MODULAR SYSTEM AND PROCESS FOR THE CONTINUOUS, MICROBE-REDUCED PRODUCTION AND/OR PROCESSING OF A PRODUCT

Abstract

The invention provides a method for the continuous, microbe-reduced production and/or processing of a biopharmaceutical, biological macromolecular product from a heterogeneous cell culture-fluid mixture, comprising the steps of: (a) providing a particle-free fluid from a heterogeneous cell culture-fluid mixture containing the product, in the form of a product stream, (b) at least one filtration, providing a filtrate, (c) at least two chromatography steps for purifying the product, (d) at least one virus depletion, (e) at least one ultrafiltration and/or at least one diafiltration of the product stream of steps (b), (c) and/or (d), characterized in that the at least two chromatography steps from (c) comprise a purification via at least two chromatography columns and/or membrane adsorbers in each case and that the process is carried out in a closed and modular manner The invention further provides a corresponding modular system for carrying out said method.

Claims

1. Method for the continuous, microbe-reduced production and/or processing of a biopharmaceutical, biological macromolecular product from a heterogeneous cell culture-fluid mixture, comprising: (a) providing a particle-free fluid from a heterogeneous cell culture-fluid mixture containing the product, in the form of a product stream, (b) at least one filtration, providing a filtrate, (c) at least two chromatography steps for purifying the product, (d) at least one virus depletion, (e) at least one ultrafiltration and/or at least one diafiltration of the product stream of (b), (c) and/or (d), wherein the at least two chromatography steps from (c) comprise a purification via at least two chromatography columns and/or membrane adsorbers and wherein the method is carried out in a closed and modular manner.

2. Method according to claim 1, wherein one or more further steps for adjusting the pH and/or the conductivity and/or filtration steps and/or concentration steps and/or a buffer exchange are carried out between the at least two chromatography steps in (c) and/or after the virus inactivation in (d).

3. Method according to claim 1, wherein all elements used in (a) to (e) that come into contact with the product are subjected to microbe reduction via a suitable microbe-reduction method, the microbe-reduction method optionally being selected from the group consisting of gamma irradiation, beta irradiation, autoclaving, ethylene oxide (ETO) treatment, ozone treatment (O.sub.3), hydrogen peroxide treatment (H.sub.2O.sub.2) and steam-in-place (SIP) treatment.

4. Method according to claim 1, wherein all the elements that are used from (b) onwards and which come into contact with product are disposable articles or are used as disposable articles.

5. Method according to claim 1, wherein all inlet fluids are filtered through a microbe-reduction filter and all outlets are optionally protected by a microbe barrier preventing a back-growth.

6. Method according to claim 1, wherein the modular process steps are carried out in modules, the modules being connected to one another, the modules optionally being connected to one another by welding or by aseptic connectors.

7. Method according to claim 1, wherein all liquids, gases and solids used in (a) to (e), are subjected to microbe reduction the microbe reduction optionally being achieved by a filtration through a filter having a pore size of optionally ?0.45 ?m, and, in-process sterilization is optionally not carried out during the process.

8. Method according to claim 1, wherein a degassing of all fluids which come onto the at least two chromatography columns is carried out before (c), the degassing optionally being achieved by at least one bubble trap and/or by at least one hydrophobic microfiltration membrane via vacuum and/or by treatment with ultrasound and/or by sparging with helium.

9. Method according to claim 1, wherein the particle-free fluid from a) is subjected to at least one ultrafiltration against a microbicide-containing buffer, the microbicide optionally being selected from the group consisting of imidazole, benzoic acid, sorbic acid, para-hydroxybenzoic esters, sulphites, disulphites, azides, ortho-phenylphenol, nisin, natamycin, hexamethylenetetramine, dimethyl dicarbonate, nitrites, nitrates, acetic acid, ascorbic acid, isoascorbic acid, L-lactic acid, propionic acid, boric acid and lysozyme.

10. Method according to claim 1, wherein the biopharmaceutical, biological macromolecular product is a protein, peptide or comprises a DNA or RNA, the protein or peptide being selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant proteins and protein vaccines, and the DNA or RNA being part of a DNA and/or RNA vaccine.

11. Method according to claim 1, wherein the at least two chromatography columns and/or membrane adsorbers of (c) bind product in accordance with the principle of affinity, via ionic interactions, via metal chelate binding, via hydrophobic interactions or via van der Waals forces, the at least two chromatography columns and/or membrane adsorbers in the case of binding in accordance with the principle of affinity comprising a ligand optionally selected from the group consisting of protein A, protein G, protein L, IgM, IgG and a recombinant protein which is different from protein A, protein G, protein L, IgM and IgG and which has an affinity for the product.

12. Method according to claim 1, wherein the method of (a) to (e) has a run time of at least 4 hours, optionally of at least 8 hours, optionally of at least 12 hours, optionally of at least 24 hours, optionally of at least 48 hours, optionally of at least 7 days, optionally of at least 4 weeks, and optionally of at least 8 weeks.

13. Method according to claim 1, wherein at least one filtration step comprising at least one filter is carried out between a) to e) and/or thereafter.

14. Method according to claim 13, wherein the filter is automatically changed under microbe-reduced conditions, the automatic filter change optionally comprising: (i) switching of the flow path to a new filter in the event of exceeding of a threshold at a pressure sensor on the non-filtrate side with closing of the flow path, the product in the used filter optionally being pushed into the filtrate side by a gas or a liquid, or in the event of exceeding of a maximum time of the used filter in the flow path, or in the event of exceeding of a maximum volume of filtrate through the used filter, (ii) venting of the new filter via an air filter having a pore size of optionally ?0.25 ?m at the venting valve of the new filter, optionally with conveyance of product into the new filter by means of a feed pump, or into a closed bag connected in a microbe-reduced manner, (iii) detecting the completion of venting of the new filter on the non-filtrate side by means of the pressure sensor or a fill-level sensor or a balance or a liquid detector, (iv) opening the filtrate outlet and closing the flow path between the venting valve (20) and the air filter by means of a valve, and (v) exchanging the used filter for a new filter.

15. Modular system for continuous, microbe-reduced production and/or processing of a biopharmaceutical, biological macromolecular product from a heterogeneous cell culture-fluid mixture, comprising the following modules: (a) at least one filtration module, (b) at least one chromatography module, comprising at least two chromatography columns and/or membrane adsorbers, (c) at least one ultrafiltration module and/or at least one diafiltration module and/or at least one dialysis module, and (d) at least one module for continuous virus depletion, wherein the modular system is closed and microbe-reduced.

16. Modular system according to claim 15, wherein all elements used in modules (a) to (d) that come into contact with the product are subjected to microbe reduction by means of a microbe-reduction method, the microbe-reduction method optionally being selected from the group consisting of gamma irradiation, beta irradiation, autoclaving, ethylene oxide (ETO) treatment, ozone treatment (O.sub.3), hydrogen peroxide treatment (H.sub.2O.sub.2) and steam-in-place (SIP) treatment.

17. Modular system according to claim 15, wherein all elements used in modules (a) to (d) that come into contact with the product are disposable articles or are used as disposable articles and that the modules are optionally connected to one another by welding or by aseptic connectors.

18. Modular system according to claim 15, wherein all inlet fluids pass through a microbe-reduction filter and that all outlets are optionally protected by a microbe barrier preventing a back-growth of microorganisms.

Description

[0137] The following are shown:

[0138] FIG. 1 shows schematically a process diagram of one embodiment of the process according to the invention. The numbers between parentheses are references to the mass balance, as listed in Example 1 and Table 1.

[0139] FIG. 2 shows schematically the operating principle of the filtration step of the process and of the system with replacement of a used filter 17 by welding-in of a new filter 16.

[0140] FIG. 3 shows exemplarily the two process steps of virus inactivation and neutralizationtwo units which are set up in a modular manner, with the pH probes pH0501 and pH0502 being autoclaved and the rest being gamma-irradiated. The pH probes pH0501 and pH0502 are then welded into an assembly. The bags are connected via aseptic GE ReadyMate? connectors. The connection to Prot-A and to the filtration unit is at first weldedshut and is then welded to the various units.

[0141] FIG. 4 shows exemplarily the modular structure of the system, with all inlet streams and outlet streams being connected to the environment via a microbe barrier 10, 13. The exemplary modular system 1 consists of three filtration modules 2 each having two filters 13, which are operated alternatively, two chromatography modules 3 having two chromatography columns 4 or two membrane adsorbers 5, a virus depletion step, for example virus inactivation 9, an ultrafiltration module 6 and a diafiltration module 7. Gas bubbles are removed from the buffers via a hydrophobic filter 15 or bubble trap 14.

[0142] Table 1 shows the averaged flow rates and antibody concentrations of the positions shown in FIG. 1.

[0143] The reference numerals used are:

[0144] 1=Modular system

[0145] 2=Filtration module

[0146] 3=Chromatography module

[0147] 4=Chromatography column

[0148] 5=Membrane adsorber

[0149] 6=Ultrafiltration module

[0150] 7=Diafiltration module

[0151] 8=Dialysis module

[0152] 9=Virus depletion

[0153] 10=Microbe-reduction filter

[0154] 11=Microbe barrier

[0155] 12=Aseptic connector

[0156] 13=Filter having a pore size of preferably ?0.45 ?m

[0157] 14=Bubble trap

[0158] 15=Hydrophobic microfiltration membrane

[0159] 16=New filter

[0160] 17=Used filter

[0161] 18=Pressure sensor

[0162] 19=Air filter, pore size preferably ?0.25 ?m

[0163] 20=Venting valve of the new filter 16

[0164] 21=Feed pump

[0165] 22=Fill level sensor

[0166] 23=Balance

[0167] 24=Valve

[0168] 25=Waste stream

[0169] 26=Product stream

[0170] 27=Buffer

[0171] 28=Liquid detector

[0172] 29=Bag

EXAMPLE 1

[0173] To purify a protein in a continuous and microbe-reduced manner from a heterogeneous cell culture/fluid mixture, a miniplant having the following modules and associated process steps was set up:

[0174] Unless otherwise noted, MasterFlex peristaltic pumps having an EasyLoad II pump head were used in the process. The tubing used was Masterflex LS16 or Cflex or Sanipure. All used components coming into contact with product were subjected to 25 kGy gamma irradiation. In exceptional cases where gamma irradiation was not feasible because of the material, components were autoclaved at 121? C. for 20 min, e.g.sub-assemblies having pH probes or virus filters. Where possible, ready-to-use disposable articles were used as gamma-irradiated modules. Without exception, this was the case for all bags. Said bags were generally connected to the modules using ReadyMate? connectors from General Electric (GE). Between each module, a single-use gamma-irradiated bag (ReadyCircuit 1 litre, GE) was placed as compensation tank between the outlet stream of module n-1 and the inlet stream of module n. Generally, there was an inlet stream and an outlet stream at that point in time in each module. Where a venting of the product liquid was advantageous, the tanks were sealed off from the environment via a hydrophobic 0.2 ?m filter.

[0175] A. Upstream

[0176] i) Perfusion Reactor

[0177] For the continuous production of an IgG monoclonal antibody, a 10 litre perfusion reactor was used. The viable cell density was 60-70 million cells/ml in the steady state. The titre was ?115 mg/l. Production was carried out for 28 days using two parallel perfusion reactors.

[0178] ii) Cell Retention System

[0179] The product was continuously discharged across an inclined plates separator (settler), by means of which the majority of cells were retained.

[0180] B. Downstream DSP-1

[0181] i) Cell Clarification

[0182] Clarification was carried out using Sartoguard NF 0.2 ?m filters (T-style, MaxiCap, 0.65 m.sup.2) operated in parallel. FIG. 2 shows how a closed low-microbe process was realized here. Both the filters and the tubing assembly were gamma-irradiated. The inlet and outlet lines were connected via aseptic connectors to gamma-irradiated bags (GE ReadyCircuit 1 litre), which were used as compensatory volumes for fluctuating flow rates. For the purpose of venting, the filters were coupled to hydrophobic 0.2 ?m air filters, and as a result, the module was closed in the meaning of the invention (FIG. 2). The air filter was either an Emflon II from Pall Corp. or a Midisart 2000 from Sartorius Stedim. The venting valves were modified such that they were permeable even in the closed state, but still reliably sealed off the environment. To this end, the inner sealing ring of the venting valve was removed on the Sartoguard NF and the valve was closed prior to gamma irradiation. Said valve was additionally secured against opening. As a result, the valve was closed in the safe state, and this was characterized by a tight fit. The venting valve was connected to the air filter via a length of tubing. Between the venting valve and the air filter, the length of tubing was inserted into a tubing pinch valve. The filter was filled until liquid entered the air filter by way of the venting valve and the tubing. The hydrophobic venting filter then blocked the liquid. At the same time, the process system blocked the production stream by ways of a valve on the filtrate side, and so there was a pressure rise in front of the filter. Said pressure rise was detected by means of a Pendotech pressure sensor. If a threshold of 0.5 bar was exceeded, the pinch valve of the length of venting tubing was closed, and the valve on the filtrate side was opened.

[0183] ii) Concentration and Rebuffering

[0184] The filtrate from the i) cell clarification was firstly continuously concentrated by a factor of 10 using an ultrafiltration hollow-fibre membrane (GE Healthcare ReadytoProcess, 0.2 m.sup.2, gamma-irradiated). The circulation pump used was a disposable QuattroFlow 1200 SU pump, the pump head of which was integrated into the tubing assembly prior to gamma irradiation.

[0185] The media constituents of the concentrated product were then exchanged for a 50 mM imidazole/NaCl buffer across a Gambro Revaclear 300 dialysis membrane. The module is provided sterile-packed by the manufacturer and was connected to the gamma-irradiated tubing assembly in a biological safety cabinet. The permeate from the concentration and the media-containing waste stream were conducted into a gamma-irradiated 200 litre Sartorius Flexboy. The Flexboy was exchanged by rewelding using a Sartorius welder.

[0186] 0.2 ?m Filtration

[0187] Prior to filling, the product was continuously filtered into a 200 litre Flexboy using gamma-irradiated Sartoguard NF filters (MaxiCap size 8) operating alternatively. The setup and operation were similar to B. Downstream DSP-1-i) Cell clarification.

[0188] C. Downstream DSP-II

[0189] 0.2 ?m Filtration

[0190] After storage, the product from DSP-1 was filtered again in order to protect the downstream chromatography columns from particles.

[0191] 1. Capture Chromatography

[0192] Mabselect Sure (GE) was used as Prot-A resin to isolate the IgG. The IgG was concentrated by up to a factor of 10 and the majority of the contaminants was removed. A continuous BioSMB system from Tarpon Biosystems, Inc. was used with 12 columns (ID 16 mm, L 80 mm), with 8 columns being in the loading zone (2 columns in series and 4 parallel series). The entire flow path including the columns was rendered microbe-reduced by sanitization or gamma irradiation. The load per cycle was 32 column volumes per column. The buffers used were acetate buffers with differing molarity, pH and conductivities. All buffers were filtered into a gamma-irradiated bag using a gamma-irradiated or autoclaved 0.2 ?m filter. The outlet tubing of the buffer bags was welded to the inlets of the BioSMB system. Said system had at each of its inlets a gamma-irradiated degasser membrane (Liquicell Micro Module, Membrana). Similarly, the product line was welded to the inlet of the BioSMB system via such a degasser. All inlet streams were then degassed by means of a vacuum pump at 50 mbar.

[0193] 1. Virus Inactivation and Neutralization

[0194] Virus inactivation and neutralization consisted of three modules and was situated between the capture chromatography and a 0.2 ?m filtration: (a) a homogenization loop with a peristaltic pump M0502; (b) a residence-time loop shown schematically as coiled tubing; (c) a neutralization bag in which the pH could be adjusted to 7.5. The modules were individually prefabricated and gamma-irradiated in line with the welding points in FIG. 3, with the ends being welded closed in each case.

[0195] Line segments with pH probes, in this case pH0501 and pH0502, were autoclaved. pH probe segments were then welded into assemblies. As shown, bags were connected via aseptic GE ReadyMate? connectors. The connections to the Prot-A eluate line and the filtration module were firstly welded shut and were then welded to the various modules.

[0196] 0.2 ?m Filtration

[0197] As it was possible for proteins to precipitate after a pH shift, the precipitated protein were filtered off.

[0198] Chromatography (Intermediate and Polish)

[0199] The product of the above 0.2 ?m filtration was purified via two chromatography steps by means of four sequentially (4-PCC) operated 2.5 ml Capto Adheres (2.5 ml GE) and then two alternatingly operated 20 ml anion exchangers (Pall Hypercel StarAX). In this purification, Prot-A leachables, DNA, HCP and aggregates were removed. The two chromatography steps were connected to one another via a gamma-irradiated bag (GE ReadyCircuit? 1 litre), in which conductivity was adjusted to 7.5 mS/cm by the supply of water in line with the requirements of the anion exchanger.

[0200] The entire flow path including the columns was sanitized or gamma-irradiated. The load per cycle was 50 column volumes per column. The buffers used were acetate buffers with differing molarity, pH and conductivities. All buffers were filtered into a gamma-irradiated bag using a gamma-irradiated or autoclaved 0.2 ?m filter. The outlet tubing of the buffer bags was welded to the inlets of the BioSMB system. Said system had at each of its inlets a gamma-irradiated degassing membrane (Liquicell Micro Module, Membrana). Similarly, the product line of the above 0.2 ?m filtration was welded to the inlet of the BioSMB system via such a degasser. All inlet streams were then degassed by means of a vacuum pump at 50 mbar. The waste stream was conducted into a gamma-irradiated 200 litre Sartorius Flexboy.

[0201] The Flexboy was exchanged by rewelding using a Sartorius welder.

[0202] The product stream was again collected in a gamma-irradiated product bag (GE ReadyCircuit 1 litre).

[0203] Prefiltration

[0204] From the product bag of the polishing chromatography, the product solution was firstly prefiltered using a 0.1 ?m capsule (Sartopore2, MidiCap size 9, 0.2 m.sup.2). The procedure and setup were similar to B. Downstream DSP-1-i) Cell clarification.

[0205] Virus Filtration

[0206] The outlet line from the prefiltration was directly connected by welding via a peristaltic pump to the inlet of the virus filtration from C. Downstream DSP-II. Otherwise, the setup and operation of the virus filtration from C. Downstream DSP-II were similar to C. Downstream DSP-II-0.2 ?m Filtration. However, the virus filter used was a Virosart CPV filter (MidiCap size 9, 0.2 m.sup.2), which was rinsed and autoclaved according to the manufacturer's instructions. Again, the filters were welded into the assembly. The product stream was again pumped into a gamma-irradiated product bag (GE ReadyCircuit 1 litre).

[0207] Final Concentration and Rebuffering

[0208] The final concentration and rebuffering was set up similarly to the above B. DSP-I-ii) Concentration and rebuffering and differed only in that an autoclaved UV cell was integrated into the concentration loop for monitoring of the product concentration. The rebuffering was carried out similarly to the above B. DSP-I-ii) Concentration and rebuffering, with a 50 mM phosphate buffer, pH 7.5 being used in this case. The product stream was again pumped into a gamma-irradiated product bag (GE ReadyCircuit 1 litre).

[0209] 0.2 ?m Filtration

[0210] The final filtration was carried out as described above in B. DSP-1 i) across gamma-irradiated Sartopore 2 capsules (Midicap size 7, 0.05 m.sup.2) into a gamma-irradiated 5 litre GE ReadCircuit bag. When the fill level of the final bag was acceptable, said bag was welded off and a new bag was welded to the process.

[0211] A regularly performed run time of the process according to the invention, as described in Example 1, was 3 days with no microbial growth, with the chromatography columns being sanitized by 40% isopropanol+0.5 M NaOH. In the case of run times of over 3 days for the process according to the invention, the chromatography columns were gamma-irradiated.

[0212] The averaged flow rates and antibody concentrations of the positions shown in FIG. 1 are summarized in Table 1.

TABLE-US-00001 TABLE 1 Process Volumetric Antibody Antibody stream flow rate concentration flow rate ml min.sup.?1 g l.sup.?1 g d.sup.?1 Upstream & 1 33.3 0.0 0.00 Downstream I 2 33.3 0.1 5.5 3 33.3 0.1 5.5 4 5.3 0.7 5.4 5 5.3 0.7 5.4 Downstream II 6 30 0.7 30.0 7 30 0.7 30.0 8 4.2 4.6 28.0 9 4.6 4.2 28.0 10 9.3 1.8 23.6 11 9.3 1.7 23.3 12 2.0 8.0 22.8 13 2.0 8.0 22.7

[0213] The work which led to this application was funded in accordance with the Bio.NRW: MoBiDiKModulare BioproduktionDisposable and Kontinuierlich [Bio.NRW: MoBiDiKmodular bioproductiondisposable and continuous] grant agreement as part of the European Regional Development Fund (ERDF).