阅读说明：本技术 引导分子药物缀合物的连续制造 (Continuous manufacture of guided molecular drug conjugates ) 是由 H.保尔森 J.克吕格 K.凯泽 V.林根 M.戈特弗里德 于 2018-07-12 设计创作，主要内容包括：本文描述了用于连续、病原体减少地处理引导分子(例如肽或蛋白或核酸和接头的缀合物)的修饰单元(37),其包括以下组件：至少一个含有在缓冲溶液中的引导分子的储液器(1)和/或至少一个用于含有在缓冲溶液中的引导分子的产物流的入口,至少一个含有在溶液中的接头的储液器(2),至少一个混合装置(3),至少两个阀(7,8),一个用于引导分子的计量加入且一个用于接头分子的计量加入,至少一个用于包含引导分子-接头复合物的产物流的出口和/或用于容纳引导分子-接头复合物的储液器(5),进一步包括至少一个停留时间装置以确保限定的停留时间,即,确保在混合后,引导分子和接头分子总是在连续处理中度过相似的时间量。(Described herein is a modification unit (37) for the continuous, pathogen-reducing treatment of a guide molecule (e.g. a peptide or protein or a conjugate of a nucleic acid and a linker) comprising the following components: at least one reservoir (1) containing a guide molecule in a buffer solution and/or at least one inlet for a product stream containing a guide molecule in a buffer solution, at least one reservoir (2) containing a linker in a solution, at least one mixing device (3), at least two valves (7,8), one for the metered addition of a guide molecule and one for the metered addition of a linker molecule, at least one outlet for a product stream comprising a guide molecule-linker complex and/or a reservoir (5) for containing a guide molecule-linker complex, further comprising at least one residence time device to ensure a defined residence time, i.e. to ensure that after mixing the guide molecule and linker molecule are always treated continuously for a similar amount of time.)

1. A modification unit (37) for the sequential, pathogen-reducing treatment of guide molecules, such as peptides or proteins or nucleic acids, and linkers, said modification unit (37) comprising the following components:

-at least one reservoir (1) containing the guide molecule in a buffer solution and/or at least one inlet for a product stream containing the guide molecule in a buffer solution,

-at least one reservoir (2) containing said adapter in solution,

-at least one mixing device (3),

-at least two valves (7,8), one for the metered addition of the guide molecule and one for the metered addition of the linker molecule,

-at least one outlet for a product stream comprising the guide molecule-linker complex and/or a reservoir (5) for containing the guide molecule-linker complex, and

-further comprising at least one residence time means to ensure a defined residence time, i.e. to ensure that after mixing, the guide molecule and the linker molecule are always in continuous treatment for a similar amount of time.

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

3. The modification unit according to any one of the preceding claims, wherein the residence time means is CFI or HFI.

4. The modifying unit according to any of the preceding claims, wherein the guide molecule is a protein and the protein is an antibody.

5. The modifying unit according to any one of the preceding claims, further comprising at least one pump.

6. The modifying unit according to any one of the preceding claims, comprising at least two pumps in place of the at least two valves.

7. The modification unit according to any one of the preceding claims, further comprising at least one waste outlet (6).

8. Modular system for the continuous, pathogen-reduced production and/or processing of guide molecule drug conjugates, such as conjugates of peptides or proteins or nucleic acids with drugs, comprising the following units:

-at least one modification unit as claimed in claims 1 to 6,

at least one unit for conjugation comprising at least one mixing device, at least one residence time device and at least one reservoir containing biologically active substances, the residence time device ensuring a defined residence time, i.e. ensuring that after mixing the guide molecule and the linker molecule are always in continuous processing for a similar amount of time,

at least one unit for concentration and rebuffering comprising at least one buffer reservoir and at least one ultrafiltration device,

at least one unit for concentration and rebuffering comprising at least one buffer reservoir and at least one diafiltration device,

-at least one filter.

9. The modular system of claim 7, wherein the modular system is closed.

10. A method for the continuous, pathogen-reduced production and/or processing of guide molecule drug-conjugates, such as conjugates of peptides or proteins or nucleic acids with drugs, comprising the steps of:

providing a guide molecule, such as a peptide or protein or a nucleic acid,

-providing a joint, the joint being,

-attachment of a linker and a guide molecule,

directing the conjugation of the molecule-linker complex to the biologically active substance,

-at least one continuous ultrafiltration,

-at least one continuous diafiltration of the product stream,

-at least one filtration of the product stream,

wherein the process is carried out in a modular fashion.

11. The method according to claim 10, wherein the method is performed in a closed manner.

12. The method according to claim 11, wherein the method is performed using the modification unit of claims 1-7.

13. Use of a method according to claims 9-11 in a method for continuous, pathogen-reduced production and/or processing of antibody-drug-conjugates from a heterogeneous cell culture fluid mixture, the method comprising the steps of:

-preparing a particle-free fluid from a heterogeneous cell culture fluid mixture comprising antibodies in the form of a product stream,

-at least one filtration is carried out,

at least one chromatography step for washing the antibodies, comprising washing via at least two chromatography columns and/or membrane adsorbers, respectively,

-at least one viral clearance is performed,

-at least one continuous ultrafiltration,

-at least one continuous diafiltration being effected,

-processing the antibody obtained in said continuous diafiltration using the method of claims 9-11 to obtain an antibody-drug-conjugate, wherein said processing is performed in a continuous, closed and modular manner.

Examples

To summarize:

to provide antibody-drug-conjugates under continuous, pathogen-reduced conditions in a modular and preferably closed process, a production facility with the following units and accompanying process steps was constructed:

unless otherwise stated, a MasterFlex peristaltic pump with an EasyLoad II pump head was used in the treatment. Masterflex or Cflex or Sanipure were used as tubes. All elements of the contact product were gamma irradiated with 25 kGy. In exceptional cases, if gamma irradiation is not allowed for reasons related to material technology, the components are autoclaved at 121 ℃ for at least 20 minutes. Where possible, a ready-to-use disposable ("disposable," "ready-to-use") is used as the unit for gamma irradiation. Typically, all bags are connected to the unit. Between each cell, a gamma-irradiated single-use bag was placed as an equalization tank between the outlet stream of cell n-1 and the inlet stream of cell n. Typically, at that time, there are inlet and outlet flows in each cell. In case the discharge of product liquid is advantageous, the vessel is isolated from the external environment via a 0.2 μm hydrophobic filter. Further, the processing is preferably controlled by the PCS7 processing system.

A storage container containing an antibody solution (e.g., IgG) is provided. Further, a storage bag containing the adapter solution is provided.

The antibody solution and the linker solution are pumped from their reservoirs (also referred to as storage bags) and allowed to mix and react, thereby modifying the two components and producing the antibody-linker substrate. The conjugation is performed in the next step, wherein a solution comprising the biologically active substance (e.g. a solution comprising the toxic groups) is added to the fluid stream comprising the antibody-linker complex and all components are mixed to produce the antibody-linker-toxic group conjugate, e.g. using a static mixer. Toxic group loading was measured via UV-absorption at two specific wavelengths. Optionally, the resulting product stream comprising the antibody drug conjugate and unbound toxic groups is passed through a depth filter and then subjected to ultrafiltration to concentrate the antibody. The product stream is then subjected to diafiltration, for example by washing the product stream 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 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, so that no undesired net flow can flow from the inside of the capillary to the outside of the capillary, or vice versa, and all fluid leaving the at least one capillary ultrafiltration unit in the direction of the product stream is passed through the at least one protective purifier. In order to convey the product stream (= feed stream) into the capillary and the washing fluid (= permeate) on the outside of the capillary of the ultrafiltration unit, one pump is used in each case. For the controlled removal of the product stream (= retentate) from the capillary, a further pump is used. By using this pump (= retentate pump), the control of retentate removal is simplified. This is advantageous because there is no qualified flow sensor that reliably measures the low flow rates (≦ 100 ml/min) typically used in this continuous process. Particularly preferably, peristaltic pumps can be used here, with the advantage that disposable peristaltic pumps are commercially available, so that sterility is available, and disposable technology is also available. In addition, a pump is used for controlled removal of the washing fluid (permeate).

Alternatively, a system comprising a filter disk in an ultrafiltration/diafiltration system may be used instead of the continuous diafiltration described above.

Finally, the product stream was filtered using two 0.2 μm filters operated in parallel. Both the filter and the tube assembly were gamma irradiated. The inlet and outlet lines are connected to the gamma-irradiated bag by sterile connectors that act as equalizing volumes for fluctuating flow rates. For the purpose of venting, the filter is coupled with a hydrophobic 0.2 μm air filter, thereby closing the unit in the sense of the invention. The air filter was either Emflon II from Pall corp. or midiart 2000 from Sartorius Stedim. The venting valve is modified such that it is permeable even when closed, but is nevertheless reliably sealed with respect to the environment.