety has the formula:

[0071] In some embodiments, the fatty acid moiety has the formula:

[0072] In some embodiments, the fatty acid moiety has the formula:

[0073] In some embodiments, the fatty acid moiety has the formula:

[0074] In some embodiments, the target peptide sequences are interspaced Tag amino acid sequence having cleavage sites, which, for instance, can be cleaved by proteolytic enzymes and / or chemicals.

[0075] In some embodiments, to improve the solubility of the multimer comprising a plurality of target peptide sequences, the intrinsic characteristics of the multimer such as pl and / or hydrophobicity are altered. In some instances, the building blocks of peptides, i.e., amino acids fall under certain categories such as acidic, basic, hydrophilic, and hydrophobic. In some instances, the Tag amino acid sequences can modulate overall pl and / or hydrophobicity of the multimer. In some instances, the Tag amino acid sequence can increase expression of the target peptide sequence. In some instances, the Tag amino acid sequence can improve solubility and ease of downstream processing. In some instances, the addition of Tag amino acid sequence into the multimer improve the modulation of intrinsic properties of the expressed multimer and can achieve high expression levels and improved solubility.

[0076] In some embodiments, the Tag amino acid sequence can be designed using a combination of amino acids which can alter intrinsic properties of the multimer such as pl and / or hydrophobicity. In some instances, the Tag amino acid sequence comprises aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), or arginine (R) to alter the pl of the multimer. In some instances, the Tag amino acid sequence comprises Aspartic acid (D) and / or glutamic acid (E) to reduce the pl of the multimer. In some instances, the Tag amino acid sequence comprises lysine and / or arginine to increase the pl of the multimer. In some instances, the Tag amino acid sequence comprises serine (S). threonine (T) asparagine (N), glutamine (Q) and other hydrophilic amino acids to make the multimer more hydrophilic. In some instances, the Tag amino acid sequence comprises leucine (L), isoleucine (I) alanine (A), valine (V), proline (P), Glycine (G), Tryptophan (W), Phenylalanine (F) and other hydrophobic amino acids to make the multimer more hydrophobic.

[0077] In one embodiment, the C-terminus of the multimer (after the target peptide sequence closest to the C-terminus in the multimer) includes a Tag amino acid sequence which is capable of being cleaved from the target peptide sequence by a protease and / or chemical treatment.

[0078] In some instances, the amino acid at the N-terminus of the multimer is methionine. In some instances, the amino acid at the N-terminus of the multimer is a histidine tag. In some instances, the amino acid sequence at the N-terminus of the multimer is M(H)P, where p is 1 to 8 (such as MHHHHHH).

[0079] In some embodiments, one or more of the Tag amino acid sequence comprises one or more amino acids to alter the pl of the multimer, the hydrophobicity of the multimer, or both. In some instances, one or more of the Tag amino acid sequence comprise aspartic acid (D), glutamic acid (E), or any combination of any of the foregoing to reduce the pl of the multimer. In some instances, one or more of the Tag amino acid sequence comprise histidine (H), lysine (K), arginine (R), or any combination of any of the foregoing to increase the pl of the multimer. In some instances, one or more of the Tag amino acid sequence comprise serine (S), threonine (T) asparagine (N), glutamine (Q) or any other hydrophilic amino acid to increase the hydrophilicity of the multimer. In some instances, one or more Tag amino acid sequence selected from leucine (L), isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F), or any other hydrophobic amino acid or any combination of any of the foregoing to increase the hydrophobicity of the multimer. In some instances, the Tag amino acid sequence, comprises one or more aspartic acid residues, one or more glutamic acid residues, or both.

[0080] In some embodiments, the multimer comprises one or more Tag amino acid sequence, wherein the Tag amino acid sequence comprises an amino acid group with a proteolytic and / or chemical cleavage site at both the N and C terminus of the target peptide sequence for cleaving the multimer to generate the monomer of the target peptide sequence. In some instances, the amino acids in the Tag amino acid sequence may act as both a modulator of pl and / or hydrophobicity as well as a proteolytic and / or chemical cleavage site.

[0081] In some instances, the multimer comprises one or more Tag amino acid sequence which include only one or more proteolytic and / or chemical cleavage sites. In some instances, each Tag amino acid sequence is KR.

[0082] In some instances, one or more of the Tag amino acid sequence comprise (or are selected from) - DSSTTDSSTTDDDDK (SEQ ID NO: 7), DSSDTTDDDDDK (SEQ ID NO: 8), DSSTTDSSTTDDDDDK (SEQ ID NO: 9), SSTTDDDDK (SEQ ID NO: 11), DSSTTDSSTTSSTT (SEQ ID NO: 10), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 14), SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 15), SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 16), SDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 17), SSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 18), SDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 19), SSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 38), SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 39), SSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 40), or SSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 41), or any combination of any of the foregoing. In some embodiments, one or more of the aforementioned Tag amino acid sequence further comprise one or more additional amino acids, such as one or more protease or chemical cleavage sites (at the N-terminus or C-terminus). In some embodiments, each of the Tag amino acid sequence are selected from DSSTTDSSTTDDDDK (SEQ ID NO: 7), DSSDTTDDDDDK (SEQ ID NO: 8), DSSTTDSSTTDDDDDK (SEQ ID NO: 9), SSTTDDDDK (SEQ ID NO: 11), DSSTTDSSTTSSTT (SEQ ID NO: 10), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 14), SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 15), SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 16), SDSTTSSTTSSTTSSTTSSTT(SEQ ID NO: 17), SSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 18), SDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 19), SSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 38), SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 39), SSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 40), or SSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 41). In some embodiments, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids (such as one or more protease or chemical cleavage sites).

[0083] In some embodiments, one or more of the Tag amino acid sequences comprise (or are selected from) - KRDSSTTDSSTTDDDDDK (SEQ ID NO: 28), KRDSSDTTDDDDDK (SEQ ID NO: 29), KRSSTTDDDDK (SEQ ID NO: 30), KRDSSTTDSSTTSSTT (SEQ ID NO: 31), KRDSSTTDSSTTDDDDK (SEQ ID NO: 32), or any combination of any of the foregoing. In some instances, each of the Tag amino acid sequence are selected from -KRDSSTTDSSTTDDDDDK (SEQ ID NO: 28), KRDSSDTTDDDDDK (SEQ ID NO: 29), KRSSTTDDDDK (SEQ ID NO: 30), KRDSSTTDSSTTSSTT (SEQ ID NO: 31), or KRDSSTTDSSTTDDDDK (SEQ ID NO: 32). In some embodiments, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids (such as one or more protease or chemical cleavage sites).

[0084] In some embodiments, one or more of the Tag amino acid sequences comprise KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 33), KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 34), KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 35), KRSSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 36), KRSSTTSSTTRSSTTSSTTSSTTR (SEQ ID NO: 37), KR, or any combination of any of the foregoing. In some embodiments, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids (such as one or more protease or chemical cleavage sites). In some embodiments, each of the Tag amino acid sequence are selected from KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 33), KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 34), KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 35), KRSSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 36), oKRSSTTSSTTRSSTTSSTTSSTTR (SEQ ID NO: 37), or any combination of any of the foregoing. In some embodiments, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids (such as one or more protease or chemical cleavage sites).

[0085] In some embodiments, one or more of the Tag amino acid sequence include one or more additional amino acids in combination with any of the foregoing Tag amino acid sequences (such as to change the pl and / or hydrophobicity of the multimer).

[0086] In some embodiments, the Tag amino acid sequence can be any combination of naturally occurring amino acids targeted to change intrinsic characteristics of the multimer, such as pl and / or hydrophobicity.

[0087] In some embodiments, the Tag amino acid sequence lowers the pl of the multimer. In some instances, the Tag amino acid sequence lowers the hydrophobicity of the multimer. In some instances, the Tag amino acid sequence is selected from SEQ ID NOs. 7-11, 14-19, 28-41, 43, or any combination of any of the foregoing.

[0088] In some instances, every Tag amino acid sequence in the multimer is the same.

[0089] In some embodiments, the multimer comprises two or more different Tag amino acid sequences.

[0090] In some embodiments, the N-terminus of the multimer includes a methionine and histidine tag. In some instances, the N-terminus of the multimer may include (or is), but is not limited to, a methionine-poly histidine (such as -MHHHHHH (SEQ ID NO. 42).

[0091] In some embodiments, the C-terminus of the multimer comprises a histidine tag followed by a stop codon. In some instances, the C-terminus of the multimer includes a polyhistidine.

[0092] In some embodiments, the poly-histidine tag can be within the multimer, i.e. not at the N- or C- terminus.

[0093] In some embodiments, the multimer comprises at least 2 copies of the target peptide sequence, such as 2 to 50 copies or 2 to 20 copies, In some instances, the multimer comprises greater than 2 copies of the target peptide sequence. In some instances, the multimer comprises 5 to 40 copies of the target peptide sequence. In some instances, the multimer comprises 10 copies of the target peptide sequence. In another embodiment, the multimer comprises 20 copies of the target peptide sequence.

[0094] In some embodiments, each occurrence of CS or the cleavage site is independently KR or any other proteolytic or chemical cleavage site. In one embodiment of the multimer described herein, each occurrence of CS or the cleavage site is KR.

[0039] In some embodiments, each occurrence of a CS or the cleavage site is independently DDDDK (SEQ ID NO: 53) or any other proteolytic or chemical cleavage site. In some embodiment In some embodiments, the multimer has a pl from about 4 to about 12. In some embodiments, the multimer has a hydrophobicity of from about 20 to about 50%. In In some embodiments, the multimer has a hydrophobicity range of from about 24 to about 42%. In some embodiments, the multimer has a molecular weight more than 2 kilodalton.

[0095] s, each occurrence of a CS is DDDDK (SEQ ID NO: 53).

[0096] In some embodiments, the multimer has a molecular weight more than 2 kilodalton. In some instances, the multimer has a molecular weight of from about 5 to about 250 kilodaltons. In some instances, the multimer has a molecular weight of from about 30 to about 60 kilodaltons.

[0097] In In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 4), a fragment of semaglutide (excluding the first four amino acids of semaglutide).

[0098] In some embodiments, the target peptide sequence is 10- 29 amino acids. In some instances, the target peptide sequence is 1-50 amino acids.

[0099] In some embodiments, one or more of the target peptide sequences comprise (or are selected from) but not limited to EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 44) (a fragment of the unmodified peptide sequence for semaglutide, excluding the first two ammo acids of semaglutide), GTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 45) (a fragment of the unmodified peptide sequence for semaglutide excluding the first three amino acids of semaglutide), FTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 46) (a fragment of the unmodified peptide sequence for semaglutide excluding the first five amino acids of semaglutide), TSD VS SYLEGQAAKEFI AWL VRGRG (SEQ ID NO. 47) (a fragment of the unmodified peptide sequence for semaglutide excluding the first six amino acids of semaglutide), SDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 48) (a fragment of the unmodified peptide sequence for semaglutide excluding the first seven amino acids of semaglutide), DVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 49) (a fragment of the unmodified peptide sequence for semaglutide), VSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 50) (a fragment of the unmodified peptide sequence for semaglutide), SSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 51) (a fragment of the unmodified peptide sequence for semaglutide), SYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 52) (a fragment of the unmodified peptide sequence for semaglutide), YLEGQAAKEFIAWLVRGRG (SEQ ID NO. 53) (a fragment of the unmodified peptide sequence for semaglutide), LEGQAAKEFIAWLVRGRG (SEQ ID NO. 54) (a fragment of the unmodified peptide sequence for semaglutide), EGQAAKEFIAWLVRGRG (SEQ ID NO. 55) (a fragment of the unmodified peptide sequence for semaglutide), GQAAKEFIAWLVRGRG (SEQ ID NO. 56) (a fragment of the unmodified peptide sequence for semaglutide), QAAKEFIAWLVRGRG (SEQ ID NO. 57) (a fragment of the unmodified peptide sequence for semaglutide), AAKEFIAWLVRGRG (SEQ ID NO. 58) (a fragment of the unmodified peptide sequence for semaglutide), AKEFIAWLVRGRG (SEQ ID NO. 59) (a fragment of the unmodified peptide sequence for semaglutide), KEFIAWLVRGRG (SEQ ID NO. 60) (a fragment of the unmodified peptide sequence for semaglutide), EFIAWL VRGRG (SEQ ID NO. 61) (a fragment of the unmodified peptide sequence for semaglutide), FIAWLVRGRG (SEQ ID NO. 62) (a fragment of the unmodified peptide sequence for semaglutide), (a fragment of the unmodified peptide sequence for semaglutide), or (a fragment of the unmodified peptide sequence for semaglutide). The target peptide sequence may be converted to semaglutide, such as for example, by conjugating a fragment of the remaining semiglutide and acylating the lysine (in AAKE) is acylated with a spacer consisting of two 8-amino-3,6-dioxaoctanoic acid (ADO) moieties, a glutamic acid moiety, and a Cis fatty diacid side chain.

[00100] In some embodiments, the target peptide sequence is HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 64), which is the unmodified amino acid sequence for semaglutide . In some embodiments, after the peptide HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 64) is obtained from the multimer, the peptide is modified at the lysine at position 20 to be Lys-(y-Glu-palmitoyl) to obtain semaglutide.

[00101] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1).

[00102] In some embodiments, the target peptide sequence is EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 44).

[00103] In some embodiments, the target peptide sequence is TSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 47).

[00104] In some embodiments, one or more of the target peptide sequences comprise, but are not limited to EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 44), GTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 45), FTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 46), TSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 47), SDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 48), DVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 49), VSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 50), SSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 51), SYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 52), YLEGQAAKEFIAWLVRGRG (SEQ ID NO. 53), LEGQAAKEFIAWLVRGRG (SEQ ID NO. 54), EGQAAKEFIAWLVRGRG (SEQ ID NO. 55), GQAAKEFIAWLVRGRG (SEQ ID NO. 56), QAAKEFIAWLVRGRG (SEQ ID NO. 57), AAKEFIAWLVRGRG (SEQ ID NO. 58), AKEFIAWLVRGRG (SEQ ID NO. 59), KEFIAWLVRGRG (SEQ ID NO. 60), EFIAWLVRGRG (SEQ ID NO. 61), FIAWLVRGRG (SEQ ID NO. 62), and any combination of any of the foregoing.

[00105] In some embodiments, the target peptide sequence is SEQ ID NO. 1. In some instances, the target peptide sequence is SEQ ID NO. 4. In some instances, the target peptide sequence is SEQ ID NO. 22. In some instances, the target peptide sequence is SEQ ID NO. 25. In some instances, the target peptide sequence is any one of SEQ ID NO. 43-84.

[00106] In some embodiments, the target peptide sequence can be but not limited to 10- 30 amino acids. In some instances, the target peptide sequence is 1-50 amino acids.

[00107] In some embodiments, one or more of the target peptide sequences comprise, but are not limited to, EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 22), GTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 67), FTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 68), TSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 25), SDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 69), DVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 70), VSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 71), SSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 72), SYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 73), YLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 74), LEGQAAKEFIAWLVRGRGL (SEQ ID NO. 75), EGQAAKEFIAWLVRGRGL (SEQ ID NO. 76), GQAAKEFIAWLVRGRGL (SEQ ID NO. 77), QAAKEFIAWLVRGRGL (SEQ ID NO. 78), AAKEFIAWLVRGRGL (SEQ ID NO. 79), AKEFIAWLVRGRGL (SEQ ID NO. 80), KEFIAWLVRGRGL (SEQ ID NO. 81),

[00108] In some embodiments, the multimer sequence of a target peptide sequence is SEQ ID NO. 2. In some instances, the multimer sequence of a target peptide sequence is SEQ ID NO. 5. In some instances, the multimer sequence of a target peptide sequence is SEQ ID NO. 12. In some instances, the multimer sequence of a target peptide sequence is SEQ ID NO. 20. In some instances, the multimer sequence of a target peptide sequence is SEQ ID NO. 23. In some instances, the multimer sequence of a target peptide sequence is SEQ ID NO. 26.

[00109] In one embodiment of the multimer described herein, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO.3. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 6. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 13. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 21. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 24. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 27.

[00110] Yet another embodiment is a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[00111] Yet another embodiment is a host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[00112] Yet another embodiment is an expression vector comprising a DNA sequence encoding the multimer as described herein. In some instances, the expression vector may have any promoter. In one preferred embodiment, the promoter is T7.

[00113] Yet another embodiment is a host cell comprising the expression vector described herein. In some instances, the expression vector may have any promoter. In one preferred embodiment, the promoter is T7.

[00114] Yet another embodiment is a method of preparing a multimer comprising expressing a DNA construct comprising a nucleic acid sequence encoding the multimer described herein in a host cell. In some instances, the method may further comprise digesting the multimer with one or more enzymes to obtain a plurality of the target peptide sequences in free form. The peptide target sequence may then be converted to the final GLP1 analogue.

[00115] Yet another embodiment is a method of preparing a peptide comprising: (a) obtaining a multimer as described herein, where the target peptide sequence in the multimer has the sequence of the peptide or is a fragment thereof; (b) digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. In some embodiments, step (a) comprises expressing the multimer in a host and isolating the multimer. In another embodiment, prior to performing step (a), the host may be transfected with an expression vector for expressing the multimer (such as that described herein). In some embodiments, the N-terminus of the multimer includes a methionine, with or without an affinity tag, such as a poly-histidine tag (for example, M(H)n where n is 1 to 10, such as 4 to 8). In some embodiments, the multimer can be isolated in a Nickel column through use of the poly-histidine tag or another affinity tag. In some embodiments, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with a second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with a third protease enzyme or chemical to form the target peptide. One or more of these digestion steps may be performed simultaneously (e.g., the second and third protease enzymes or chemicals may be applied to the peptide mixture in the same step (i.e., at the same time)). In an alternative embodiment, these digestion steps are performed sequentially. In another embodiment, the multimer can be digested with a single protease to obtain the target peptide sequences in free form. In some embodiments, the method may further comprise step (c) isolating the peptide. In another embodiment, the multimer can be digested with a single protease to obtain the target peptide sequences. In another embodiment, the multimer can be digested with two different proteases, in a single or multiple steps. In some embodiments, the method may further comprise converting the target peptide sequence into the desired GLP1 analogue, such as semaglutide or liraglutide.

[00116] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4), which is obtained by (a) obtaining a multimer described herein, where the target peptide sequence in the multimer is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4); and (b) digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) may comprise expressing the multimer in a host cell and isolating the multimer. Prior to performing step (a), the host may be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and polyhistidine tag. The multimer is isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with a second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with a third protease enzyme or chemical to form the target peptide. One or more of these digestion steps may be performed simultaneously (e.g., the second and third protease enzymes or chemicals may be applied to the peptide mixture in the same step (i.e., at the same time)). In an alternative embodiment, these digestion steps are performed sequentially. In one embodiment, the method further comprises step (c) isolating the peptide. In another embodiment, the multimer can be digested with a single protease to obtain the target peptide sequences in free form.

[00117] In some embodiments, the multimer are digested with two different proteases. In some instances, the proteases digest the multimer in a single step. In some instances, the proteases digest the multimer in separate steps.

[00118] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4).

[00119] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) is obtained by a method of preparation as described herein.

[00120] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1).

[00121] In some embodiments, the method further comprises conjugating a second peptide to the target peptide sequence to form a third peptide and optionally, conjugating one or more chemical moieties to the third peptide.

[00122] In some embodiments, the target peptide sequence is a Glucagon-like Peptide-1 (GLP-1) agonist peptide.

[00123] In some embodiments, the target peptide sequence is converted to a Glucagon-like Peptide-1 (GLP-1) agonist peptide comprising the steps (i) conjugating a protected and / or activated amino acid fragment to a target peptide sequence prepared according to a method described herein, (ii) conjugating a fatty acid moiety, and (iii) optionally purifying the Glucagon-like Peptide-1 (GLP-1) agonist peptide.

[00124] In some embodiments, the protected and / or activated amino acid fragment thereof may be made by a method described herein, another recombinant method, or synthetically. In another embodiment, the protected and / or activated amino acid fragment is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. In an embodiment, the fatty acid moiety is conjugated at the E amino group of lysine in step (ii) as described herein. The fatty acid moiety may be prepared synthetically.

[00125] In some embodiments, the target peptide sequence (TP) is TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO.l). In a preferred embodiment of the multimer described herein, one or more of the target peptide sequences comprise (or are selected from) EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 22), GTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 67), FTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 68), TSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 25), SDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 69), DVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 70), VSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 71), SSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 72), SYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 73), YLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 74), LEGQAAKEFIAWLVRGRGL (SEQ ID NO. 75), EGQAAKEFIAWLVRGRGL (SEQ ID NO. 76), GQAAKEFIAWLVRGRGL (SEQ ID NO. 77), QAAKEFIAWLVRGRGL (SEQ ID NO. 78), AAKEFIAWLVRGRGL (SEQ ID NO. 79), AKEFIAWLVRGRGL (SEQ ID NO. 80), KEFIAWLVRGRGL (SEQ ID NO. 81),

[00126] In some embodiments, the protected and / or activated amino acid fragment is His-Aib-Glu-Gly or protected and / or activated derivative thereof.

[00127] In some embodiments, the fatty acid moiety is -^-U-W-Y-Z (I) wherein U represents -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-} wherein “}” is the point of attachment to W; W represents -C(O)-NH-(CH2)3-4-NH-], -C(O)-C(CH3)2-NH-], or wherein “]” is the point of attachment to Y; Y represents -C(O)-(CH2)2-CH(COOH)NH— and “—" is the point of attachment to Z; Z represents -C(0)-(CH2)n-C00H or -C(O)-(CH2)n-CH3 wherein n is an integer from 14-20. In a preferred embodiment, U-W-Y-Z represents a group of the formula: wherein n is an integer from 14-20 and Ro is CO2H or CH3. In another preferred embodiment, U-W-Y-Z represents a compound of the formula: 0

[00128] The term “GLP-1 agonist” is described in Gilroy et al., Set. Adv. 2020, 6, 1-12; Nauck et al., Molecular Metabolism, 46 (2021) 101102. In some embodiments, Glucagon-like Peptide-1 (GLP-1) agonist peptides comprising a modification, wherein the modification is a group of formula (I) attached to an amino acid residue in the peptide -^-U-W-Y-Z (I) wherein U represents -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-} wherein “}” is the point of attachment to W; W represents -C(O)-NH-(CH2)3-4-NH-], -C(O)-C(CH3)2-NH-], or -C(O)-N wherein “]” is the point of attachment to Y; Y represents -C(O)-(CH2)2-CH(COOH)NH— and “—" is the point of attachment to Z; Z represents -C(0)-(CH2) n-COOH or -C(0)-(CH2) n-CHs wherein n is an integer from 14-20.

[00129] In a preferred embodiment, U-W-Y-Z represents a group of the formula: wherein n is an integer from 14-20 and Ro is CO2H or CH3. In another preferred embodiment, U-W-Y-Z represents a compound of the formula: 0

[00130] In some embodiments, the target peptide sequence is a Glucagon-like Peptide-1 (GLP-1) agonist. In some embodiments, the present invention provides a Glucagon-like Peptide-1 (GLP-1) agonist peptide obtained by a process comprising the steps (i) conjugating a protected and / or activated amino acid fragment to a target peptide sequence prepared according to the methods described herein, (ii) conjugating a fatty acid moiety, and (iii) optionally purifying the crude a Glucagon-like Peptide-1 (GLP-1) agonist peptide. In some instances, the protected and / or activated amino acid fragment thereof may be made by a method described herein, another recombinant method, or synthetically. In some embodiments, the protected and / or activated amino acid fragment is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. In an embodiment, the fatty acid moiety is conjugated at the E amino group of lysine in step (ii) as described herein. The fatty acid moiety may be prepared synthetically.

[00131] According to any one of the embodiments described herein, the protected and / or activated amino acid fragment is His-Aib-Glu-Gly or protected and / or activated derivative thereof.

[00132] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4). In one embodiment of any of the methods of preparing this target peptide sequence, the method further comprises (i) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence, and (ii) conjugating the fatty acid moiety N-(17-carboxy-l-oxoheptadecyl)-L-y-glutamyl-2-[2-(2-aminoethoxy)ethoxyl]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl at the E amino group of lysine at position 26 to form semaglutide. The His-Aib-Glu-Gly or protected and / or activated derivative thereof may be made by the method described herein, another recombinant method, or synthetically. In an additional embodiment, His-Aib-Glu-Gly or protected and / or activated derivative thereof is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. The fatty acid moiety may be prepared synthetically.

[00133] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRGX (SEQ ID NO. 84), wherein X is absent or leucine.

[00134] In one embodiment of any of the methods for preparing this target peptide sequence, the method further comprises (i) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence, and (ii) conjugating a fatty acid moiety at the E amino group of lysine to form a GLP1 receptor agonist peptide. The His-Aib-Glu-Gly or protected and / or activated derivative thereof peptide may be prepared by any of the methods known in the art or described herein such as recombinant methods, or synthetically. In an additional embodiment, His-Aib-Glu-Gly or protected and / or activated derivative thereof is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof.

[00135] The fatty acid moiety listed herein may be prepared by any of the methods known in the art. Fatty acids moieties listed hereinbelow is incorporated for references, however not limited to these fatty acid moieties. Designation Moiety Moiety A H              ? J                  H              O HOA O                                  -OH O Moiety B o ( 0 V ^NH 8 Y Moiety C O. -OH o H      o Moiety D T o ^o o        f o        \ Z-T       ( T-\ * / X—i   z—K o '—( o >° o T Moiety E o Ck / NH            / O.            \ NH               o       J r                             ° HO. ^NH O J. o

[00136] Wherein, the fatty acid moieties as described above, may be prepared by any of the methods known in the art. For the purpose of the present disclosure, moiety D may be prepared according to the process described in either of WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00137] In some embodiments, the present disclosure relates to a process for the preparation of a GLP1 receptor agonist of following formula: E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G-L .. ■ . „zNH Moiety D wherein the said process comprises: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) from a multimer of SEQ ID 2; (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00138] In some embodiments, the present disclosure relates to a process for the preparation of a GLP1 receptor agonist of following formula: H °                            H 9 H-N'Jl—E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-N   K-E-F-l-A-W-L-V-R-G-R-G-L Moiety D comprising: (i) preparing a target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1); (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00139] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) is prepared by a process comprising the steps of: (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequence along with protease or chemical cleaving site; (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide, iv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00140] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) is prepared from multimer of SEQ ID 2.

[00141] In some embodiments, the present disclosure relates to a GLP1 receptor agonist compound of formula: H  °                            H  2 H— N   IL. E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-N   K-E-F-l-A-W-L-V-R-G-R-G-L Moiety D wherein the compound is obtained by a process comprising the steps of: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) from a multimer of SEQ ID 2; (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00142] In some embodiments, the present invention relates to a GLP1 receptor agonist compound of formula: E-F-l-A-W-L-V-R-G-R-G-L H E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-N Moiety D wherein the compound is obtained by a process comprising the steps of: (i) preparing a target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1); (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00143] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) is prepared by a process comprising the steps of: (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site; (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide. liv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprise step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00144] In some embodiments, His-Aib-Glu-Gly or protected and / or activated derivative thereof as used herein, may be prepared by any of the methods known in the art or described herein such as recombinant methods or synthetically. In some embodiments, His-Aib-Glu-Gly or protected and / or activated derivative thereof is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. Wherein, the conjugation may be carried out by any known methods known in the art, such as but not limited to: solid phase peptide synthesis, liquid phase peptide synthesis or combination thereof, also known as hybrid approach.

[00145] The fatty acid moiety D, as used according to any one of the embodiments described herein, may be prepared by any of the methods known in the art. For the purpose of the present disclosure, moiety D may be prepared according to the process described in either of WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00146] In some embodiments, wherein conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine of target peptide sequence may carried out according to the process described in the example, by any general standard methods known in the art, such as those described in WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00147] In some embodiments, the present disclosure relates to a process for the preparation of Semaglutide of following formula: E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G ■ * a-NH Moiety A wherein the said process comprises: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) from multimer of SEQ ID 5; (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety A at the E amino group of lysine.

[00148] In some embodiments, the present disclosure relates to a process for the preparation of Semaglutide of following formula: E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G Moiety A ^NH comprising: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4); (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety A O selected from hydrogen or a carboxylic acid activating group wherein R is at the E amino group of lysine. In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) is prepared by a process comprising the steps of: (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving sites, (b) digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide, (iv). Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00149] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 4) is prepared from multimer of SEQ ID 5.

[00150] In some embodiments, the present disclosure relates to Semaglutide of formula: H E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A—N E-F-l-A-W-L-V-R-G-R-G ■ * a-NH Moiety A wherein Semaglutide is obtained by a process comprising the steps of: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) from multimer of SEQ ID 5; (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety A at the E amino group of lysine.

[00151] In some embodiments, the present disclosure relates to Semaglutide of formula: E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G .. ■ x a-NH Moiety A wherein Semaglutide is obtained by a process comprising the steps of: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4); (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety A at the E amino group of lysine. Wherein, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) is prepared by a process comprising the steps of: (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site (b) digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide. liv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00152] In some embodiments, His-Aib-Glu-Gly or protected and / or activated derivative thereof as used herein, may be prepared by any of the methods known in the art or described herein such as recombinant methods or synthetically. In an additional embodiment, His-Aib-Glu-Gly or protected and / or activated derivative thereof is conjugated to the target peptide sequence by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. Wherein, the conjugation may be carried out by any known methods known in the art, such as but not limited to: solid phase peptide synthesis, liquid phase peptide synthesis or combination thereof, also known as hybrid approach.

[00153] The fatty acid moiety A, as used according to any one of the embodiments described herein, may be prepared by any of the methods known in the art. For the purpose of the present disclosure, moiety D may be prepared according to the process described in either of WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00154] In some embodiments, wherein conjugating a fatty acid moiety of formula: Moiety A H              ? h%-L O                                 ^OH °       , wherein R is selected from hydrogen or a carboxylic acid activating group at the E amino group of lysine of target peptide sequence may carried out according to the process described in the example, by any general standard methods known in the art, such as that disclosed in WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639. BRIEF DESCRIPTION OF THE FIGURES

[00155] Figure 1: The SDS-PAGE analysis of multimer of target peptide 1 (SEQ ID NO: 2). Lane 1: Molecular wt marker; Lane 2: multimer of target peptide 1 (SEQ ID NO: 2) (34.9 kDa).

[00156] Figure 2: Ni-NTA purification sample run on SDS PAGE of multimeric target peptide 1(SEQ ID NO: 2); Lane 1: Molecular weight marker, Lane 2: Load sample of multimeric target peptide 1, Lane 3: Flow through sample of multimeric target peptide 1, Lane 4: Wash of multimeric target peptide 1, Lane 5: Eluate of multimeric target peptide 1.

[00157] Figure 3: The SDS-PAGE analysis of multimer of target peptide 2 (SEQ ID NO: 5). Lane 1: Molecular wt marker; Lane 2: multimer of target peptide 2 (SEQ ID NO: 5) (33.8 KDa)

[00158] Figure 4: Ni-NTA purification sample run on SDS PAGE of multimeric target peptide 2 (SEQ ID NO: 5); Lane 1: Molecular weight marker, Lane 2: Load sample of multimeric target peptide 2, Lane 3: Flow through sample of multimeric target peptide 2, Lane 4: Wash of multimeric target peptide 2, Lane 5: Eluate of multimeric target peptide 2.

[00159] Figure 5: RPHPLC analysis of multimer and after enzymatic digestion with Kex2 and CpB protease to generate monomer of target peptide 2. Black peak indicates multimer (SEQ ID NO: 5), blue peak indicates intermediate after Kex2 digestion and red peak indicates monomer of target peptide 2 (SEQ ID NO:4) after CpB digestion.

[00160] Figure 6: The SDS-PAGE analysis of multimer of target peptide 2 (SEQ ID 12) having improved solubility. Lane 1: Molecular wt. marker; Lane 2: multimer of target peptide 2 (SEQ ID 12) (48.5 kDa)

[00161] Figure 7: Solubilization of multimer of target peptide 2 in different solubilization buffers. Multimer of target peptide 2 (SEQ ID NO:5) is insoluble in 8M Urea (1), whereas multimer of target peptide 2 with improved solubility (SEQ ID NO: 12) is easily soluble in mild solubilizing agent such as 6M Urea (2).

[00162] Figure 8: SDS-PAGE analysis of multimer of target peptide 2 (SEQ ID 20). Lane 1: Molecular wt. marker; Lane 2: multimer of target peptide 2 (SEQ ID 20) (51.3 kDa)

[00163] Figure 9: Comparative SDS-PAGE analysis of multimer of target peptide 2 (SEQ ID NO:20) and SEQ ID NO:5. Lane 1: Molecular wt. marker; Lane 2: multimer of target peptide 2 (SEQ ID NO: 20) (51.3 kDa). Lane 3: multimer of target peptide 2 (SEQ ID NO: 5) (33.8Kda)

[00164] Figure 10: Ni-NTA purification sample run on SDS PAGE of multimeric target peptide 2 SEQ ID NO: 20; Lane 1: Load sample of multimeric target peptide 2, Lane 2: Molecular weight marker, Lane 3: Flow through sample of multimeric target peptide 2, Lane 4: Wash 1 of multimeric target peptide 2, Lane 5: Wash 2 of multimeric target peptide 2. Lane 6: Eluate 1 of multimeric target peptide 2. Lane 7: Eluate 2 of multimeric target peptide 2.

[00165] Figure 11: Elute sample of SEQ ID NO:20 was buffer exchanged in different molar of Urea concentration. Tube 1: Sample dissolved in 2M Urea. Lane 2: Sample dissolved in 3M Urea. Lane 3: Sample dissolved in 4M Urea.

[00166] Figure 12: SDS-PAGE analysis of multimer of target peptide 3 (SEQ ID NO: 23). Lane 1: Molecular wt. marker; Lane 2: multimer of target peptide 3 (SEQ ID 23) (36.9 kDa).

[00167] Figure 13: Ni-NTA purification sample run on SDS PAGE of multimeric target peptide 3; Lane 1: Molecular weight marker, Lane 2: Load sample of multimeric target peptide 3, Lane 3: Flow through sample of multimeric target peptide 3, Lane 4: Wash 1 of multimeric target peptide 3, Lane 5: Wash 2 of multimeric target peptide 3, Lane 6: Fraction 1 of Eluate of multimeric target peptide 3, Lane 7: Fraction 2 of Eluate of multimeric target peptide 3.

[00168] Figure 14: Mass and sequence coverage analysis of multimer and after enzymatic digestion with Kex2 and CpB protease to generate monomer of target peptide 3. Figure indicates mass confirmation of monomer for target peptide 3 (SEQ ID NO:22) after Kex2 and CpB digestion.

[00169] Figure 15: SDS-PAGE analysis of multimer of target peptide 4 (SEQ ID 26). Lane 1: Molecular wt. marker; Lane 2: multimer of target peptide 4 (SEQ ID 26) (32.4 kDa).

[00170] Figure 16: Ni-NTA purification sample run on SDS PAGE of multimeric target peptide 4 (SEQ ID NO: 26; Lane 1: Molecular weight marker, Lane 2: Load sample of multimeric target peptide 4, Lane 3: Flow through sample of multimeric target peptide 4, Lane 4: Wash 1 of multimeric target peptide 4, Lane 5: Wash 2 of multimeric target peptide 4, Lane 6: Fraction 1 of Eluate of multimeric target peptide 4, Lane 7: Fraction 2 of Eluate of multimeric target peptide 4,

[00171] Figure 17: Mass and sequence coverage analysis of multimer and after enzymatic digestion with Kex2 and CpB protease to generate monomer of target peptide 4. Figure indicates mass confirmation of monomer for target peptide 4 (SEQ ID NO:25) after Kex2 and CpB digestion. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

[00172] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Disclosed and described, it is to be understood that this invention is not limited to the specific examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing specific embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[00173] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[00174] In the present context the terms "polypeptide", "protein" and "peptide" may be used interchangeably to designate a polypeptide. It is to be understood that the particular term used has no limitation as to the size of the molecule (unless directly stated in the particular context). Amino acid residues are generally designated according to single letter abbreviation according to 1UPAC nomenclature, e.g., D meaning aspartic acid (Asp) and G meaning glycine.

[00175] The term “promoter” generally refers to a regulatory region of DNA usually located upstream of the inserted gene of interest providing a control point for regulated gene transcription.

[00176] The term "host cell" as used herein is intended to mean a microorganism which is used for the expression of a peptide of interest. A host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. Host cell can be but not limited to bacteria, Yeast, CHO.

[00177] The hydrophobicity of a peptide may be calculated by methods known in the art, such as by Peptide 2.0 (www.peptide2.com / N_peptide_hydrophobicity_hydrophilicity.php) or the use of the Monera scale (Monera et al., 1995, J. Protein Sci., 1:319-329). In one embodiment, each amino acid in the peptide is assigned a hydrophobicity value based on established scales, such as the Monera scale. The scale quantifies the hydrophobic nature of each amino acid. The hydrophobicity values of all amino acids in the peptide are summed up. This total may then be divided by the number of amino acids in the peptide to get an average hydrophobicity value. The average hydrophobicity value can be normalized to a percentage scale. This involves comparing the peptide's hydrophobicity to a reference value, which could be the maximum hydrophobicity value on the scale used.

[00178] Throughout this specification and the examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term comprising will also be understood to include “consisting of’ and “consisting essentially of.”

[00179] Digestion of the multimer can be performed by proteases (for example, the Kex2 protease and carboxypeptidase B) and chemical reactions. The Kex2 protease specifically hydrolyzes a carboxyl terminal peptide bond in an alpha factor precursor, in particular a carboxyl terminal peptide bond of two consecutive basic amino acids, such as Lys-Arg, Lys-Lys, or ArgArg. Recombinant carboxypeptidase B (CpB) can selectively hydrolyze arginine or lysine at the carboxyl terminus of a peptide. While proteases are referenced for cleaving Tag amino acid sequences, other enzymes may be used.

[00180] Affinity tag: Short amino acid sequence attached to protein / molecule which is capable of binding to specific metal / surfaces, enabling protein purification.

[00181] Tag amino acid sequence: Amino acid sequence ranging from (~ 4-30 amino acids), capable of modulating the pl and hydrophobicity of the proteins.

[00182] Monomer: Single unit, comprising of combination of amino acid sequence having target peptide, protease / chemical cleavage sequence and or Tag amino acid sequence.

[00183] Multimer: Recombinant protein molecule comprising of multiple repeats of the monomer along with or without affinity tag.

[00184] One of the major challenges for improving the efficiency of production of recombinant peptide or protein from a target peptide sequence is the solubility of the inclusion body which requires strong chaotropic agents such as guanidine HC1. To mitigate this insolubility issue some of the characteristics of the multimeric peptide such as pl and / or hydrophobicity are / is modified using intervening specific Tag amino acid sequences which aides in higher solubility and expression of the multimeric peptide. This invention provides a technology platform that enables small peptide (recombinant peptide of interest) having more than 3 amino acids in length to be produced in the form of multimers in microbial cells such as E. coli or yeast

[00185] The present invention provides a method for enhanced expression of a multimer containing a plurality of target peptide sequences, wherein each target peptide sequence is a GLP-1 analogue or a fragment thereof. Furthermore, the solubility of the multimer allows for convenient downstream processing, including digestion and purification, including to purity levels of at least 95%.

[00186] The present invention is directed to peptide of interest using efficient process of a multimers containing a plurality of the recombinant peptide of interest derived from target peptide sequences (TP), wherein to improve the solubility of the multimer of the recombinant peptide of interest, the intrinsic characteristics of the multimer such as pl and / or hydrophobicity can be altered. Disclosed are specific Tag amino acid sequences sequence (Tag) which can modulate overall pl and / or hydrophobicity of the multimer of recombinant peptide of interests (TP) enabling high expression, improved solubility and ease of downstream processing. Specific Tag amino acid sequences in combination with CS within the MO of a multimeric approach will modulate intrinsic properties of the expressed multimer.

[00187] In some embodiments, the Tag amino acid sequence can be designed using a combination of amino acids which can alter intrinsic properties of the multimer such as pl and / or hydrophobicity. In some instances, the Tag amino acid sequence comprises aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), or arginine (R) to alter the pl of the multimer. In some instances, the Tag amino acid sequence comprises Aspartic acid (D) and / or glutamic acid (E) to reduce the pl of the multimer. In some instances, the Tag amino acid sequence comprises lysine and / or arginine to increase the pl of the multimer. In some instances, the Tag amino acid sequence comprises serine (S). threonine (T) asparagine (N), glutamine (Q) and other hydrophilic amino acids to make the multimer more hydrophilic. In some instances, the Tag amino acid sequence comprises leucine (L), isoleucine (I) alanine (A), valine (V), proline (P), Glycine (G), Tryptophan (W), Phenylalanine (F) and other hydrophobic amino acids to make the multimer more hydrophobic.

[00188] In some embodiments, the C-terminus of the multimer (after the last target peptide in the multimer) includes an Tag amino acid sequence which is capable of being cleaved from the target peptide sequence by a protease and / or chemical cleavage. In some instances, the one or more of the Tag amino acid sequences comprise the amino acid sequence KR. In some instances, the R of the KR amino acid sequence is directly bound to the N-terminus of one of the target peptide sequences, and the K of the KR amino acid sequence is directly bound to the C-terminus of one of the target peptide sequences.

[00189] In some embodiments, one or more of the Tag amino acid sequence comprises one or more amino acids to alter the pl of the multimer, the hydrophobicity of the multimer, or both. In some instances, one or more of the Tag amino acid sequence comprise aspartic acid (D), glutamic acid (E), or any combination of any of the foregoing to reduce the pl of the multimer. In some instances, one or more of the Tag amino acid sequence comprise histidine (H), lysine (K), arginine (R), or any combination of any of the foregoing to increase the pl of the multimer. In some instances, one or more of the Tag amino acid sequence comprise serine (S), threonine (T) asparagine (N), glutamine (Q) or any other hydrophilic amino acid to increase the hydrophilicity of the multimer. In some instances, one or more Tag amino acid sequence selected from leucine (L), isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F), or any other hydrophobic amino acid or any combination of any of the foregoing to increase the hydrophobicity of the multimer. In some instances, the Tag amino acid sequence, comprises one or more aspartic acid residues, one or more glutamic acid residues, or both.

[00190] In some embodiments, the multimer comprises one or more Tag amino acid sequence, wherein the Tag amino acid sequence comprises an amino acid group with a proteolytic and / or chemical cleavage site at both the N and C terminus of the target peptide sequence for cleaving the multimer to generate the monomer of the target peptide sequence. In some instances, the amino acids in the Tag amino acid sequence may act as both a modulator of pl and / or hydrophobicity as well as a proteolytic and / or chemical cleavage site.

[00191] In some instances, the multimer comprises one or more Tag amino acid sequence which include only one or more proteolytic and / or chemical cleavage sites. In some instances, each Tag amino acid sequence is KR.

[00192] In some instances, one or more of the Tag amino acid sequence comprise (or are selected from) DSSTTDSSTTDDDDK (SEQ ID NO: 7), DSSDTTDDDDDK (SEQ ID NO: 8), DSSTTDSSTTDDDDDK (SEQ ID NO: 9), SSTTDDDDK (SEQ ID NO: 11), DSSTTDSSTTSSTT (SEQ ID NO: 10), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 14), SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 15), SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 16), SDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 17), SSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 18), SDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 19), SSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 38), SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 39), SSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 40), or SSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 41), or any combination of any of the foregoing. In some embodiments, one or more of the aforementioned Tag amino acid sequence further comprise one or more additional amino acids, such as one or more protease or chemical cleavage sites (at the N-terminus or C-terminus). In some embodiments, each of the Tag amino acid sequence are selected from DSSTTDSSTTDDDDK (SEQ ID NO: 7), DSSDTTDDDDDK (SEQ ID NO: 8), DSSTTDSSTTDDDDDK (SEQ ID NO: 9), SSTTDDDDK (SEQ ID NO: 11), DSSTTDSSTTSSTT (SEQ ID NO: 10), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 14), SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 15), SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 16), SDSTTSSTTSSTTSSTTSSTT(SEQ ID NO: 17), SSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 18), SDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 19), SSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 38), SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 39), SSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 40), or SSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 41). In some embodiments, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids (such as one or more protease or chemical cleavage sites).

[00193] In some embodiments, one or more of the Tag amino acid sequences comprise (or are selected from) KRDSSTTDSSTTDDDDDK (SEQ ID NO: 28), KRDSSDTTDDDDDK (SEQ ID NO: 29), KRSSTTDDDDK (SEQ ID NO: 30), KRDSSTTDSSTTSSTT (SEQ ID NO: 31), KRDSSTTD SSTTDDDDK (SEQ ID NO: 32), or any combination of any of the foregoing. In some instances, each of the Tag amino acid sequence are selected from KRDSSTTDSSTTDDDDDK (SEQ ID NO: 28), KRDSSDTTDDDDDK (SEQ ID NO: 29), KRSSTTDDDDK (SEQ ID NO: 30), KRDSSTTDSSTTSSTT (SEQ ID NO: 31), or KRDSSTTDSSTTDDDDK (SEQ ID NO: 32). In some embodiments, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids (such as one or more protease or chemical cleavage sites).

[00194] In some embodiments, one or more of the Tag amino acid sequences comprise KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 33), KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 34), KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 35), KRSSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 36), KRSSTTSSTTRSSTTSSTTSSTTR (SEQ ID NO: 37), KR, or any combination of any of the foregoing. In some embodiments, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids (such as one or more protease or chemical cleavage sites). In some embodiments, each of the Tag amino acid sequence are selected from KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 33), KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 34), KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 35), KRSSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 36), or KRSSTTSSTTRSSTTSSTTSSTTR (SEQ ID NO: 37), KR, or any combination of any of the foregoing. In some embodiments, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids (such as one or more protease or chemical cleavage sites).

[00195] In some embodiments, one or more of the Tag amino acid sequence include one or more additional amino acids in combination with any of the foregoing Tag amino acid sequences (such as to change the pl and / or hydrophobicity of the multimer).

[00196] In some embodiments, the Tag amino acid sequence can be any combination of naturally occurring amino acids targeted to change intrinsic characteristics of the multimer, such as pl and / or hydrophobicity.

[00197] In some instances, every Tag amino acid sequence in the multimer is the same.

[00198] In some embodiments, the multimer comprises two or more different Tag amino acid sequences.

[00199] In some embodiments, the N-terminus of the multimer includes a methionine and histidine tag. In some instances, the N-terminus of the multimer may include (or is), but is not limited to, a methionine-poly histidine (such as -MHHHHHH (SEQ ID NO. 42).

[00200] In some embodiments, the C-terminus of the multimer comprises a histidine tag followed by a stop codon. In some instances, the C-terminus of the multimer includes a polyhistidine.

[00201] In some embodiments, the poly-histidine tag can be within the multimer, i.e. not at the N- or C- terminus.

[00202] In some embodiments, the multimer comprises at least 2 copies of the target peptide sequence, such as 2 to 50 copies or 2 to 20 copies, In some instances, the multimer comprises greater than 2 copies of the target peptide sequence. In some instances, the multimer comprises 5 to 40 copies of the target peptide sequence. In some instances, the multimer comprises 10 copies of the target peptide sequence. In another embodiment, the multimer comprises 20 copies of the target peptide sequence.

[00203] In some instances, the multimer comprises a Tag amino acid sequence to alter the hydrophobicity, pl, or both of the multimer or a cleaved product of the multimer. In some instances, Tag amino acid sequence comprises one or more amino acids selected from aspartic acid (D), glutamic acid (E), serine (S), threonine (T), arginine (R), glutamine (Q), lysine (K), histidine (H), leucine (L) and isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F), tyrosine (Y), cysteine (C), and methionine (M).

[00204] In some embodiments, the multimer comprises an affinity tag, for example, to facilitate purification of the multimer. In some embodiments, the N-terminus of the multimer comprises Met-A-, where the variable A is an affinity tag. In some instances, the variable A may comprise a histidine tag (e.g., poly histidine). In some embodiments, the variable A is absent (i.e., there is no affinity tag in the sequence at the N-terminus).

[00205] In some embodiments, each occurrence of CS or the cleavage site is independently KR or any other proteolytic or chemical cleavage site. In one embodiment of the multimer described herein, each occurrence of CS or the cleavage site is KR.

[00206] In some embodiments, each occurrence of CS is independently DDDDK or any other proteolytic / chemical cleavage site. In some embodiments, each occurrence of CS is DDDDK (SEQ ID NO. 43). In some instances, the Tag amino acid sequence comprises the sequence DDDDK (SEQ ID NO: 43). In some instances, the K of the DDDDK sequence is directly bound to the N-terminus of one of the target peptide sequences.

[00207] In some embodiments, the multimer has a pl from about 4 to about 12.

[00208] In some embodiments, the multimer has a hydrophobicity of from about 20 to about 50%.

[00209] In some embodiments, the multimer has a hydrophobicity range of from about 24 to about 42%.

[00210] In some embodiments, the multimer has a molecular weight more than 2 kilodalton.

[00211] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4).

[00212] In some embodiments, the target peptide sequence can be but not limited to 10- 29 amino acids.

[00213] In some embodiments, one or more of the target peptide sequences comprise EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 44), GTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 45), FTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 46), TSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 47), SDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 48), DVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 49), VSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 50), SSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 51), SYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 52), YLEGQAAKEFIAWLVRGRG (SEQ ID NO. 53), LEGQAAKEFIAWLVRGRG (SEQ ID NO. 54), EGQAAKEFIAWLVRGRG (SEQ ID NO. 55), GQAAKEFIAWLVRGRG (SEQ ID NO. 56), QAAKEFIAWLVRGRG (SEQ ID NO. 57), AAKEFIAWLVRGRG (SEQ ID NO. 58), AKEFIAWLVRGRG (SEQ ID NO. 59), KEFIAWLVRGRG (SEQ ID NO. 60), EFIAWLVRGRG (SEQ ID NO. 61), and FIAWLVRGRG (SEQ ID NO. 62),.

[00214] In some embodiments, the target peptide sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 65).

[00215] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1)

[00216] In some embodiments, the target peptide sequence can be but not limited to 10- 30 amino acids.

[00217] In some embodiments, one or more of the target peptide sequences comprise EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 22), AEGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 66),GTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 67), FTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 68), TSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 25), is SDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 69), DVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 70), VSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 71), SSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 72), SYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 73), YLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 74), LEGQAAKEFIAWLVRGRGL (SEQ ID NO. 75), EGQAAKEFIAWLVRGRGL (SEQ ID NO. 76), GQAAKEFIAWLVRGRGL (SEQ ID NO. 77), QAAKEFIAWLVRGRGL (SEQ ID NO. 78), AAKEFIAWLVRGRGL (SEQ ID NO. 79), AKEFIAWLVRGRGL (SEQ ID NO. 80), KEFIAWLVRGRGL (SEQ ID NO. 81), or and any combination of any of the foregoing.

[00218] In some embodiments, the invention is directed towards a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[00219] In some embodiments, a host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[00220] In some embodiments, the invention is directed towards an expression vector comprising a DNA sequence encoding the multimer as described herein.

[00221] In some embodiments, is a host cell comprising the expression vector described herein. In some instances, the expression vector may have any promoter. In one preferred embodiment, the promoter is T7.

[00222] In some embodiments, the invention is directed towards a method of preparing a multimer comprising expressing a DNA construct comprising a nucleic acid sequence encoding the multimer described herein in a host cell. In some instances, the method may further comprise digesting the multimer with one or more enzymes to obtain a plurality of the target peptide sequences in free form. The peptide target sequence may then be converted to the final GLP1 analogue.

[00223] In some embodiments, the invention is directed towards is a method of preparing a peptide comprising: (a) obtaining a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and; with or without not limited to poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide. liv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00224] In some embodiments, a target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) is obtained by (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide. liv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00225] In some embodiments, a target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) is obtained by (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide, (iv) Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00226] In some embodiments, the target peptide sequence is not limited to TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1)

[00227] In some embodiments, the method further comprises conjugating a second peptide to the target peptide sequence to form a third peptide and optionally, conjugating one or more chemical moieties to the third peptide.

[00228] In some embodiments, the target peptide sequence is a Glucagon-like Peptide-1 (GLP-1) agonist peptide.

[00229] In some embodiments, the target peptide sequence is converted to a Glucagon-like Peptide-1 (GLP-1.) agonist peptide comprising the steps (i) conjugating a protected and / or activated amino acid fragment to a target peptide sequence prepared according to the methods described herein, (ii) conjugating a fatty acid moiety, and (iii) optionally purifying the crude a Glucagon-like Peptide-1 (GLP-1) agonist peptide.

[00230] In some embodiments, the protected and / or activated amino acid fragment thereof may be made by a method described herein, another recombinant method, or synthetically. In another embodiment, the protected and / or activated amino acid fragment is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. In an embodiment, the fatty acid moiety is conjugated at the E amino group of lysine in step (ii) as described herein. The fatty acid moiety may be prepared synthetically.

[00231] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) from multimer of SEQ ID 2. In some embodiments, the target peptide sequence can be but not limited to 10-30 amino acids. In a preferred embodiment of the multimer described herein, one or more of the target peptide sequences comprise EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 22), AEGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 66), GTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 67), FTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 68), TSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 25), SDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 69), DVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 70), VSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 71), SSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 72), SYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 73), YLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 74), LEGQAAKEFIAWLVRGRGL (SEQ ID NO. 75), EGQAAKEFIAWLVRGRGL (SEQ ID NO. 76), GQAAKEFIAWLVRGRGL (SEQ ID NO. 77), QAAKEFIAWLVRGRGL (SEQ ID NO. 78), AAKEFIAWLVRGRGL (SEQ ID NO. 79), AKEFIAWLVRGRGL (SEQ ID NO. 80), KEFIAWLVRGRGL (SEQ ID NO. 81), or and any combinations of any of the foregoing.

[00232] In some embodiments, the protected and / or activated amino acid fragment is His-Aib-Glu-Gly or protected and / or activated derivative thereof.

[00233] In some embodiments, the fatty acid moiety is -^-U-W-Y-Z (I) Wherein U represents -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-} wherein} is point of attachment to W; W represents -C(O)-NH-(CH2)3-4-NH-], -C(O)-C(CH3)2-NH-], or -C(0)-NH- / ~^N—] wherein ] is point of attachment to Y; Y represents -C(O)-(CH2)2-CH(C00H)NH— and — is point of attachment to Z; Z represents -C(0)-(CH2)n-C00H or -C(O)-(CH2)n-CH3 wherein n is an integer from 14-20. In a preferred embodiment, U-W-Y-Z represents a group of the formula: Wherein n is an integer from 14-20 and Ro is CO2H or CH3. More preferably, U-W-Y-Z represents a compound of the formula: O

[00234] In some embodiments, the Glucagon-like Peptide-1 (GLP-1) agonist peptides those skilled in the art understand what is meant by the term “GLP-1 agonist” and would certainly understand what is meant by the term Glucagon-like Peptide-1 (GLP-1) agonist peptides as used herein. For the purpose of the present disclosure, the term “GLP-1 agonist” is commonly described in literature such as Gilroy et al., Sci. Adv. 2020, 6, 1-12; Nauck et al., Molecular Metabolism, 46 (2021) 101102. In an embodiment, Glucagon-like Peptide-1 (GLP-1) agonist peptides comprising a modification, wherein the modification is a group of formula (I) attached to an amino acid residue in the peptide: -^-U-W-Y-Z (I) wherein U represents -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-} wherein} is point of attachment to W; W represents -C(O)-NH-(CH2)3-4-NH-], -C(O)-C(CH3)2-NH-], or -C(0)-NH-^~^N-] wherein] is point of attachment to Y; Y represents -C(O)-(CH2)2-CH(COOH)NH— and — is point of attachment to Z; Z represents -C(0)-(CH2)n-C00H or -C(O)-(CH2)n-CH3 wherein n is an integer from 14-20. In a preferred embodiment, U-W-Y-Z represents a group of the formula: wherein n is an integer from 14-20 and Ro is CO2H or CH3. More preferably, U-W-Y-Z represents a compound of the formula: O

[00235] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4). In one embodiment of any of the methods of preparing this target peptide sequence, the method further comprises (i) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence, and (ii) conjugating the fatty acid moiety N-(17-carboxy-l-oxoheptadecyl)-L-y-glutamyl-2-[2-(2-aminoethoxy)ethoxyl]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl at the E amino group of lysine at position 26 to form semaglutide. The His-Aib-Glu-Gly or protected and / or activated derivative thereof may be made by the method described herein, another recombinant method, or synthetically. In an additional embodiment, His-Aib-Glu-Gly or protected and / or activated derivative thereof is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. The fatty acid moiety may be prepared synthetically.

[00236] In some embodiments, the target peptide sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRGX (SEQ ID NO. 84), wherein X is absent or leucine.

[00237] In one embodiment of any of the methods for preparing this target peptide sequence, the method further comprises (i) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence, and (ii) conjugating a fatty acid moiety at the E amino group of lysine to form a GLP1 receptor agonist peptide. The His-Aib-Glu-Gly or protected and / or activated derivative thereof peptide may be prepared by any of the methods known in the art or described herein such as recombinant methods, or synthetically. In an additional embodiment, His-Aib-Glu-Gly or protected and / or activated derivative thereof is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof.

[00238] The fatty acid moiety listed herein may be prepared by any of the methods known in the art. Fatty acids moieties listed hereinbelow is incorporated for references, however not limited to these fatty acid moieties. Designation Moiety Moiety A H              ? J                  H              O HYf O                                 ^OH V o Moiety B o n ,NH             JL        _O.              X r                                          ° HO. ,..-L Y 1 o A J Moiety C oVH H      o Moiety D T o \=o E o        \ o           / Z-T       ( \ T-\     * / X—1   z \\ o  '— /  o >° o T Moiety E o °\ / NH JL                 A NH               O       J r                               ° HO. ^NH v          >r O Wherein, the ?atty acid moieties as described above, may be prepared by any of the methods known in the art. For the purpose of the present disclosure, moiety D may be prepared according to the process described in either of WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00239] In some embodiments, the present disclosure relates to a process for the preparation of a GLP1 receptor agonist of following formula: H—N E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G-L .. ■ . „zNH Moiety D wherein the said process comprises: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) from multimer of SEQ ID 2; (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00240] In some embodiments, the present disclosure relates to a process for the preparation of a GLP1 receptor agonist of following formula: H °                            H 9 H-N'Jl—E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-N   K-E-F-l-A-W-L-V-R-G-R-G-L Moiety D comprising: (i) preparing a target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1); (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00241] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) is prepared by a process comprising the steps of: (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site; (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide. liv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00242] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) is prepared from multimer of SEQ ID 2.

[00243] In some embodiments, the present disclosure relates to a GLP1 receptor agonist compound of formula: H  °                            H  2 H— N   IL. E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-N   K-E-F-l-A-W-L-V-R-G-R-G-L Moiety D wherein the compound is obtained by a process comprising the steps of: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) from multimer of SEQ ID 2; (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00244] In some embodiments, the present invention relates to a GLP1 receptor agonist compound of formula: E-F-l-A-W-L-V-R-G-R-G-L H E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-N Moiety D wherein the compound is obtained by a process comprising the steps of: (i) preparing a target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1); (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00245] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1) is prepared by a process comprising the steps of: (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site; (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide. liv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00246] In some embodiments, His-Aib-Glu-Gly or protected and / or activated derivative thereof as used herein, may be prepared by any of the methods known in the art or described herein such as recombinant methods or synthetically. In an additional embodiment, His-Aib-Glu-Gly or protected and / or activated derivative thereof is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. Wherein, the conjugation may be carried out by any known methods known in the art, such as but not limited to: solid phase peptide synthesis, liquid phase peptide synthesis or combination thereof, also known as hybrid approach.

[00247] The fatty acid moiety D, as used according to any one of the embodiments described herein, may be prepared by any of the methods known in the art. For the purpose of the present disclosure, moiety D may be prepared according to the process described in either of WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00248] According to any one of the embodiments described herein, wherein conjugating a fatty acid moiety of formula: Moiety D from hydrogen or a carboxylic acid activating group at the E amino group of lysine of target peptide sequence may carried out according to the process described in the example, by any general standard methods known in the art, such as that disclosed in WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00249] In some embodiments, the present disclosure relates to a process for the preparation of Semaglutide of following formula: E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G ■ * a-NH Moiety A wherein the said process comprises: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) from multimer of SEQ ID 5; (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety A at the E amino group of lysine.

[00250] In some embodiments, the present disclosure relates to a process for the preparation of Semaglutide of following formula: E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G Moiety A ^NH comprising: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4); (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety A °       , wherein R is selected from hydrogen or a carboxylic acid activating group at the E amino group of lysine.

[00251] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) is prepared by a process comprising the steps of: (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site (b) digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide. liv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00252] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) is prepared from multimer of SEQ ID 5.

[00253] In some embodiments, the present disclosure relates to Semaglutide of formula: H E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A—N E-F-l-A-W-L-V-R-G-R-G ■ * a-NH Moiety A wherein Semaglutide is obtained by a process comprising the steps of: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) from multimer of SEQ ID 5; (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety A at the E amino group of lysine.

[00254] In another aspect, the present disclosure relates to Semaglutide of formula: E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G .. ■ x a-NH Moiety A wherein Semaglutide is obtained by a process comprising the steps of: (i) preparing the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4); (ii) conjugating His-Aib-Glu-Gly or protected and / or activated derivative thereof to the target peptide sequence; and (iii) conjugating a fatty acid moiety of formula: Moiety A at the E amino group of lysine.

[00255] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO. 4) is prepared by a process comprising the steps of: (a) a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site (b) digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form. Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide. liv. Or subjecting all three or a combination of any tow enzymes together to achieve the final target peptide in free form. The method further comprises step (c) isolating the peptide. In another embodiment, multimer can be digested with single protease. In another embodiment, the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and / or chemicals in a single step.

[00256] According to any one of the embodiments described herein, His-Aib-Glu-Gly or protected and / or activated derivative thereof as used herein, may be prepared by any of the methods known in the art or described herein such as recombinant methods or synthetically. In some embodiments, His-Aib-Glu-Gly or protected and / or activated derivative thereof is conjugated to the target peptide by any methods known in the art, such as but not limited to: single amino acid coupling, direct fragment coupling or combination thereof. In some embodiments, the conjugation may be carried out by any known methods known in the art, such as but not limited to: solid phase peptide synthesis, liquid phase peptide synthesis or combination thereof, also known as hybrid approach.

[00257] The fatty acid moiety A, as used according to any one of the embodiments described herein, may be prepared by any of the methods known in the art. For the purpose of the present disclosure, moiety D may be prepared according to the process described in either of WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00258] According to any one of the embodiments described herein, wherein conjugating a fatty acid moiety of formula: Moiety A at the E amino group of lysine of target peptide sequence may carried out according to the process described in the example, by any general standard methods known in the art, such as that disclosed in WO2019 / 193576, WO2021 / 260530 or WO2022 / 079639.

[00259] The sequences referenced by sequence number herein are provided in Table A at the end of the specification.

[00260] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following nonlimiting examples. EXAMPLES EXAMPLE 1 PREPARATION OF A MULTIMER

[00261] For producing target peptide 1 (SEQ ID NO: 1) a multimer of target peptide 1 was constructed (SEQ ID NO: 2). The multimer included a methionine and a histidine at the N-terminus followed by repeating units of the target peptide 1 conjugated to a KR tag. The N-terminus of the first iteration of target peptide 1 in the multimer includes a KR tag. The Histidine tag was added to facilitate purification by Nickel column chromatography. A KR site has been added as a tag between iterations of target peptide 1 to generate monomers of target peptide 1 after enzymatic cleavage using Kex2 and CpB protease. This approach increases the efficiency of peptide expression as it eliminates the usage of fusion partner tags. The monomeric target peptide 1 is obtained by cleavage of the multimer of target peptide 1 using Kex2 and CpB proteases.

[00262] To express the multimer (SEQ ID NO:2) of target peptide 1 in E. coli, the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO:3. The nucleotide sequence (SEQ ID NO:3) was cloned in a suitable expression vector with a T7 promoter and transformed inE. coli ToplO cells to generate the expression vector.

[00263] The cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 optical density (OD), induced with isopropyl P-D-l-thiogalactopyranoside (IPTG) and harvested 9 hours post induction. Expression of the multimer (SEQ ID NO:2, molecular weight 34.9 kDa) was confirmed by SDS PAGE analysis as shown in Figure 1.

[00264] The harvested pellet was weighed and resuspended in 20X 8M guanidinium hydrochloride (GuCl) solubilization buffer. The sample was sonicated for 15 minutes and incubated at shaking overnight till the solution became clear. The sample was filtered and processed through a Ni-NTA (nickel nitrilotriacetic acid) column to capture the multimer (SEQ ID NO:2). The captured multimer was eluted and further analyzed for protein expression by SDS PAGE, which confirmed preparation and purification of the multimer as shown in Figure 2.

[00265] After purification of the multimer, the sample is subjected to cleavage by Kex2 and CpB protease to obtain the target peptide 1 monomer (SEQ ID NO:1). Kex2 protease specifically recognizes the amino acid sequence KR and cleaves at the C-terminus of R. CpB protease is an exopeptidase which subsequently removes R and K leaving behind the target peptide 1 (SEQ ID NO:1). EXAMPLE 2 PREPARATION OF A MULTIMER OF TARGET PEPTIDE 2

[00266] For producing target peptide 2 (SEQ ID NO: 4), a multimer comprising target peptide 2 was constructed (SEQ ID NO: 5). The multimer, like in Example 1, included a methionine and a histidine tag at the N-terminus followed by repeating units of the target peptide 2 conjugated to a KR tag. The N-terminus of the first target peptide 2 in the multimer also includes a KR tag. The monomeric target peptide 2 is obtained by cleavage of the multimer of target peptide 2 using Kex2 and CpB proteases.

[00267] A multimer of target peptide 2 was constructed (SEQ ID NO: 5) where monomeric target peptide 2 units were connected by specific amino acid residues. For example, these amino acid residues could be lysine (K) and arginine (R) which are cleavage site for proteases Kex2 and CpB. This approach significantly increases the efficiency of peptide expression as it eliminates the usage of fusion partner tags. The monomeric target peptide 2 is obtained by cleavage of multimer of target peptide 2 using Kex2 and CpB protease.

[00268] To express the multimer (SEQ ID NO: 5) of target peptide 2 in E. colt, the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO:6. The nucleotide sequence (SEQ ID NO:6) was cloned into a suitable expression vector with a T7 promoter and transformed inE. coli ToplO cells to generate expression vector.

[00269] The cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 OD, induced with IPTG and harvested 9 hours post induction. Expression of the multimer SEQ ID NO: 5 (molecular weight 33.8 kDa) was confirmed by SDS PAGE as shown in Figure 3.

[00270] The harvested pellet was weighed and resuspended in 20X 8M GuCl solubilization buffer. Sample was sonicated for 15 minutes and incubated overnight under shaking conditions till the solution became clear. The sample was filtered and processed through a Ni-NTA column for capturing the multimer (SEQ ID NO:5). The captured multimer was eluted and further analyzed for protein expression by SDS PAGE as shown in Figure 4.

[00271] After purification of the multimer, the sample was subjected to cleavage by Kex2 and CpB protease to obtain the target peptide 2 monomer (SEQ ID NO:4). Kex2 protease specifically recognizes the amino acid sequence KR and cleaves at the C-terminus of R. CpB protease is an exopeptidase which subsequently removes R and K leaving behind the target peptide 2 (SEQ ID NO:4). Reverse phase HPLC (RP-HPLC) analysis was performed for confirmation of target peptide 2 monomer as shown in Figure 5. EXAMPLE 3 PREPARATION OF MULTIMER WITH IMPROVED SOLUBILITY

[00272] In Example 2, the multimer (SEQ ID NO: 5) had high expression level but the inclusion bodies formed were difficult to solubilize, requiring 8M GuCl. The addition of 8M GuCl increases the cost of production and is un-favorable for downstream enzymatic processing. The multimer (SEQ ID NO:5) had a pl of 10.28 and hydrophobicity of 37.1% which are unfavorable parameters for solubility. To overcome these issues, specific Tag amino acid sequences SEQ ID NOs:7-ll were incorporated into the multimer resulting in the pl decreasing from 10.28 to 4.49 and the hydrophobicity decreasing from 37.1% to 25.4%. The multimer has been designed in such a way that monomers of target peptide 2 are interspaced with the specific Tag amino acid sequences SEQ ID Nos: 7-11. Protease cleavage sites were added at the N and C terminus of the target peptide 2 to ensure generation of the monomer of target peptide 2 after enzymatic digestion with one or more proteases. The resulting multimer was SEQ ID NO: 12, which had improved solubility.

[00273] The Tag amino acid sequences include aspartic acid (D), serine (S) and threonine (T). Aspartic acid (D) helps in lowering the pl of the multimer. Serine (S) and threonine (T) help in lowering the overall hydrophobicity of the multimer. In order to increase the number of D (aspartic acid) and decrease the overall pl of the multimer, the sequence DDDDK (SEQ ID NO:43) was incorporated into the multimer. DDDDK additionally provides a cleavage site that can be digested with an enterokinase enzyme (EK).

[00274] To express the multimer (SEQ ID NO: 12) in E. coli, the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO: 13. The nucleotide sequence (SEQ ID NO: 13) was cloned in a suitable expression vector with a T7 promoter and transformed into E. coli Top 10 cells to generate the expression vector.

[00275] The cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 OD, induced with IPTG and harvested 9 hours post induction. Expression of the multimer SEQ ID NO: 12, molecular weight 48.5 kDa) was confirmed by SDS PAGE analysis as shown Figure 6.

[00276] Inclusion bodies or cell pellet formed by the multimer SEQ ID NO: 12 were soluble in mild solubilizing agents such as urea as compared to multimer of Example 2 (SEQ ID NO: 5) which was insoluble in 8M Urea and required strong chaotropic agent for solubilization as shown in Figure 7. This modulation of pl and hydrophobicity enabled improved solubility and ease of downstream processing, however multimer expression level is lower as shown in Figure 6. EXAMPLE 4 PREPARATION OF MULTIMER WITH IMPROVED SOLUBILITY AND EXPRESSION

[00277] For producing target peptide 2 (SEQ ID NO: 4), a multimer of target peptide 2 was constructed SEQ ID NO: 20 . As shown in example 3, a multimer (SEQ ID NO: 12) of target peptide 2 with improved solubility (but low expression) had low pl 4.49 and low hydrophobicity of 25.4%. To enhance expression level, SEQ ID NO:20 was designed where the hydrophobicity was kept low (23.77%), however, the pl was increased from 4.49 to 10.3. The hydrophobicity was reduced by incorporating S (Serine) and T (Threonine) amino acids. To increase the pl to 10.3, R (Arginine) was added to the tag sequence. Lower hydrophobicity is required to enhance solubility of the multimer, whereas pl modulation is required for enhanced multimer expression.

[00278] To express the multimer (SEQ ID NO:20) of target peptide 2 in E. coli, the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO:21. The nucleotide sequence (SEQ ID NO:21) was cloned into a suitable expression vector with a T7 promoter and transformed into E. coli Top 10 cells to generate expression vector.

[00279] The cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 OD, induced with IPTG and harvested 9 hours post induction. Expression of the multimer SEQ ID NO:20 (molecular weight 51.3 kDa) was confirmed by SDS PAGE analysis as shown in Figure 8. The expression level was improved to be better than expression level of multimer (SEQ ID NO: 5) as shown in Figure 9.

[00280] The inclusion bodies or cell pellet obtained after flask study of the multimer (SEQ ID NO:20) is easily soluble in urea buffer. The samples were purified using Ni-NTA column where eluates were in 4M urea buffer (mild solubilizing agent). Refer Figure 10. The eluate containing multimer of target peptide 2 is soluble in mild urea concentrations of 2-4M (Refer Figure 11). This improved solubility of the high expressing multimer enable ease of enzymatic digestion and further downstream processing. EXAMPLE 5 PREPARATION OF COMPOUND OF FORMULA: H  9                             H H—N   11—E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A —N E-F-l-A-W-L-V-R-G-R-G-L .. ■ . „zNH Moiety D The preparation involved independent preparation of (i) Moiety D-OSu and (ii) protected tetramer of formula: Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OSu (Intermediate B). Preparation of fatty acid moiety of Moiety D-OSu of following formula: O The fatty acid side chain was prepared using solid phase synthesis using 2-chlorotrityl chloride resin as schematically represented below. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in presence of DIPEA to yield 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc protecting group was removed by selective de-blocking of amino group using piperidine followed by coupling with Fmoc-Aib-OH in THF using DIPC and HOBt which yielded 2-[2-[2-[(2-Fmoc-amino-2-methyl-propanoyl)amino] ethoxy] ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group was removed by selective de-blocking using piperidine and the free amino group was coupled with Fmoc-Glu-OtBu using HOBt and DIPC to yield 2-[2-[2-[[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-2-methyl-propanoyl] amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group of the resultant compound was selectively de-blocked using piperidine and the free amino group was then coupled with octadecanedioic acid mono tert butyl ester using HOBt and DIPC to give 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]- amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The intermediate was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol:DCM (1:1) to obtain 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid (LCMS= m / z: 786.39 (M+H+)). Which on the treatment with DCC in THF / MDC to yield active ester on further treatment with TFA to yield C18-yGlu-Aib-AEEA-OSu. Which on the treatment with n-hydroxysuccinamide and DCC in THF / MDC to yield active ester on further treatment with TFA to yield C18-yGlu-Aib-AEEA-OSu. Preparation of protected and / or activated tetramer of formula Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OH: The protected tetrapeptide was synthesized by solid-phase method. The starting resin used for synthesis was 2-chlorotrityl chloro (2-CTC) resin. Fmoc protected glycine was used for coupling with the 2-chlorotrityl chloro resin. The coupling was performed by using diisopropylethylamine in Dichloromethane solvent which yielded Fmoc-Gly-2-CTC Resin. Methanol was used to terminate uncoupled active sites of resin. Selective de-blocking of amino group of Fmoc-Gly-2-CTC Resin using piperidine followed by coupling with Fmoc-Glu(OtBu)-OH using HOBt and DIPC yielded Fmoc-Glu(OtBu)-Gly-2-CTC Resin. Selective de-blocking of amino group of Fmoc-Glu(OtBu)-Gly-2-CTC Resin using piperidine followed by coupling with Fmoc-Aib-OH using HOBt and DIPC yielded Fmoc-Aib-Glu(OtBu)-Gly-2-CTC Resin. Selective de-blocking of amino group of Fmoc-Aib-Glu(OtBu)-Gly-2-CTC Resin using piperidine followed by coupling with Fmoc-His(Boc)-OH using HOBt and DIPC yielded Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-2-CTC Resin. Deprotection of Fmoc using piperidine followed by protection using Boc anhydride to yield Boc-His(Boc)-Aib-Glu(OtBu)-Gly-2-CTC-resin. Protected tetrapeptide on resin was cleaved from 2-CTC-Resin using trifluoroethanol:DCM (1:1) to obtain Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OH. Preparation of Intermediate B: Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OH was treated with n-hydroxysuccinamide and DCC in THF / DCM to yield Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OSu (Intermediate B). Preparation of Intermediate B’: Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OH was treated with pentafluorophenol(PFP) and DCC in DCM to yield Boc-His(Boc)-Aib-Glu(OtBu)-Gly-PFP (Intermediate B’). Method A for the preparation of compound of formula: H ° H—N   JJ—E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G-L Moiety D

[00281] The fatty acid moiety of Moiety D-OSu was grafted on the Target Peptide 1 (SEQ ID NO: 1) in the presence of water : acetonitrile at pH about 11. The resulted crude peptide fragment was purified by preparative HPLC followed by freeze-drying to yield Intermediate A. H ° T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A—N      E-F-l-A-W-L-V-R-G-R-G-L ■ x n^H Moiety D Intermediate A

[00282] Intermediate A was coupled with Intermediate B in water: acetonitrile at pH about 8, followed by freeze-drying the solution. The resulted protected peptide compound was deprotected using TFA and TIPS to yield desired compound in crude form. The crude was purified using preparative HPLC to yield desired compound in pure form. Method B for the preparation of compound of formula: H O                               H H—N   11—E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A—N E-F-l-A-W-L-V-R-G-R-G-L .. ■ . „zNH Moiety D

[00283] The fatty acid moiety of Moiety D-OSu was grafted on the Target Peptide 1 (SEQ ID NO: 1) in the presence of water : acetonitrile at pH about 11. The resulted crude peptide fragment was purified by preparative HPLC followed by freeze-drying to yield Intermediate A.

[00284] Intermediate A was coupled with Intermediate B’ in NMP at pH of approximately 8 in DIPEA, followed by precipitating the compound using diethyl ether. The resulted protected peptide compound was deprotected using TFA and TIPS to yield desired compound in crude form. The crude was purified using preparative HPLC to yield desired compound in pure form.

[00285] Mass (LCMS): m / z = 1042.60 (MH4 4+), Calculated Mass= 4166.368, HPLC Purity: 93.86%. EXAMPLE 6 PREPARATION OF SEMAGLUTIDE: E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A E-F-l-A-W-L-V-R-G-R-G Moiety A'

[00286] Preparation of fatty acid moiety of Moiety A-OSu of following formula: Moiety A-OSu was prepared according to the same process as described in Example 5 for Moiety D-OSu, wherein 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-[2-(2-Fmoc- aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin, instead of Fmoc-Aib-OH. PREPARATION OF INTERMEDIATE C:

[00287] Target Peptide 2 was suspended in water with added triethylamine to obtain a clear solution, followed by addition of 1.2 molar equivalent of Moiety A-OSu solution in methanol. The reaction mixture was allowed to stir for 30 minutes to Ihour at room temperature. The reaction mixture was lyophilised for 48 to 84 hours to yield the residues (Intermediate C).

[00288] Intermediate C was coupled with Intermediate B in N-methyl pyrrolidone at basic pH. The reaction mixture was added diethyl ether and filtered to get protected peptide. The resulting protected peptide was deprotected using TFA to obtain desired compound in crude form. The crude was purified using preparative HPLC and lyophilization to yield pure Semaglutide. EXAMPLE 7 PREPARATION OF A MULTIMER OF TARGET PEPTIDE 3

[00289] Target peptide 3 (SEQ ID NO: 22) was designed with two additional amino acids at the N-terminus of target peptide 2 to demonstrate that the multimer strategy can be successfully applied to different peptide lengths. For producing target peptide 3 (SEQ ID NO: 22), a multimer (SEQ ID NO: 23) of target peptide 3 (SEQ ID NO: 22) was constructed. The multimer, as done in Examples 1 and 2, included a methionine and a histidine tag at the N-terminus followed by repeating units of the target peptide 3 conjugated to a KR tag. Similar to target peptides 1 and 2, it also includes a KR tag at the N-terminus of the first target peptide 3 in the multimer. The monomeric target peptide 3 is obtained by cleavage of the multimer of target peptide 3 using Kex2 and CpB proteases.

[00290] A multimer (SEQ ID NO: 23) of target peptide 3 (SEQ ID NO: 22) was constructed where monomeric peptide units were connected by specific amino acid residues such as lysine (K) and arginine (R) which are cleavage sites for proteases Kex2 and CpB. Kex2 and CpB proteases were used to cleave the multimer and obtain the monomeric target peptide 3 (SEQ ID NO: 22).

[00291] To express the multimer (SEQ ID NO: 23) of target peptide 3 (SEQ ID NO:22) in E. coli, the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO: 24. The nucleotide sequence (SEQ ID NO: 24) was cloned in a suitable expression vector with a T7 promoter and transformed into E. coli ToplO cells to generate expression vector.

[00292] The cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 OD, induced with IPTG and harvested 9 hours post induction. Expression of the multimer of SEQ ID NO:23 (molecular weight 36.9 kDa) was confirmed by SDS PAGE as seen in Figure 12.

[00293] The harvested pellet was weighed and resuspended in 20X 8M GuCl solubilization buffer. The sample was sonicated for 15 minutes and incubated overnight under shaking conditions till the solution became clear. The sample was filtered and processed through a Ni-NTA column for capturing the multimer (SEQ ID NO: 23). The captured multimer was eluted and further analyzed for protein expression by SDS PAGE as seen in Figure 13.

[00294] After purification of the multimer through Ni-NTA purification, the sample was subjected to cleavage by Kex2 and CpB protease to obtain the target peptide 3 monomer (SEQ ID NO: 22). Kex2 protease specifically recognizes the amino acid sequence KR and cleaves at the C-terminus of R. CpB protease is an exopeptidase which subsequently removes R and K leaving behind the target peptide 3 (SEQ ID NO: 22). Intact mass and sequence analysis was performed for confirmation of target peptide 3 monomer as shown in Figure 14. EXAMPLE 8 PREPARATION OF A MULTIMER OF TARGET PEPTIDE 4

[00295] Target peptide 4 (SEQ ID NO: 25) was designed with two amino acids deleted from the N-terminus of the target peptide 2 to demonstrate that the multimer strategy can be successfully applied to different peptide lengths. For producing target peptide 4 (SEQ ID NO: 25), a multimer of target peptide 4 was constructed (SEQ ID NO: 26). Similar to the multimers mentioned above, it included a methionine and a histidine tag at the N-terminus followed by repeating units of the target peptide 4 conjugated to a KR. Further it includes a KR tag at the N-terminus of the first target peptide 4 in the multimer. The monomeric target peptide 4 is obtained by cleaving the multimer of target peptide 4 using Kex2 and CpB proteases.

[00296] A multimer (SEQ ID NO: 26) of target peptide 4 (SEQ ID NO: 25) was constructed where monomeric peptide units were connected by specific amino acid residues like lysine (K) and arginine (R) which are cleavage site for proteases Kex2 and CpB. Kex2 and CpB protease were used to cleave the multimer and obtain the monomeric target peptide 4 (SEQ ID NO: 25).

[00297] To express the multimer (SEQ ID NO: 26) of target peptide 4 (SEQ ID NO: 25) in E. coli, the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO: 27. The nucleotide sequence (SEQ ID NO: 27) was cloned into a suitable expression vector with a T7 promoter and transformed in E. coli ToplO cells to generate expression vector.

[00298] The cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 OD, induced with IPTG and harvested 9 hours post induction. Expression of the multimer SEQ ID NO: 26 (molecular weight 32.4 kDa) was confirmed by SDS PAGE as shown in Figure 15.

[00299] The harvested pellet was weighed and resuspended in 20X 8M GuCl solubilization buffer. The sample was sonicated for 15 minutes and incubated overnight under shaking conditions till the solution became clear. The sample was filtered and processed through a Ni-NTA column for capturing the multimer (SEQ ID NO: 26). The captured multimer was eluted and further analyzed for protein expression by SDS PAGE as shown in Figure 16.

[00300] After purification through Ni-NTA purification of the multimer, the sample was subjected to cleavage by Kex2 and CpB protease to obtain the target peptide 4 monomer (SEQ ID NO: 25). Kex2 protease specifically recognizes the amino acid sequence KR and cleaves at the C-terminus of R. CpB protease is an exopeptidase which subsequently removes R and K leaving behind the target peptide 4 (SEQ ID NO: 25). Intact mass and sequence analysis was performed for confirmation of target peptide 3 monomer as shown in Figure 17. Table A Description SEQID NO: Sequence Target Peptide 1 in Example 1 1 TFTSDVSSYLEGQAAKEFIAWLVRGRGL Multimer of Target Peptide 1 in Example 1 2 MHHHHHHKRTFTSDVSSYLEGQAAKEFIAWLVRGRGLKRTFTS DVSSYLEGQAAKEFIAWLVRGRGLKRTFTSDVSSYLEGQAAKE FIAWLVRGRGLKRTFTSDVSSYLEGQAAKEFIAWLVRGRGLKR TFTSDVSSYLEGQAAKEFIAWLVRGRGLKRTFTSDVSSYLEGQ AAKEFIAWLVRGRGLKRTFTSDVSSYLEGQAAKEFIAWLVRGR GLKRTFTSDVSSYLEGQAAKEFIAWLVRGRGLKRTFTSDVSSY LEGQAAKEFIAWLVRGRGLKRTFTSDVSSYLEGQAAKEFIAWL VRGRGLKR Nucleotide sequence for Multimer of Example 1 3 ATGCATCATCACCATCATCATAAACGCACCTTTACCTCAGATG TTTCAAGCTATCTGGAAGGTCAGGCAGCCAAAGAATTTATTGC ATGGCTGGTTCGTGGTCGTGGTCTGAAACGTACATTTACCAGT GATGTTAGCAGTTACCTGGAAGGCCAAGCCGCAAAAGAGTTTA TCGCCTGGTTAGTGCGTGGTCGCGGTTTAAAAAGAACCTTTAC CAGCGACGTGAGTAGCTATTTAGAAGGACAAGCTGCGAAAGAA TTCATAGCGTGGTTAGTACGCGGACGCGGACTGAAACGCACGT TCACATCTGATGTTAGTTCATATTTAGAGGGCCAAGCGGCTAA agaatttatcgcgtggcttgtcagaggccgtggtttaaaacgt ACCTTCACATCAGACGTGTCATCATATCTTGAGGGTCAAGCAG CAAAAGAATTCATAGCCTGGCTGGTGAGAGGTAGAGGCCTGAA GCGTACTTTCACGTCAGATGTAAGCTCTTATCTTGAAGGCCAG GCTGCTAAAGAGTTCATTGCTTGGCTTGTGCGTGGAAGAGGCT TAAAACGGACGTTTACAAGTGACGTGTCTAGCTACCTTGAAGG GCAAGCAGCGAAAGAGTTCATTGCCTGGTTGGTACGTGGCCGT GGCCTTAAGCGCACCTTCACAAGTGATGTGTCCAGTTACTTAG AGGGACAAGCAGCCAAAGAGTTTATCGCTTGGCTTGTACGCGG TAGAGGTCTGAAAAGAACGTTCACGAGTGACGTTAGCTCATAC TTAGAAGGCCAGGCAGCAAAAGAATTCATTGCGTGGCTTGTTC GAGGACGTGGGCTGAAGCGGACCTTCACTTCTGATGTCAGTAG CTATTTGGAGGGGCAAGCCGCAAAAGAATTTATTGCGTGGTTG GTAAGAGGTAGAGGTCTTAAACGTTAATGA Target Peptide 2 in Example 2 4 TFTSDVSSYLEGQAAKEFIAWLVRGRG Multimer of Target Peptide 2 in Example 2 5 MHHHHHHKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRTFTSD VS SYLEGQAAKE FIAWLVRGRGKRTFTSDVS S YLEGQAAKE FI AWLVRGRGKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRTFTS DVSSYLEGQAAKEFIAWLVRGRGKRTFTSDVSSYLEGQAAKEF IAWLVRGRGKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRTFT SDVSSYLEGQAAKEFIAWLVRGRGKRTFTSDVSSYLEGQAAKE FIAWLVRGRGKRTFTSDVS S YLEGQAAKE FIAWLVRGRGKR Nucleotide sequence for Multimer of Example 2 6 ATGCATCATCACCATCATCATAAACGCACCTTTACCTCAGATG TTTCAAGCTATCTGGAAGGTCAGGCAGCCAAAGAATTTATTGC ATGGCTGGTTCGTGGTCGTGGTAAACGTACATTTACAAGTGAT GTTAGCAGTTACCTGGAAGGCCAAGCCGCAAAAGAGTTTATCG CCTGGTTAGTGCGTGGCCGTGGCAAAAGAACCTTTACCAGCGA CGTTAGCAGCTATTTAGAAGGACAAGCTGCGAAAGAATTTATA GCGTGGTTGGTACGCGGACGCGGTAAGCGTACCTTCACATCTG ATGTTAGTTCATATTTAGAGGGCCAAGCGGCAAAAGAATTTAT TGCGTGGCTTGTGAGAGGCCGTGGAAAACGCACGTTCACCAGT GATGTGTCATCATATCTTGAGGGTCAAGCAGCGAAAGAGTTCA TAGCCTGGCTTGTCCGTGGTAGAGGCAAACGGACGTTTACATC CGATGTGAGTAGTTACTTGGAGGGACAAGCAGCTAAAGAATTT ATCGCATGGTTAGTTAGAGGTCGCGGTAAAAGAACATTCACGT CAGATGTAAGCTCTTATCTTGAAGGCCAGGCTGCAAAAGAATT TATAGCCTGGCTGGTGCGTGGACGGGGAAAGCGCACTTTTACA TCAGACGTTAGCTCGTACTTAGAAGGGCAAGCAGCAAAAGAAT TTATCGCTTGGCTGGTAAGAGGAAGAGGAAAGCGGACCTTTAC GAGTGACGTGAGTTCATACCTTGAAGGACAGGCAGCGAAAGAG tttattgcttggttagttcgcggtagaggaaaacgaaccttca CTTCGGATGTGAGCAGCTACTTAGAAGGTCAGGCAGCGAAAGA ATTTATCGCGTGGCTTGTCCGTGGTCGCGGTAAACGTTAATAA Tag + CS in Example 3 to lower pl and / or hydrophobicity of multimer, where Tag is DSSTTDSSTT and CS is DDDDK 7 DSSTTDSSTTDDDDK Tag + CS in Example 3 to lower pl and / or hydrophobicity of multimer where Tag is DSSDTTD and CS is DDDDK 8 DSSDTTDDDDDK Tag + CS in Example 3 to lower pl and / or hydrophobicity of multimer where Tag is DSSTTDSSTTD and CS is DDDDK 9 DSSTTDSSTTDDDDDK Tag in Example 3 to lower pl and / or 10 DSSTTDSSTTSSTT hydrophobicity of multimer, Tag + CS in Example 3 to lower pl and / or hydrophobicity of multimer, where Tag is SSTT and CS is DDDDK 11 SSTTDDDDK Multimer of Target Peptide 2 in Example 3 12 MHHHHHHKRDSSTTDSSTTDDDDKTFTSDVSSYLEGQAAKEFI AWLVRGRGKRDSSTTDSSTTDDDDDKTFTSDVSSYLEGQAAKE FIAWLVRGRGKRDSSDTTDDDDDKTFTSDVSSYLEGQAAKEFI AWLVRGRGKRDSSDTTDDDDDKTFTSDVSSYLEGQAAKEFIAW LVRGRGKRDSSDTTDDDDDKTFTSDVSSYLEGQAAKEFIAWLV RGRGKRSSTTDDDDKTFTSDVSSYLEGQAAKEFIAWLVRGRGK RDSSDTTDDDDDKTFTSDVSSYLEGQAAKEFIAWLVRGRGKRD SSDTTDDDDDKTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDSS DTTDDDDDKTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDSSDT TDDDDDKTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDSSTTDS STTSSTT Nucleotide sequence for Multimer of Example 3 13 ATGCATCATCACCATCATCATAAACGTGATAGCAGCACCACCG ATAGCTCAACCACCGATGATGATGATAAAACCTTTACCAGTGA TGTGAGCAGCTATCTGGAAGGTCAGGCAGCAAAAGAATTTATT GCATGGCTGGTTCGTGGTCGTGGTAAACGCGATTCAAGTACCA CCGATTCTAGCACAACGGATGATGACGATGACAAAACATTCAC CTCAGATGTTAGCTCATACCTGGAAGGCCAAGCTGCCAAAGAG TTTATCGCCTGGTTAGTGCGTGGCCGTGGCAAACGTGACAGCT CAGATACGACCGATGACGACGACGATAAGACGTTTACATCTGA TGTTTCCAGCTATTTAGAAGGGCAAGCTGCGAAAGAATTTATA GCGTGGCTTGTACGCGGACGCGGAAAACGTGATTCTAGCGATA CTACCGACGACGATGATGACAAGACTTTTACGTCCGATGTTAG TAGTTATCTTGAAGGACAAGCAGCCAAAGAATTTATTGCGTGG TTAGTCAGAGGTCGGGGTAAGCGTGATAGTAGCGATACGACAG ATGATGATGACGACAAGACCTTCACCAGCGACGTTTCAAGTTA CTTAGAGGGTCAAGCCGCTAAAGAGTTCATTGCTTGGTTGGTC CGTGGAAGAGGTAAACGTAGCAGTACCACAGACGATGACGACA AAACATTTACAAGTGACGTTTCTAGTTATTTGGAAGGCCAAGC CGCAAAAGAATTTATCGCTTGGTTAGTTCGCGGAAGAGGCAAG CGTGATTCAAGTGATACCACTGATGATGACGACGATAAAACTT TTACGAGCGACGTTAGTTCATATTTGGAGGGTCAAGCAGCTAA agaatttatcgcctggctggtaagaggcagaggtaagcgcgac TCATCAGACACCACAGATGACGATGATGATAAGACATTCACAT CAGACGTGTCCTCATACTTAGAAGGCCAGGCAGCCAAAGAATT TATCGCGTGGCTTGTGCGTGGTAGAGGCAAACGCGATAGTTCT GACACTACCGACGATGACGACGACAAAACCTTCACAAGTGATG TTAGTTCTTACTTGGAAGGACAGGCTGCTAAAGAGTTCATAGC CTGGCTGGTTCGCGGACGTGGGAAGCGTGATAGTTCTGATACC ACGGACGACGATGACGATAAAACCTTCACTTCTGATGTGTCAA GTTACCTTGAAGGTCAAGCGGCAAAAGAGTTCATTGCCTGGCT TGTGCGTGGTCGCGGTAAGCGTGATTCATCAACCACCGATAGC AGCACAACCAGCAGCACCACATAA Tag in Example 4 to lower hydrophobicity and to maintain pl of multimer 14 SDSTTRSDSTTSDSTTRSDSTTSDSTTR Tag in Example 4 to lower hydrophobicity and to maintain pl of multimer 15 SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR Tag in Example 4 to lower hydrophobicity and to maintain pl of multimer 16 SDSTTSDSTTRSSTTSSTTRSSTTR Tag in Example 4 to lower hydrophobicity and to maintain pl of multimer 17 SDSTTSSTTSSTTSSTTSSTT Tag in Example 4 to lower hydrophobicity and to maintain pl of multimer 18 SSTTSDSTTRSDSTTSSTTRSDSTTR Tag in Example 4 to lower hydrophobicity and to maintain pl of multimer 19 SDSTTSDSTTRSDSTTSDSTTSDSTTR Multimer of Target Peptide 2 in Example 4 20 MHHHHHHKRSDSTTRSDSTTSDSTTRSDSTTSDSTTRKRTFTS DVSSYLEGQAAKEFIAWLVRGRGKRSDSTTSSTTRSDSTTSDS TTRSDSTTSSTTRKRTFTSDVSSYLEGQAAKEFIAWLVRGRGK RSDSTTSDSTTRSSTTSSTTRSSTTRKRTFTSDVSSYLEGQAA KEFIAWLVRGRGKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKR TFTSDVSSYLEGQAAKEFIAWLVRGRGKRTFTSDVSSYLEGQA AKEFIAWLVRGRGKRTFTSDVSSYLEGQAAKEFIAWLVRGRGK RTFTSDVSSYLEGQAAKEFIAWLVRGRGKRSDSTTSSTTSSTT SSTTSSTTKRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRSSTT SDSTTRSDSTTSSTTRSDSTTRKRTFTSDVSSYLEGQAAKEFI AWLVRGRGKRSDSTTSDSTTRSDSTTSDSTTSDSTTR Nucleotide sequence for Multimer of Example 4 21 ATGCATCATCACCATCATCATAAACGTAGCGATAGCACAACCC GTAGCGATTCAACCACCAGTGATAGTACCACACGTTCAGATAG CACCACCTCAGATAGTACGACCCGTAAACGTACCTTTACCAGT GATGTTAGCAGCTATCTGGAAGGTCAGGCAGCAAAAGAATTTA TTGCATGGCTGGTTCGTGGTCGTGGTAAACGCAGTGATTCTAC CACCTCAAGCACCACACGCTCTGATAGTACAACCAGTGATTCA ACAACCCGTTCAGATTCTACAACCAGCTCAACCACACGCAAAC GCACATTTACCTCAGATGTTAGTTCATACCTGGAAGGCCAAGC TGCCAAAGAGTTTATCGCCTGGTTAGTGCGTGGCCGTGGCAAA CGTTCTGATTCAACGACCTCTGATTCCACCACACGTAGCAGCA CCACCAGTAGCACTACCCGTTCAAGTACAACGCGCAAGCGCAC GTTTACAAGCGACGTTTCATCATATTTAGAAGGGCAAGCAGCG AAAGAGTTCATAGCTTGGCTGGTACGCGGACGCGGAAAAAGAA CCTTCACATCAGATGTTTCATCCTATCTTGAGGGTCAAGCTGC GAAAGAATTTATAGCGTGGCTTGTGAGAGGTCGCGGAAAACGT ACATTCACCAGCGACGTGTCAAGTTATCTTGAAGGACAAGCAG CCAAAGAGTTCATTGCCTGGCTTGTCAGAGGCCGTGGGAAACG CACTTTCACAAGTGATGTGTCCAGCTACTTAGAGGGCCAAGCC GCTAAAGAATTTATTGCTTGGTTAGTAAGAGGCCGTGGTAAGC GTACATTTACATCCGATGTGAGTAGTTACTTGGAGGGACAAGC GGCAAAAGAGTTTATAGCCTGGCTTGTTCGCGGTAGAGGTAAA AGAACATTTACGTCTGATGTCTCAAGCTACCTTGAAGGCCAGG CTGCTAAAGAATTTATCGCGTGGTTGGTTCGCGGAAGAGGCAA ACGCTCCGATAGCACGACCAGCAGCACAACCTCATCAACCACA AGCTCAACAACCAGCAGTACCACCAAACGGACGTTTACGTCAG ACGTGTCCTCTTACTTAGAAGGCCAGGCAGCCAAAGAATTTAT AGCCTGGTTAGTACGCGGTCGGGGAAAACGCAGTAGCACCACA AGCGACAGTACTACCCGCAGCGACAGCACCACGAGCAGTACAA CACGTAGTGACTCTACAACTCGCAAACGGACCTTCACGAGTGA CGTGAGCAGTTATTTAGAAGGTCAAGCCGCAAAAGAATTTATC GCTTGGCTGGTAAGAGGTAGAGGTAAACGTTCTGATAGCACAA CAAGCGATAGCACCACTCGCTCAGATTCTACCACATCTGATTC AACCACATCCGATAGTACAACGCGGTAATAA Target Peptide 3 in Example 7 22 EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL Multimer of Target Peptide 3 in Example 7 23 MHHHHHHKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGLKREG TFTSDVSSYLEGQAAKEFIAWLVRGRGLKREGTFTSDVSSYLE GQAAKEFIAWLVRGRGLKREGTFTSDVSSYLEGQAAKEFIAWL VRGRGLKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGLKREGT FTSDVSSYLEGQAAKEFIAWLVRGRGLKREGTFTSDVSSYLEG QAAKEFIAWLVRGRGLKREGTFTSDVSSYLEGQAAKEFIAWLV RGRGLKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGLKREGTF TSDVSSYLEGQAAKEFIAWLVRGRGLKR Nucleotide sequence for Multimer of Example 7 24 ATGCATCATCACCATCATCATAAACGTGAAGGCACCTTTACCA GTGATGTTAGCAGCTATCTGGAAGGTCAGGCAGCAAAAGAATT TATTGCATGGCTGGTTCGTGGTCGTGGTCTGAAACGCGAAGGT ACATTTACCTCAGATGTTTCCAGTTACCTGGAAGGCCAAGCTG CCAAAGAGTTTATCGCCTGGTTAGTGCGTGGTCGCGGTTTAAA ACGTGAGGGTACGTTTACATCTGATGTGAGTAGCTATTTAGAA GGGCAAGCTGCGAAAGAATTTATAGCGTGGCTTGTACGCGGAC GCGGACTGAAGCGCGAGGGCACCTTCACGTCAGATGTTAGTTC ATATCTTGAAGGACAAGCAGCCAAAGAATTTATTGCGTGGTTA GTCAGAGGCCGTGGCCTGAAGAGAGAAGGTACGTTCACAAGTG ATGTGTCAAGTTACTTAGAGGGTCAAGCCGCTAAAGAGTTCAT TGCTTGGTTGGTAAGAGGTAGAGGCCTGAAGCGTGAGGGAACA TTTACGAGCGACGTTTCATCATACTTAGAAGGCCAAGCCGCAA AAGAATTTATAGCATGGTTAGTCCGTGGAAGAGGTCTTAAGCG CGAAGGCACGTTTACAAGCGACGTGTCTAGCTACCTTGAAGGC CAGGCTGCTAAAGAATTTATCGCTTGGTTGGTACGCGGTCGTG GCTTAAAAAGAGAAGGGACTTTTACATCAGACGTAAGCTCATA TTTGGAGGGCCAAGCAGCTAAAGAATTTATAGCCTGGTTGGTG CGTGGCCGTGGCCTTAAACGAGAGGGAACCTTTACTTCCGATG TTTCCTCATACCTTGAGGGTCAAGCGGCAAAAGAATTTATCGC CTGGCTTGTCAGAGGTCGGGGATTAAAGAGAGAGGGCACATTC ACCTCTGACGTTTCGTCATATTTGGAAGGACAGGCAGCGAAAG AGTTCATAGCCTGGCTGGTTCGAGGAAGAGGACTGAAACGTTA ATAA Target Peptide 4 in Example 8 25 TS DVS SYLEGQAAKEFIAWLVRGRGL Multimer of Target Peptide 4 in Example 8 26 MHHHHHHKRTSDVSSYLEGQAAKEFIAWLVRGRGLKRTSDVSS YLEGQAAKEFIAWLVRGRGLKRTSDVSSYLEGQAAKEFIAWLV RGRGLKRTSDVSSYLEGQAAKEFIAWLVRGRGLKRTSDVSSYL EGQAAKEFIAWLVRGRGLKRTSDVSSYLEGQAAKEFIAWLVRG RGLKRTSDVSSYLEGQAAKEFIAWLVRGRGLKRTSDVSSYLEG QAAKE FIAWLVRGRGLKRTS DVS SYLEGQAAKEFIAWLVRGRG LKRTSDVSSYLEGQAAKEFIAWLVRGRGLKR Nucleotide sequence for Multimer of Example 8 27 ATGCACCACCACCACCACCATAAACGTACTTCCGATGTAAGCT CTTACCTGGAAGGTCAGGCTGCAAAAGAATTTATCGCTTGGCT GGTTCGCGGCCGTGGTCTGAAACGTACCAGCGACGTGAGCAGC TACCTGGAAGGCCAAGCGGCAAAAGAATTCATCGCATGGCTGG TTCGTGGTCGTGGTCTGAAACGCACCTCTGATGTGTCCTCCTA TCTGGAGGGTCAGGCGGCTAAAGAGTTCATCGCATGGCTGGTG CGTGGTCGCGGTCTGAAACGTACCTCCGATGTTAGCTCTTACC TGGAAGGCCAGGCTGCCAAAGAATTCATCGCGTGGCTGGTCCG TGGTCGTGGCCTGAAACGCACGAGCGACGTCAGCAGCTATCTG GAAGGCCAGGCGGCGAAAGAGTTCATTGCTTGGCTGGTTCGCG GTCGCGGCCTGAAGCGTACCAGCGATGTGAGCTCTTATCTGGA AGGTCAGGCGGCCAAGGAATTCATCGCTTGGCTGGTACGTGGC CGTGGTCTGAAGCGTACCTCCGACGTTTCTAGCTATCTGGAGG GCCAGGCCGCGAAAGAATTTATTGCTTGGCTGGTCCGTGGCCG CGGTCTGAAACGTACGAGCGACGTCTCCAGCTACCTGGAAGGT CAGGCAGCAAAAGAATTTATTGCCTGGCTGGTTCGTGGCCGCG GCCTGAAACGCACCTCTGACGTTTCCTCCTACCTGGAGGGCCA GGCAGCCAAAGAGTTTATCGCTTGGCTGGTGCGTGGCCGTGGT CTGAAACGCACGTCTGACGTATCTTCTTATCTGGAAGGCCAGG CTGCGAAAGAATTTATCGCGTGGCTGGTACGCGGTCGTGGTCT GAAACGTTAATAA CS + Tag amino acid Sequence + CS, where Tag is DSSTTDSSTTD and CS is KR and DDDDK 28 KRDSSTTDSSTTDDDDDK CS + Tag amino acid Sequence + CS, where Tag is DSSDTTD and CS is KR and DDDDK 29 KRDSSDTTDDDDDK CS + Tag amino acid Sequence + CS, where Tag is SSTT and CS is KR and DDDDK 30 KRSSTTDDDDK CS + Tag amino acid Sequence , where Tag is DSSTTDSSTTSSTT and CS is KR 31 KRDSSTTDSSTTSSTT CS + Tag amino acid Sequence + CS, where Tag is DSSTTDSSTT and CS is KR and DDDDK 32 KRDSSTTDSSTTDDDDK CS + Tag amino acid Sequence + CS, where Tag is SSTTRSSTTSSTTRS STTSSTTR and CS is KR and DDDDK 33 KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK CS + Tag amino acid Sequence + CS, where Tag is SSTTSSTTRSSTTSS TTRSSTTSSTTR and CS is KR and DDDDK 34 KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK CS + Tag amino acid Sequence + CS, where Tag is SSTTSSTTRSSTTSS TTRSSTTR and CS is KR and DDDDK 35 KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK CS + Tag amino acid Sequence + CS, where Tag is 36 KRSSTTSSTTSSTTSSTTSSTTDDDDK SSTTSSTTSSTTSST TSSTT and CS is KR and DDDDK CS + Tag amino acid Sequence , where Tag is SSTTSSTTRSSTTSS TTSSTTR and CS is KR 37 KRSSTTSSTTRSSTTSSTTSSTTR Tag amino acid Sequence + CS where Tag is SSTTRSSTTSSTTRS STTSSTTR and CS is DDDDK 38 SSTTRSSTTSSTTRSSTTSSTTRDDDDK Tag amino acid Sequence + CS where Tag is SSTTSSTTRSSTTSS TTRSSTTSSTTR and CS is DDDDK 39 SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK Tag amino acid Sequence + CS where Tag is SSTTSSTTRSSTTSS TTRSSTTR and CS is DDDDK 40 SSTTSSTTRSSTTSSTTRSSTTRDDDDK Tag amino acid Sequence + CS where Tag is SSTTSSTTSSTTSST TSSTT and CS is DDDDK 41 SSTTSSTTSSTTSSTTSSTTDDDDK Met-His Tag. 42 MHHHHHH Cleavage Site Sequence 43 DDDDK Target Peptide Sequence 44 EGTFTSDVSSYLEGQAAKEFIAWLVRGRG Target Peptide Sequence 45 GTFTSDVSSYLEGQAAKEFIAWLVRGRG Target Peptide Sequence 46 FTSDVSSYLEGQAAKEFIAWLVRGRG Target Peptide Sequence 47 TSDVSSYLEGQAAKEFIAWLVRGRG Target Peptide Sequence 48 SDVSSYLEGQAAKEFIAWLVRGRG Target Peptide Sequence 49 DVS S YLEGQAAKE FIAWLVRGRG Target Peptide Sequence 50 VSSYLEGQAAKEFIAWLVRGRG Target Peptide Sequence 51 S SYLEGQAAKE FIAWLVRGRG Target Peptide Sequence 52 SYLEGQAAKEFIAWLVRGRG Target Peptide Sequence 53 YLEGQAAKE FIAWLVRGRG Target Peptide Sequence 54 LEGQAAKEFIAWLVRGRG Target Peptide Sequence 55 EGQAAKEFIAWLVRGRG Target Peptide Sequence 56 GQAAKE FIAWLVRGRG Target Peptide Sequence 57 QAAKEFIAWLVRGRG Target Peptide Sequence 58 AAKEFIAWLVRGRG Target Peptide Sequence 59 AKEFIAWLVRGRG Target Peptide Sequence 60 KEFIAWLVRGRG Target Peptide Sequence 61 EFIAWLVRGRG Target Peptide Sequence 62 FIAWLVRGRG Target Peptide Sequence 63 IAWLVRGRGL Target Peptide Sequence 64 HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG Target Peptide Sequence 65 HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGL Target Peptide Sequence 66 AEGTFTSDVSSYLEGQAAKEFIAWLVRGRGL Target Peptide Sequence 67 GTFTSDVSSYLEGQAAKEFIAWLVRGRGL Target Peptide Sequence 68 FTSDVSSYLEGQAAKEFIAWLVRGRGL Target Peptide Sequence 69 SDVSSYLEGQAAKEFIAWLVRGRGL Target Peptide Sequence 70 DVS S YLEGQAAKE FIAWLVRGRGL Target Peptide Sequence 71 VSSYLEGQAAKEFIAWLVRGRGL Target Peptide Sequence 72 SSYLEGQAAKEFIAWLVRGRGL Target Peptide Sequence 73 SYLEGQAAKEFIAWLVRGRGL Target Peptide Sequence 74 YLEGQAAKE FIAWLVRGRGL Target Peptide Sequence 75 LEGQAAKEFIAWLVRGRGL Target Peptide Sequence 76 EGQAAKEFIAWLVRGRGL Target Peptide Sequence 77 GQAAKE FIAWLVRGRGL Target Peptide Sequence 78 QAAKEFIAWLVRGRGL Target Peptide Sequence 79 AAKEFIAWLVRGRGL Target Peptide Sequence 80 AKEFIAWLVRGRGL Target Peptide Sequence 81 KEFIAWLVRGRGL Target Peptide Sequence 82 EFIAWLVRGRGL Target Peptide Sequence 83 FIAWLVRGRGL Target Peptide Sequence 84 TFTSDVSSYLEGQAAKEFIAWLVRGRGX

[00301] All publications, patents and patent applications cited herein are hereby incorporated by reference as if set forth in their entirety herein. While this invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to the description. It is therefore intended that the appended claims encompass such modifications and enhancements.

Claims

1. High expression of the target peptide (TP) by constructing multiple repeats of the TP along with flanking amino acids that constitute a protease / chemical cleavage site (CS) depicted as CS-TP-CS and referred to as Monomer (MO).i. Each MO has the formula -MO = (CS-TP-CS) and / orMO = (Tag-CS-TP-CS) and / orMO = (CS-TP-CS-Tag),where Tag is a sequence of amino acids to modulate pl and / or hydrophobicity of MO and MU.ii. Multiple units of MO are arranged in tandem to make a Multimer (MU), which is represented as MU = (MO)n, the plurality of MO is defined by n which is an integer ranging from 2-50, preferably 2-20 and more precisely 10.iii. If TP does not have methionine at the N-terminus, the MU will have Met at the N-terminus, represented as MU = Met - (MO)n,iv. To modulate the hydrophobicity and / or pl of the TP or the MU, additional amino acids are added, referred as a Tag,v. The Tag is not positioned between TP and CS,vi. The number of Tag moieties to be included will depend on the degree of change in the pl and / or hydrophobicity desired for the MU,vii. The choice of amino acids used in the tags shall be based on whether the expression of the MU is desired in the soluble form or as insoluble inclusion bodies,viii. The choice of the CS between the TP sequences shall be selected such that similar sites are not present within the TP.ix. The choice of the CS should be such that upon cleavage, by chemicals or one or more proteases, shall leave no additional amino acids at either the N or the C terminus of the TP.x. The multimer of any one of the preceding claims, wherein the target peptide (TP) sequence is exemplified, but not restricted to the following sequences -TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 1), EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 22), GTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 67), FTSDVSSYLEGQAAKEFIAWLVRGRGL tSEO ID NO: 68), TSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 25), SDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 69), DVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 70), VSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 71), SSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 72), SYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 73), ■ YLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 74), LEGQAAKEFIAWLVRGRGL (SEQ ID NO: 75), EGQAAKEFIAWLVRGRGL (SEQ ID NO: 76), GQAAKEFIAWLVRGRGL (SEQ ID NO: 77), QAAKEFIAWLVRGRGL (SEQ ID NO: 78), AAKEFIAWLVRGRGL (SEQ ID NO: 79), AKEFIAWLVRGRGL (SEQ ID NO: 80), KEFIAWLVRGRGL (SEQ ID NO: 81),xi. Conjugating any of the TP sequences as shown in claim 1 (x) with a fatty acid moiety to give an intermediate product.

2. A Multimer (MU) comprising a plurality of target peptide (TP) sequences connected in series, wherein -i. each target peptide sequence is a GLP-1 analogue or a fragment thereof,ii. every two adjacent TP sequences are flanked on the N and C terminus by a cleavage site (CS), which is capable of being cleaved by one or more chemicals or proteasesto form the TP, and shall leave no additional amino acids at either the N or the C terminus of the TP.iii. the amino acid sequence that make up the CS site shall not be present within the TP,iv. additional amino acid sequence (referred as Tag) may or may not be present, to modulate the pl and hydrophobicity of the Multimer (MU) as well as Monomer (MO),v. the number of Tag moieties to be included will depend on the degree of change desired in the pl and / or hydrophobicity of the MU and TP,vi. the Tag moieties will not be positioned between TP and CS sites,vii. the first target peptide sequence at the N-terminal of MU is preceded by methionine in the absence of a methionine at the N-terminal of TP.viii. Additional amino acid sequence (such as poly-histidine) to facilitate purification of the MU, may or may not be added at the N and / or C-terminus of the MU.

3. The MU in any of the preceding claims, wherein one or more of the Tag amino acid sequences comprise one or more aspartic acid (D), lysine (K) and arginine (R) residues.

4. Tag sequence in any of the preceding claims comprises of one or more of aspartic acid (D), lysine (K), arginine (R), glutamic acid (E), and histidine (H) amino acid in any order to modulate the pl of the MO and / or MU.

5. Tag sequence in any of the preceding claims comprises of one or more serine (S), threonine (T), glutamine (Q), leucine (L) and isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F), tyrosine (Y), cysteine (C), and methionine (M) residues to modulate the hydrophobicity of the MO and / or MU.

6. The MU in any one of the preceding claims, wherein one or more of the CS comprise of the amino acid KR wherein the R of the KR sequence is directly bound to the N-terminus of the TP while the K of the KR sequence is directly bound to the C-terminus of one of the preceding TP. The MU in any of the preceding claims, wherein one or more of the CS comprise of the amino acid DDDDK (SEQ ID NO: 53) where the K ofthe DDDDK sequence is directly bound to the N-terminus of the TP. The MU in any of the preceding claims, wherein the multimer includes one or more Tag amino acid sequences to alter the hydrophobicity, pl, or both of the multimer or a cleaved product of the multimer.

7. The MU in any of the preceding claims has a pl ranging from about 3 to 12, dependingon the desired pl of the MU.

8. The MU in any of the preceding claims has a hydrophobicity ranging from 20% to 50% depending on the desired hydrophobicity of the MU9. The MU in any of the preceding claims, wherein each Tag amino acid sequence between two target peptides is the same or different.

10. The MU in any of the preceding claims, wherein the multimer includes 2 to 50 copies of a target peptide sequence.

11. The MU in any of the preceding claims include 2 to 20 copies of the TP, more specifically 10 copies of the TP sequence.

12. The MU in any of the preceding claims, wherein the MU has a molecular weight of from about 5 to about 200 kilodaltons, more precisely from about 30 to about 60 kilodaltons.

13. The MU in any one of the preceding claims is designed using a DNA construct comprising a nucleic acid sequence encoding the MU as described below, but not restricted to the following steps -i. The nucleic acid sequence is inserted in to an expression vector / plasmid DNA.ii. The expression vector used contains a T7 promoter.iii. The plasmid DNA is transformed in to a host cell (e.g. E. coli) to express the desired MU, wherein the E. coli strain used is BL21, BL21(DE3), ToplO, or DH5a cells.iv. Optimize the growth conditions to maximize production of the MU either in the soluble or insoluble form.v. The MU is extracted from the cells either in the insoluble form (as inclusion bodies) or in the soluble form,vi. The insoluble form of MU (inclusion bodies) are extracted from cell lysate and then solubilized using Urea and GuCl in to a soluble form,vii. The soluble form of MU bearing the affinity tag as described in claim 2 is extracted by loading the cell lysate on an affinity column via the affinity tag,14. The digestion of the MU as described in claim 13 is done using Kex2 and / or enterokinase followed by carboxypeptidase B.

15. The MU as described in claim 13 can also be digested using Kex2 followed by carboxypeptidase B.

16. The digestion as described in claim 14 and claim 15 when taken to completion will result in TP.

17. The resulting TP from the preceding claim (claim 16) may or may not be further subjected to further purification by any of the standard purification methods, such as one or more chromatography steps or solvent fractionation.

18. The purified TP as described in claim 17 may or may not be conjugated to a second peptide to form a third peptide and, optionally, conjugating one or more moieties to the third peptide.

19. The method of any one of claims 13-18, further comprising converting the TP sequence to a GLP-1 analogue.

20. The method of claim 19, the method comprises converting the TP sequence to semaglutide.

21. The method of claim 19, the method comprises converting the TP sequence to liraglutide.

22. The method of any one of claims 13-21, further comprising conjugating one or more chemical moieties to the TP sequence.

23. The method of any one of claims 13-22, further comprising conjugating a second peptide to the TP sequence to form a TP and, optionally, conjugating one or more moieties to the third peptide.

24. A method for preparing a glucagon-like peptide-1 (GLP-1) agonist peptide comprising: (a) conjugating a protected activated amino acid fragment to a TP sequence prepared by the method of any one of claims 13-23 to form an intermediate product; and(b) conjugating a fatty acid moiety to the intermediate product.

25. The method of claim 24, wherein the activated amino acid fragment is conjugated to the TP sequence through a single amino acid coupling, direct fragment coupling, or a combination thereof.

26. The method of claim 24 or 25, wherein the fatty acid moiety is conjugated at the E of lysine.

27. The method of any one of claims 24-26 , wherein the TP sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO. 1), but not restricted to following sequences.

28. The method of any one of claims 24-26, wherein the TP sequence is selected from -EGTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 22), GTFTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 67), FTSDVSSYLEGQAAKEFIAWLVRGRGL (SEQID NO 68), TSDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 25), SDVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 69), DVSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 70), VSSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 71), SSYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 72), SYLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 73), YLEGQAAKEFIAWLVRGRGL (SEQ ID NO: 74),LEGQAAKEFIAWLVRGRGL (SEQ ID NO: 75), EGQAAKEFIAWLVRGRGL (SEQ ID NO: 76), GQAAKEFIAWLVRGRGL (SEQ ID NO: 77), QAAKEFIAWLVRGRGL (SEQ ID NO: 78), AAKEFIAWLVRGRGL (SEQ ID NO: 79), AKEFIAWLVRGRGL (SEQ ID NO: 80) and KEFIAWLVRGRGL (SEQ ID NO: 81)29. The method of any one of claims 24-28, wherein the fatty acid moiety is-^-U-W-Y-Z(I)whereinU represents -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-} wherein “}” is the point of attachment to W;W represents-C(O)-NH-(CH2)3-NH-],-C(O)-NH-(CH2)4-NH-],-C(O)-C(CH3)2-NH-], or-C(O)-N, wherein “]” is the point of attachment to Y;Y represents -C(O)-(CH2)2-CH(COOH)NH— and " is the point of attachment to Z;Z represents -C(O)-(CH2)n-COOH or -C(O)-(CH2)n-CH3, andn is an integer from 14 to 20.

30. The method of claim 31, wherein the U-W-Y-Z represents a group of the formula:wherein n is an integer from 14-20 and Ro is CO2H or CH3.

31. The method of any one of claims 24-29, wherein the fatty acid moiety has the formula:

32. The method of any one of claims 24-29, wherein the fatty acid moiety has the formula:

033. The method of any one of claims 24-29, wherein the fatty acid moiety has the formula:

34. The method of any one of claims 24-29, wherein the fatty acid moiety has the formula:O35. The method of any one of claims 24-29, wherein the fatty acid moiety has the formula:

36. The method of any one of claims 24-29, wherein the fatty acid moiety has the formula:

37. The method of any one of claims 24-29, wherein the fatty acid moiety has the formula:O38. The method of any one of claims 24-29, wherein the fatty acid moiety has the formula:

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