Production of recombinant human glu-plasminogen

Abstract

The present invention relates to methods for the production of recombinant human Glu-form of plasminogen, the isolated plasminogen, compositions comprising the isolated plasminogen as well as use of the isolated plasminogen in several medical indications.

Description

[0001] PROTEIN PRODUCTION SYSTEM

[0002] TECHNICAL FIELD

[0003] The present invention relates to methods for production of recombinant human Glu- plasminogen, the isolated plasminogen, compositions comprising the isolated plasminogen as well as use of the isolated plasminogen for the treatment of a mammal in need thereof.

[0004] BACKGROUND

[0005] Human plasminogen is a glycoprotein involved in the plasminogen activator (PA) system and is challenging to produce as Glu-form of plasminogen that is less susceptibility to degradation or conversion into plasmin. Plasminogen can be easily converted into plasmin, if exposed to serine proteases during production. Plasmin, in turn, can further degrade plasminogen into non-functional protein fragments. Additionally, plasmin can catalyse the conversion of Glu-form of plasminogen into Lys-t form of plasminogen, which accelerates the degradation of plasminogen to nonfunctional protein fragments. During recombinant plasminogen production, it is critical to prevent the premature conversion of Glu-plasminogen to Lys-plasminogen and further degradation into inactive fragments. Maintaining the Glu-form is essential for achieving a stable, functional product suitable for therapeutic applications. These issues present significant barriers to producing intact and functional Glu-plasminogen or intact Glu-plasminogen to acceptable levels, thereby limiting the availability of plasminogen-based pharmaceutical products on the market.

[0006] Plasminogen exists in two major forms: Glu-form and Lys-form. The Glu-type plasminogen, characterised by its glutamic acid residue at the N-terminus, is the more stable form under physiological conditions, with a higher half-life in vivo. In contrast, the Lys-form of plasminogen, resulting from partial proteolytic cleavage of the Glu-form, is less stable and more prone to further degradation. The stability of the Glu-form plasminogen makes it the preferred form for pharmaceutical applications, as it can maintain functionality and avoid premature degradation during storage or therapeutic use.

[0007] The production of recombinant plasminogen can be achieved in different ways, including expression in a host cell. However, the production and cultivation of such host cells for recombinant plasminogen must be carefully controlled to ensure that the protein is produced at high levels, remains intact, and is not degraded or converted into plasmin or smaller peptides. The quality and yield of the final recombinant plasminogen product depend critically on maintaining host cell viability and preventing proteolytic degradation of the protein.

[0008] W02021007612 discloses methods of producing recombinant plasminogen, not explicitly Glu-plasminogen. The inventors have surprisingly found that co-expression of plasminogen with plasminogen activator inhibitor (PAI-1) enables large quantities of full-length, functional plasminogen in recombinant mammalian cells. A person skilled in the art would therefore include that particular plasminogen activator inhibitor in the system.

[0009] Our inventors have found that it is not sufficient to co-express plasminogen and a plasminogen activator inhibitor and very difficult to control a proper, stable expression of Glu-plasminogen so that no degradation occur due to serine proteases present in the culture system. Instead, another approach was evaluated to strictly control the production process and maintain the recombinant plasminogen in its Glu-form at commercially viable levels that completely inhibits the degradation.

[0010] Surprisingly, the inventors found that by having a first N-1 stage of cell culture comprising a protease inhibitor in specific amounts prior to the fed-batch cultivation it was for the first time possible to solve the issues of proteolytic degradation of Glu- plasminogen and still maintaining sufficient cell viability to ensure production of high titres of Glu-plasminogen.

[0011] SUMMARY

[0012] The present invention relates to a method for production of recombinant plasminogen that addresses the significant challenges outlined in the background section. Specifically, it mitigates the issues of proteolytic degradation, conversion of Glu- plasminogen to Lys-plasminogen, and the loss of functionality caused by for example the action of serine proteases during production, whilst maintaining sufficient cell viability to ensure production of high titres of plasminogen. This was surprisingly found to be achieved by the addition of a N-1 stage of production, wherein a protease inhibitor was included. The invention provides a method for producing recombinant plasminogen in a host cell, wherein the process is optimised to achieve a high yield of intact and stable recombinant Glu-form plasminogen. The method utilises a fed-batch cultivation strategy optimised to control nutrient consumption and maintaining an environment conducive to the production of high-quality recombinant Glu-plasminogen. The process takes into account comprehensive factors, including culture environmental parameters, feed composition, and feeding strategy, to enhance cell growth, ensure protein stability, and maximise yields. The focus on stabilising by the use of stabilisation agents, secures that the Glu-form plasminogen is more stable throughout the production. The Glu-form plasminogen exhibits a longer half-life in vivo. By implementing this process, recombinant plasminogen is economically produced at commercially viable levels, overcoming the limitations of existing methods. The obtained product meets the stringent requirements for therapeutic use, providing stable compositions for pharmaceutical applications.

[0013] In a first aspect the invention relates to a method for production of human recombinant plasminogen comprising at least one N-1 (minus) stage of cell cultivation (precultivation) and at least one subsequent fed-batch cultivation (N stage), the method comprising the steps of: a. Providing a host cells expressing a human recombinant plasminogen, b. Culturing (preculturing) said host cells in a culture medium, wherein at the N-1 stage of production the culture medium comprises a serine protease inhibitor, in an amount of 1 KlU / mL to 150 KlU / mL c. Fed-batch (N-stage) cultivating said cultivated host cells and d. Obtaining recombinant human Glu-type plasminogen.

[0014] The disclosed method ensures efficient production of high yield and intact Glu-form of plasminogen to high levels, meeting the stringent requirements for therapeutic use while supporting scalability and robustness in manufacturing processes.

[0015] In a second aspect the invention relates to an isolated recombinant human Glu-form of plasminogen obtained by the above method and a composition comprising an isolated, purified, or substantially purified recombinant Glu-type plasminogen as well as use of the composition for the treatment of a mammal in need thereof, such as for the treatment of a disorder or disease or for the stimulation of wound healing. BRIEF DESCRIPTION OF THE DRAWINGS.

[0016] Figure 1 shows the polypeptide sequence of human Glu-type plasminogen including the signal peptide. Named SEQ ID NO:1.

[0017] Figure 2 shows the flow chart of the method to produce plasminogen

[0018] Figure 3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of batches of plasminogen with or without Aprotinin added at the N-1 stage or N stages of production. Glu-plasminogen band indicated at ~92 kDa. Lys-plasminogen band indicated at ~81 kDa.

[0019] Figure 4 Viable Cell Count (VCC) and viability profiles of production batches with or without aprotinin supplementation at the N-1 stage. (A) VCC profiles for batches with and without aprotinin. (B) Viability profiles for the same batches.

[0020] DETAILED DESCRIPTION

[0021] Definitions

[0022] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology.

[0023] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0024] The terms “cell”, “cell culture”, and “cell line” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny is exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny express the plasminogen polypeptide to acceptable stable levels and retain the same functionality as that of the original cell, cell culture, or cell line.

[0025] As used herein, “protein”, “polypeptide”, or “peptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers.

[0026] As used herein, “plasminogen”, “recombinant plasminogen”, “rec-PLG”, “human plasminogen”, “human recombinant plasminogen”, “Glu-type plasminogen” or “Glu- plasminogen” refers to one and the same protein throughout the application, and they are used interchangeably and meaning a polypeptide having the sequence as shown in SEQ ID NO:1 or a variant thereof and wherein said SEQ ID NO:1 are integrated in the host cell.

[0027] Unless specifically indicated otherwise, as used herein, a sequence identity of "at least about" an indicated percentage includes the indicated percentage ± 20% thereof, and every integer and non-integer percentage above the specific percentage. Accordingly, "at least about 85%" identity to SEQ ID NO. 1 includes about 85%, 86%, 87%, 89% 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 , and also includes all non-integer percentages in between two integer percentages (e.g.,92.5%, 99.1%, etc.).

[0028] As used herein the wording N- (minus) means a step / stage upstream of the fed-batch cultivation and may include one or more minus steps / stages, for example N-1, N-2.

[0029] The fed-batch cultivation step / stage is the N step / stage. For better understanding see figure 2. Another word for the N-minus stage of cultivation is precultivation that occurs before the fed batch cultivation stage / step.

[0030] Method for production

[0031] The invention relates to a method for the production of human recombinant Glu-form of plasminogen comprising at least one N-1 (minus) stage of production and at least one subsequent fed-batch cultivation (N stage). The method comprising the steps of: Providing a host cells expressing a human recombinant Glu-type plasminogen, Culturing said host cells in a culture medium, wherein at the N-1 stage of production the culture medium comprises at least a serine protease inhibitor, Fed-batch cultivating said cultivated host cells and Obtaining recombinant human Glu-type plasminogen.

[0032] By such a method it is for the first time possible to produce a stable Glu-type plasminogen to reasonable prices and quantities and subsequent provide a pharmaceutical composition.

[0033] The recombinant plasminogen is produced in a host cell, wherein the host cell is selected from the group consisting of, Human Embryonic Kidney (HEK) 293 cells, Baby Hamster Kidney (BHK-21) cells, Chinese Hamster Ovary (CHO) cells, NSO and Sp2 / 0 cells and Madin-Darby Canine Kidney (MDCK) cells or progenies thereof. In one example the host cell is a CHO cells. The recombinant plasminogen has a polynucleotide sequence as shown in SEQ ID NO:1 or a variant thereof as defined above.

[0034] The serine protease inhibitor present at the N-1 stage, may be aprotinin and aprotinin may be present in a concentration of at least 1 KlU / ml, such as of at least 5 KlU / mL, such as at least 10 KlU / mL, such as at least 15 KlU / mL, such as at least 20 KlU / mL, such as at least 25 KlU / mL, such as at least 30 KlU / mL, such as at least 35 KlU / mL, such as at least 40 KlU / mL, such as at least 45 KlU / mL, such as at least 50 KlU / mL, such as at least 55 KlU / mL, such as at least 60 KlU / mL, such as at least 65 KlU / mL, such as at least 70 KlU / mL, such as at least 75 KlU / mL, such as at least 80 KlU / mL or at least 150 KlU / mL, such as from 1 KlU / mL to 150 KlU / mL, 5 KlU / mL to 80 KlU / mL or 20 to 80 KlU / mL.

[0035] The N minus stages, for example N-1 stage is a cultivation stage upstream of the fed- batch cultivation step (the N stage) as shown in figure 2. The fed-batch cultivation step is subsequent the N-1 stage. There may be one or more N- (minus step / stages).

[0036] The fed-batch cultivation step comprises at least a serine protease inhibitor and / or lysine analogue or a synthetic analogue to maintain Glu-type plasminogen intact and no degradation or conversion into the Lys-type plasminogen or to non-functional parts or fragments. Examples of analogues to the amino acid lysine are Tranexamix acid (TXA) or e-aminocaproic acid (EACA) and aprotinin is an example of a serine protease inhibitor.

[0037] The fed-batch cultivation step comprises at least one serine protease inhibitor and / or lysine and / or lysine analogues selected from the group comprising aprotinin, e- aminocaproic acid, tranexamic acid, lysine and benzamidine. In one example the at least one serine protease inhibitor and / or lysine and / or lysine analogues is aprotinin and / or tranexamic acid.

[0038] If aprotinin is present in the fed-batch cultivation, aprotinin may be present at concentration of at least 1 KlU / m, such as 5 KlU / ml, such as at least 10 KlU / ml to at least 80 KIU / ML, such as at least 10 KlU / mL, such as at least 15 KlU / mL, such as at least 20 KlU / mL, such as at least 25 KlU / mL, such as at least 30 KlU / mL, such as at least 35 KlU / mL, such as at least 40 KlU / mL, such as at least 45 KlU / mL, such as at least 50 KlU / mL, such as at least 55 KlU / mL, such as at least 60 KlU / mL, such as at least 65 KlU / mL, such as at least 70 KlU / mL, such as at least 75 KlU / mL, such as at least 80 KlU / mL or from 1 KlU / mL to 150 KlU / mL or 5 KlU / mL to 100 KlU / mL or 20 to 80 KlU / mL.

[0039] The lysine and / or lysine analogue may be present at a concentration of at least 1 g / L, such as at least 2 g / L, such as at least 3 g / L, such as at least 4 g / L, such as at least 5 g / L, such as at least 6 g / L or at a concentration of at the most 6 g / L, such as at the most 5 g / L, such as at the most 4.5 g / L. For example 1 g / L to 6g / L.

[0040] The disclosed method for production of human recombinant plasminogen result in that at least 80% Glu-plasminogen of total plasminogen, such as at least 85% Glu- plasminogen of total plasminogen, 90% Glu-plasminogen of total plasminogen or 95% Glu-plasminogen of total plasminogen is obtained which is extremely good. Thereby acceptable amounts of therapeutic Glu-plasminogen is obtained in an economical manner to be able to produce a medical product. Thereby a stable Glu-form of plasminogen product is produced having a prolonged half-life in vivo compared to plasminogen in another form.

[0041] By the invented method and method steps it is for the first time possible to obtain cells that have a cell viability of at least 95% in the N-1 stage, such as at least 90%, such as at least 85%, such as at least 80%, such as at least 75%, such as at least 70%.

[0042] In addition, the cells will have a cell viability of at least 95% in the N stage, such as at least 90%, such as at least 85%, such as at least 80%, such as at least 75%, such as at least 70% and still expressing / producing plasminogen and especially the Glu-form of plasminogen. By having an increased cell viability a higher yield of the final medical polypeptide is obtained.

[0043] The invented method will further comprise one or more of the subsequent following methods: cation-exchange (CEX) capture chromatography, lysine affinity chromatography, anion exchange (AEX) chromatography, ultrafiltration, tangential flow filtration (TFF) and / or dialysis to be able to purify the recombinant human Glu-form of plasminogen.

[0044] The invention also relates to the isolated recombinant plasminogen as well as a composition comprising an isolated, purified, or substantially purified recombinant plasminogen obtained by a method as defined above as well as the use of the recombinant plasminogen for the treatment of a mammal in need thereof, for example for the treatment of a disorder or disease.

[0045] EXAMPLES

[0046] Example 1 Production of human recombinant plasminogen

[0047] The preparation of recombinant plasminogen (rec-PLG) was initiated with production method as shown in Figure 2. The cells expressing SEQ ID NO:1 was thaw and added to a viable cell count (VCC) of 0.5 x 106cells / mL of CHO cells in a conventional cell cultivation medium at a temperature of 37°C. The cells were cultivated in successive stages in shake flasks until reaching optimal cell number to be transferred to the production Stage reactor (Stage N). The fed-batch cultivation was performed in 10 L vessels, following approximately 40 cell generations.

[0048] To inhibit protease activity, 10 KlU / mL of aprotinin was added during the pre-cultivation step (N-1 ).

[0049] The process utilises conventional medium in all stages well known for a person skilled in the art faced with the problem of cultivating CHO cells for the production of a polypeptide.

[0050] In the fed-batch culture step (N), aprotinin was added to a total of 80 KlU / mL. Additionally, tranexamic acid (TXA) was added in a total amount of 4.5 g / L.

[0051] The cells were harvested using a two-step depth filtration process, yielding a clarified harvest containing glu-plasminogen with high yield. The clarified harvest involved the removal of cell debris, host cell proteins (HCP), and host cell DNA (HCD) using primary and secondary depth filters. Phosphate buffer saline at pH 7.5, supplemented with 10 KlU / mL of Aprotinin, was used in this step. The method of production combined with a downstream process, summarised in Figure 2 produced high-yield and high-purity Glu-type plasminogen.

[0052] Example 2 - Analysis of batches produced in Example 1

[0053] The batches from example 1 were analysed using SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of culture media on different days from batches without aprotinin (lanes 1-3) or with aprotinin (lanes 4-8) addition at the N-1 stage or N stages of plasminogen production. Glu-plasminogen band indicated at ~92 kDa and Lys-plasminogen band indicated at ~81 kDa.

[0054] Lane (1-3): culture media from batch 2 (without aprotinin in N-1 stage and N stages of production) from day 7 to day 9; lane (4-8): culture media from batch 1 (with aprotinin in N-1 stage and N stages of production) from day 7 to day 11 ; lane 9: human plasma derived plasminogen (hPIg) standard from plasma; lane 10: molecular weight marker (see Figure 3).

[0055] The amount of Glu-plasminogen was compared to the total amount of plasminogen and found to be at least 80 % Glu-plasminogen of total plasminogen.

[0056] The batches from example 1 was also analysed for cell viability with or without Aprotinin added at the N-1 stage of production (see Figure 4) and was found to be high enough so that the cells could be used for production of plasminogen for the batches with Aprotinin.

Claims

CLAIMS1 . A method for the production of human recombinant Glu-plasminogen comprising at least one N-1 stage of cell cultivation / precultivation and at least one subsequent fed-batch cultivation stage N-stage, the method comprising the steps of: a. Providing host cells expressing a human recombinant plasminogen, b. Culturing / preculturing said host cells in a culture medium, wherein at the N-1 stage of production the culture medium comprises at least a protease inhibitor in an amount of 1 KlU / mL to 150 KlU / mL, c. Fed-batch cultivating said host cells and d. Obtaining recombinant human Glu-plasminogen.

2. The method according to claim 1 , wherein at the N-1 stage of production the culture medium comprises aprotinin.

3. The method according to any of the preceding claims, wherein at the N-1 stage of production the culture medium comprises aprotinin at a concentration of 5 KlU / mL to 80 KlU / mL.

4. The method according to any of the preceding claims, wherein at the N-1 stage of production the culture medium comprises aprotinin at a concentration of or 20 to 80 KlU / mL.

5. The method according to any of preceding claims, wherein the fed-batch cultivation step comprises a serine protease inhibitor and / or lysine or lysine analogue.

6. The method according to claim 5, wherein the fed-batch cultivation comprises at least one serine protease inhibitor and / or lysine and / or lysine analoguesselected from the group comprising aprotinin, E-aminocaproic acid, tranexamic acid, lysine and benzamidine.

7. The method according to claim 6, wherein the inhibitor is aprotinin.

8. The method according to any one of claims 7, wherein the fed-batch cultivation comprises aprotinin at a concentration of at least 1 KlU / mL to 150 KlU / mL9. The method according to any of preceding claims, wherein the fed-batch culture medium comprises lysine and / or a lysine analogue at a concentration of 1 g / L to 6g / L.

10. The method according to claims 9, wherein lysine analogue is tranexamic acid.

11. The method according to any of the preceding claims, wherein plasminogen comprises at least 80% Glu-plasminogen of total plasminogen in step d).

12. The method according to any of the preceding claims, wherein the host cell is selected from the group comprising of, Human Embryonic Kidney (HEK) 293 cells, Baby Hamster Kidney (BHK-21 ) cells, Chinese Hamster Ovary (CHO) cells, NSO and Sp2 / 0 cells and Madin-Darby Canine Kidney (MDCK) cells or progenies thereof.

13. The method according to any of the preceding claims, wherein the host cell expresses a human plasminogen polynucleotide according to SEQ ID NO:1 or a variant thereof.

14. The method according to any of the preceding items, wherein the method further comprises recovering and / or purifying plasminogen from the host cell and / or cell culture media.

15. The method according to any of the preceding claims, wherein the method further comprises any one or more of the following methods: cation-exchange (CEX) capture chromatography, lysine affinity chromatography, anion exchange (AEX) chromatography, and tangential flow filtration (TFF).

16. A recombinant plasminogen obtained by a method according to any one of the preceding claims.

17. A composition comprising a recombinant plasminogen according to claim 16.

18. Use of the recombinant plasminogen and / or the composition according to any of claims 16-17 for the treatment of a disease or disorder.

Images