CONTINUOUS MANUFACTURE OF GUIDANCE MOLECULE DRUG CONJUGATES

Abstract

Described herein is a modification unit (37) for the continuous, pathogen reduced processing of a guidance molecule e.g. a conjugate of a peptide or a protein or a nucleic acid and a linker comprising the following components: at least one reservoir containing the guidance molecule in buffer solution (1) and/or at least one inlet for a product stream containing the guidance molecule in buffer solution, at least one reservoir containing the linker in solution (2), at least one mixing device (3), at least two valves (7, 8), one for dosage of the guidance molecule and one for dosage of the linker molecule at least one outlet for the product stream comprising the guidance molecule-linker complexes and/or a reservoir for taking up the guidance molecule-linker complexes (5)
further comprising at least one residence time device ensuring a defined residence time, i.e. ensuring that after mixing the guidance molecules and the linker molecules always spend a similar amount of time in a continuous process.

Claims

1. Modification unit for continuous, pathogen reduced processing of a guidance molecule, optionally a peptide or a protein or a nucleic acid and a linker comprising the following components: at least one reservoir containing the guidance molecule in buffer solution and/or at least one inlet for a product stream containing the guidance molecule in buffer solution, at least one reservoir containing the linker in solution, at least one mixing device, at least two valves, one for dosage of the guidance molecule and one for dosage of the linker molecule at least one outlet for the product stream comprising the guidance molecule-linker complexes and/or a reservoir for taking up the guidance molecule-linker complexes and further comprising at least one residence time device ensuring a defined residence time, optionally ensuring that after mixing the guidance molecules and the linker molecules always spend a similar amount of time in a continuous process.

2. Modification unit according to claim 1, further comprising at least three valves.

3. Modification unit according to claim 1, wherein the residence time device is a CFI or a HFI.

4. Modification unit according to claim 1, wherein the guidance molecule is a protein and said protein is an antibody.

5. Modification unit according to claim 1, further comprising at least one pump.

6. Modification unit for continuous, pathogen reduced processing of a guidance molecule, optionally a peptide or a protein or a nucleic acid and a linker comprising the following components: at least one reservoir containing the guidance molecule in buffer solution and/or at least one inlet for a product stream containing the guidance molecule in buffer solution, at least one reservoir containing the linker in solution, at least one mixing device, at least one outlet for the product stream comprising the guidance molecule-linker complexes and/or a reservoir for taking up the guidance molecule-linker complexes and further comprising at least one residence time device ensuring a defined residence time, optionally ensuring that after mixing the guidance molecules and the linker molecules always spend a similar amount of time in a continuous process further comprising at least two pumps.

7. Modification unit according to claim 1, further comprising at least one waste outlet.

8. Modular system for continuous, pathogen reduced production and/or processing of a guidance molecule drug conjugate, optionally a conjugate of a peptide or a protein or a nucleic acid and a drug, comprising the following units: at least one modification unit as claimed in claim 1, at least one unit for conjugation, comprising at least one a mixing device, at least one residence time device ensuring a defined residence time, optionally ensuring that after mixing the guidance molecules and the linker molecules always spend a similar amount of time in a continuous process and at least one reservoir containing the biologically active substance at least one unit for concentration and re-buffering, comprising at least one buffer reservoir and at least one ultrafiltration device. least one unit for concentration and re-buffering, comprising at least one buffer reservoir and at least one diafiltration device at least one filter.

9. Modular system according to claim 7, wherein the modular system is closed.

10. A method for the continuous, pathogen reduced production and/or processing of a guidance molecule drug-conjugate optionally a conjugate of a peptide or a protein or a nucleic acid and a drug, comprising: providing a guidance molecule optionally a peptide or a protein or a nucleic acid providing a linker, attachment of linker and guidance molecule, conjugation of the guidance molecule-linker complex to a biologically active substance, at least one continuous ultrafiltration, at least one continuous diafiltration of a product stream, at least one filtration of the product stream wherein the process is carried out in a modular manner.

11. Method according to claim 10, wherein the method is carried out in a closed manner.

12. Method for continuous pathogen reduced production and/or processing of a guidance molecule drug-conjugate optionally a conjugate of a peptide or a protein or a nucleic acid and a drug wherein the method is carried out using a modification unit-of claim 1.

13. A method according to claim 9 in a process for the continuous, pathogen reduced production and/or processing of an antibody-drug-conjugate from a heterogeneous cell culture fluid mixture, comprising: preparation of a particle-free fluid from a heterogeneous cell culture fluid mixture containing the antibody in the form of a product stream, at least one filtration, at least one chromatography step for cleaning the antibody comprising a cleaning via at least two chromatography columns and/or membrane absorbers, respectively at least one viral clearance, at least one continuous ultrafiltration at least at least one continuous diafiltration, processing the antibody derived in said continuous diafiltration using the method to derive an antibody-drug-conjugate wherein the process is carried out in a continuous, closed and modular manner.

Description

EXAMPLES

[0127] General Considerations:

[0128] In order to provide an antibody-drug-conjugate under continuous, pathogen reduced conditions in a modular process and preferably closed a production plant with the following units and accompanying process steps is constructed:

[0129] Unless otherwise specified, MasterFlex peristaltic pumps with an EasyLoad II pump head are used in the process. Masterflex or Cflex or Sanipure are used as tubes. All of the elements coming into contact with the product are gamma-irradiated with 25 kGy. In exceptional cases, if gamma irradiation is not allowable for reasons connected with materials technology, components are autoclaved at 121 C. for at least 20 min. When possible, ready-to-use disposable articles (disposables, ready-to-use) are used as gamma-irradiated units. As a rule, all bags are connected to the units. Between each unit, a single-use gamma-irradiated bag is placed between the outlet flow of the unit n1 and the inlet flow of the unit n as an equalizing tank. As a rule, there is an inlet and outlet flow at that time in each unit. In cases where venting of the product liquid is advantageous, the vessels are sealed off from the external environment via a 0.2 m hydrophobic filter. Moreover, the process is preferably controlled by a PCS7 process system.

[0130] A storage container comprising an antibody solution, e.g. an IgG is provided. Moreover, a storage bag comprising a linker solution is provided.

[0131] The antibody solution as well as the linker solution are pumped from their reservoirs, also termed storage bags, and allowed to mix and react, thereby modifying both components and generating an antibody-linker substrate. The conjugation is carried out in the next step, where, a solution comprising the biologically active substance, e.g. a solution comprising toxophores, is added to the fluid stream comprising the antibody-linker complex and all components are mixed, e.g. using a static mixer to generate the antibody-linker-toxophore conjugate. The toxophore load is measured via a UV-absorption at two specific wavelengths. The resulting product stream comprising antibody drug conjugates as well as unbound toxophores is optionally passed through a depth filter and then subjected to an ultrafiltration in order to concentrate the antibody. Then the product stream is subjected to a diafiltration e.g. the product stream is washed with a washing fluid via at least one capillary ultrafiltration membrane of a capillary ultrafiltration unit, wherein the product stream is conveyed into the capillary and the washing fluid is conveyed over the outside of the capillary and the product stream and the washing fluid are continuously fed into the capillary ultrafiltration unit, and are continuously removed from the capillary ultrafiltration unit and the product stream and the washing fluid are not circulated into the capillary ultrafiltration unit and the removal of the product stream is regulated such that no undesired net flows can pass from the capillary interior to the capillary exterior or vice versa and all fluid leaving the at least one capillary ultrafiltration unit in the direction of the product stream is passed through at least one guard purifier. For conveying the product stream (=feed stream) into the capillaries and the washing fluid (=permeate) on the outsides of the capillaries of the ultrafiltration unit in each case one pump is used. For the controlled removal of the product stream from the capillaries (=retentates), one further pump is used. Through the use of this pump (=retentate pump), control of the removal of the retentate is simplified. This is advantageous as there are no adequate flow sensors which reliably measure the low flow rates (<100 ml/min) usually employed in this continuous process. Particularly preferably, peristaltic pumps can be used here with the advantage that peristaltic disposable pumps are commercially available, so that sterility and also disposable technology are available. Moreover, a pump is used for the controlled removal of the wash fluid (permeate).

[0132] Alternatively, instead of the continuous diafiltration described above a system comprising diafiltration slices in an ultrafiltration/diafiltration system can be used.

[0133] Finally the product stream is filtered using two 0.2 m filters operated in parallel. Both the filter and the tube assembly are gamma-irradiated. The inlet and outlet lines are connected to gamma-irradiated bags) by means of sterile connectors, serving as an equalizing volume for fluctuating flow rates. For venting, the filters are coupled to hydrophobic 0.2 m air filters, thus closing the unit within the meaning of the invention. The air filter is either an Emflon II from Pall Corp. or a Midisart 2000 from Sartorius Stedim. The venting valves are modified so that they are permeable even when closed, but still reliably sealed with respect to the environment.

SPECIFIC EXAMPLES

Example 1: Continuous Modification

[0134] The antibody solution. which had an initial concentration of 15.3 mg/mL was diluted with 100 mM potassiumphosphat (KPi)-Buffer (pH 7.5) to a final concentration of 10.72 g/L. The Linker-solution. here a SPDB Linker (N-succinimidyl-4-(2-pyridyldithio)butanoate) in DMA. was prepared with a concentration of 1.56 g/L.

[0135] These solutions were filled in the reservoirs and pumped with specific flow rates of 284.3 mL/h for AB-Solution and 20 mL/h for Linker-solution until the system reached a steady state. This period of time required by the system to reach a steady state, e.g. essentially constant pump rates, was termed start-up phase. In this example the start-up phase was defined as the first five residence times, where one residence interval equals the amount of time that a given portion of the product stream requires to flow through the modification unit. During start-up all produced material was directed to the waste outlet.

[0136] After the start-up phase the system was switched from start-up to production mode. Thus, the AB-Solution and the Linker-solution were pumped with specific flow rates of 284.3 mL/h for AB-Solution and 20 mL/h for Linker-solution into the mixing device, here a static Cascade-Micromixer from Ehrfeld was used which had the following setting [asymmetric Cascade mixer with max. inner diameter 150 m, 11 mixing stages]. After passing the mixing device the product stream passed the residence time device here a Masterflex Tygon Tube was used with an inner-diameter of 3.1 mm having a total volume of 6.3 mL.

[0137] Thereafter the product stream was collected in a reservoir, here a Schott-Flask with Bola-Connectors.

[0138] For collection of sample material the product stream was also directed to sample reservoirs. During the experiment samples of 20 mL were taken.

[0139] For process shut down, the flow was directed again to the waste outlet, the pumps were stopped and the all interconnected parts were flushed with solvent and water, which were filled into the reservoirs that previously contained the antibody and linkers solutions, respectively, for cleaning. If single use equipment was employed, it was discarded.

[0140] To prove that the modification reaction was successful under continuous process conditions it had to be demonstrated that the generated antibody-linker complexes had the same characteristics as antibody-linker complexes generated in a batch process. In order to do so the generated antibody-linker complexes were conjugated with a toxophore under standard conditions and their monomer content as well as their dimer content and the achieved Drug-to-Antibody ratio were determined.

[0141] For said analysis. 2.25 mL aliquot of the sample was diluted with 2 mL phosphate buffer and 0.209 mL of dimethylacetamide (DMA). Afterwards 40.9 L of a 20 mM toxophore solution in DMA was added. After a reaction time of 18 h the reaction was stopped by a buffer exchange to His/Gly (pH 5.5) by a PD10 column. Size exclusion chromatography was carried out and showed a monomer content of 96.4% and a dimer content of 2.8%. An aliquot of this sample was diluted and analyzed by UV-Measurement showing a Drug-to-Antibody-Ratio (DAR) of 3.4. The ULAR (Unconjugated Linker to Antibody Ratio) was determined to be below 0.03 using a specifc HPLC analysis. in which the sample is treated with a disulphide cleaving agent. the protein is removed and the HPLC detects the released mercaptopyridine.

[0142] In other words, it was shown that the continuous process conditions did not affect the stability of the final conjugate as otherwise the monomer content would be lower. Moreover, the antibody-linker complex generated under continuous conditions requires the same amount of toxophore per antibody-linker complex as in the batch process, since otherwise the DAR and ULAR values would be higher or lower.

Example 2: Continuous Conjugation

[0143] The conjugation Reaction was used as second reaction step following the above described modification in the model process of ADC production. The device set up was similar to set up described above for the modification reaction.

[0144] The solution of modificated antibody was diluted with 100 mM KPi-Buffer (pH 7.5) to a final concentration of approx. 6 g/L. A 3.3 mM solution of Maytansinoid DM4 (14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1 (6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10.12-dien-4-yl N-(4-mercapto-4-methylpentanoyl)-N-methyl-L-alaninate) in DMA was prepared.

[0145] These solutions were filled in the reservoirs and pumped with specific flow rates of 169.6 mL/h for Antibody-Linker-Solution and 10.0 mL/h for DM4-solution. During process start up all produced material was directed to the waste outlet. For collection of sample material the flow was directed to sample reservoirs. During the experiment samples of 20 mL were taken, after the reaction time of 2 h 36 min.

[0146] For process shut down, the flow was directed to the waste outlet, pumps were stopped and reservoirs were changed to cleaning solutions (water for cleaning, 1 mol NaOH to quench DM4), the whole set up was flushed with water and NaOH-solution for cleaning. Alternatively in case of single use the set up could be discarded.

[0147] After reaction the solution was concentrated and the buffer was exchanged to His/Gly (pH 5.5) by ultrafiltration/diafiltration (UF/DF). SEC (Size Exclusion Chromatography) of the resulting mixture showed a Monomer content of 96.7% and a Dimer content of 3.03%. An aliquot of this sample was diluted and analyzed by UV-Measurement showing a Drug-to-Antibody-Ratio of 3.3. The yield of this reaction was 87% after UF/DF.

[0148] Thus, overall these results demonstrated that the antibody-linker complexes generated in a continuous process using the conjugation unit as described herein are comparable to those generated in a batch process.

FIGURES

[0149] FIG. 1 Overview of an example of a modification unit described herein for the continuous, pathogen reduced processing of a guidance molecule and a linker comprising the following components: a reservoir containing the guidance molecule (1) in buffer solution, a reservoir containing the linker in solution (2), a mixing device (3), a residence time device (4), apump (7) for dosage of the guidance molecule, a pump (8) for dosage of the linker molecule, a reservoir for taking up the guidance molecule-linker complexes (5) waste outlet (6) and a three way valve (9) for directing the product stream exciting the residence time device (4) into the waste outlet (6) or the reservoir for taking up the guidance molecule-linker complexes (5). During operation, the guidance molecules in buffer solution and the linker solution are added in a controlled fashion via the two pumps (7) and (8) to the mixing device (3). Following the mixing the product stream in this example flows through residence time device (4). Afterwards the product stream is collected in reservoir (5).

[0150] FIG. 2 depicts a schematic illustration of an example of a modification unit described herein for the continuous. pathogen reduced processing of a guidance molecule and a linker. In this example the modification unit is in fluid communication with a downstream unit. Hence, no reservoir for taking up the guidance molecule linker complexes is needed as the product stream continuously flows to the next unit operation. In this example the modification unit comprises the following components: a reservoir containing the guidance molecule (1) in buffer solution, a reservoir containing the linker in solution (2), a mixing device (3), a residence time device (4), a pump (7) for dosage of the guidance molecule, a pump (8) for dosage of the linker molecule, a waste outlet (6) and a connection to the next unit operation (10). During operation, the guidance molecules in buffer solution and the linker solution are added in a controlled fashion via the two pumps (7) and (8) to the mixing device (3). Following the mixing the product stream in this example flows through the plug-flow reactor (4). Afterwards the product stream continuously flows to the subsequent unit operation e.g. the one depicted in FIG. 3, via connection (10).

[0151] It should be noted that intermediate reservoirs/storage containers (not depicted) may be included in the connection to the next unit operation (10), if this is beneficial.

[0152] Moreover, it should be noted that as described above instead of providing an antibody in a reservoir (1) it could be provided from an upstream process continuously generating and purifying said antibody.

[0153] Furthermore, in this example two optional mass flow controls (26) were employed.

[0154] FIG: 3 depicts a schematic overview of an exemplary unit operation carrying out the conjugation. In this example, said unit operation for conjugation comprises a connection to the previous unit operation (10) and a connection to the subsequent unit operation (11), a reservoir containing buffer solution (12), a reservoir containing a toxophore (13), a mixing device (14)-here a static mixer, a residence time device (15), two homogenization loops (16) and (17) several pumps (18a-f) for controlling the dosage of the different fluid streams, two waste outlets (19a and 19b) and a reservoir containing buffer solution (20) for the subsequent unit operation.

[0155] During operation the product stream comprising the antibody-linker complexes generated in the previous unit operation, i.e. the unit for modification described herein, enters the unit operation for conjugation via connection (10) and buffer from reservoir (12) is added via the homogenization loop (16) before toxophore solution is dosed to the product stream containing the antibody-linker complexes from the reservoir containing the toxophore (13). Then the product stream comprising antibody-linker complexes and toxophore is mixed and following the mixing in device (14) the product stream in this example flows through the plug-flow reactor (15) and is optionally analyzed via UV-measurement. Afterwards buffer is added to the product stream from reservoir (20) via homogenization loop (17) in order to adjust the pH and the concentration and the antibody-linker-toxophore conjugates continuously flow to the subsequent unit operation e.g. the one depicted in FIG. 4.

[0156] FIG. 4 depicts a schematic overview of an exemplary unit for continuous ultrafiltration comprising a connection to the previous unit operation (11) and a connection to the subsequent unit operation (21), an ultrafiltration device (22), a depth filter (23), a waste outlet (24), another filter (25), a detection device (26), a surge bag (27) and several pumps (28a-d) for controlling the fluid flow.

[0157] During operation the product stream comprising the antibody-linker-toxophore conjugates in this example enters this unit operation via connection (11), flows through filter (25), ultrafiltration device (22), detection device (26), surge bag (27) and depth filter (23) and then through connection (21) to the subsequent unit operation depicted in FIG. 5.

[0158] FIG. 5: depicts a schematic overview of an exemplary unit for continuous dialysis comprising a connection from the previous unit operation (21), a reservoir containing buffer solution (29), a dialysis module (30), a detection device (31), a waste outlet (32), a filter (33), a reservoir for storing the final product (34), i.e the ultrafiltrated and dialysed antibody-linker-toxophore conjugates, as well as pumps (35-b) for controlling the fluid stream.

[0159] During operation the product stream comprising the antibody-linker-toxophore conjugates in this example enters this unit operation via connection (21) and flows through dialysis module (30), where buffer from reservoir (29) is exchanged for previous the buffer of the antibody-linker-toxophore conjugates. The previous buffer is discarded to waste outlet (32). The product stream comprising the antibody-linker-toxophore conjugates flows from the ultrafiltration module via the detection device (31) and the filter (33) to the reservoir for storing the final product (34).

[0160] As described above unit operations for continuous cell culture generating the antibody, continuous cell separation as well as continuous purification of the antibody can be added upstream to enable a continuous production of an antibody linker conjugate starting with cell fermentation.

[0161] Furthermore, a unit operation for formulation, e.g. adding of stabilizers and adjusting the pH of the final medicament, can be added downstream of the reservoir for storing the final product.

[0162] FIG. 6 Overview of an example of a conjugation unit (38) described herein for the continuous, pathogen reduced processing of a guidance molecule linker complex and a biologically active substance comprising the following components: a reservoir containing the guidance molecule linker complex (39) in buffer solution, a reservoir containing the biologically active substance, here a toxophore, in solution (40), a mixing device (3), a residence time device (4), a pump (7) for dosage of the guidance molecule linker complex, a pump (8) for dosage of the toxophore, a reservoir for taking up the guidance molecule linker toxophore conjugates (5) waste outlet (6) and a three way valve (9) for directing the product stream exciting the residence time device (4) into the waste outlet (6) or the reservoir for taking up the guidance molecule linker toxophore conjugates (5). During operation, the guidance molecule linker complexes, in buffer solution and the toxophore solution are added in a controlled fashion via the two pumps (7) and (8) to the mixing device (3). Following the mixing the product stream in this example flows through plug-flow reactor (4). Afterwards the product stream is collected in reservoir (5).

LIST OF REFERENCE SIGNS

[0163] 1. reservoir containing the guidance molecule (1) [0164] 2. reservoir containing the linker in solution (2), [0165] 3. mixing device (3), [0166] 4. residence time device (4) [0167] 5. reservoir (5) [0168] 6. waste outlet (6) [0169] 7. pump (7) [0170] 8. pump (8) [0171] 9. three way valve (9) [0172] 10. connection between units (10), [0173] 11. connection between units (11), [0174] 12. reservoir containing buffer solution (12), [0175] 13. reservoir containing a toxophore (13), [0176] 14. mixing device (14) [0177] 15. residence time device (15), [0178] 16. homogenization loop (16) [0179] 17. homogenization loop (17) [0180] 18. pumps (18a-f) [0181] 19. waste outlets (19a and 19b) [0182] 20. reservoir containing buffer solution (20) [0183] 21. connection between units (21) [0184] 22. ultrafiltration device (22), [0185] 23. depth filter (23), [0186] 24. waste outlet (24), [0187] 25. filter (25), [0188] 26. detection device (26), [0189] 27. surge bag (27) [0190] 28. pumps (28a-d) [0191] 29. reservoir containing buffer solution (29), [0192] 30. dialysis module (30), [0193] 31. detection device (31), [0194] 32. waste outlet (32), [0195] 33. filter (33), [0196] 34. reservoir for storing the final product (34) [0197] 35. pumps (35a and 35b) [0198] 36. mass flow controls (36) [0199] 37. modification unit (37) [0200] 38. conjugation unit (38) [0201] 39. reservoir containing the modified antibody (39) [0202] 40. reservoir containing a toxophore (40)