REMOVAL OF UNBOUND DRUG AFTER ANTIBODY DRUG CONJUGATE COUPLING

Abstract

Disclosed herein is a unit for ultrafiltration and purification (1) of a product stream (3) containing a biological macromolecular product as well as a substance to be separated from the product stream, comprising at least one capillary ultrafiltration module (2), characterized in that at least one pump (4) conveys the product stream into the capillaries of the at least one capillary ultrafiltration module, at least one positive displacement pump (5) conveys the product stream from the capillaries and at least one further pump (6) passes a washing fluid (9) over the outside of the capillaries, the unit includes no measure for circulating the product stream and the washing fluid into the ultrafiltration module, as well as
at least one guard filter (8), through which all fluid leaving the at least one capillary ultrafiltration module in the direction of the product stream is passed.

Claims

1. Unit for ultrafiltration and purification of a product stream comprising a biological macromolecular product as well as a substance to be separated from the product stream, said unit comprising at least one capillary ultrafiltration module, wherein said unit further comprises at least one pump that conveys the product stream into the capillaries of the at least one capillary ultrafiltration module, at least one positive displacement pump that conveys the product stream from the capillaries and at least one further pump that passes a washing fluid over the outside of the capillaries, the unit includes no measure for circulating the product stream and the washing fluid into the ultrafiltration module, and at least one guard filter, through which all fluid leaving the at least one capillary ultrafiltration module in the direction of the product stream (=ultrafiltration retentate) is passed.

2. The unit for ultrafiltration and purification according to claim 1, wherein the guard filter is a depth filter which can bind the substance to be separated from the product stream and/or a column which can bind the substance to be separated from the product stream.

3. The unit for ultrafiltration and purification according to claim 1, wherein the guard filter is an activated carbon depth filter.

4. The unit for ultrafiltration and purification according to according to claim 1, wherein the unit is a unit for continuous ultrafiltration and continuous purification.

5. The unit for ultrafiltration and purification according to claim 1, wherein the biological macromolecular product is an antibody-drug-conjugate.

6. The unit for ultrafiltration and purification according to claim 1, wherein substance to be separated from the product stream is a toxophore.

7. The unit for ultrafiltration and purification according to claim 1, wherein the unit is disposable and/or for single use.

8. Method for continuous ultrafiltration and continuous purification of a product stream comprising biological macromolecular product as well as a substance to be separated from the product stream, said method comprising washing the product stream with a washing fluid via at least one capillary ultrafiltration membrane of a capillary ultrafiltration module, the product stream is conveyed into the capillary and the washing fluid is conveyed over the outside of the capillary, the product stream and the washing fluid are continuously fed into the capillary ultrafiltration module, and are continuously removed from the capillary ultrafiltration module, the product stream and the washing fluid are not circulated into the capillary ultrafiltration module removal of the product stream is regulated such that the rate with which the product stream enters the unit for ultrafiltration and purification is in the range of 20% or is equal to the rate with which the retentate leaves the unit for ultrafiltration and purification to ensure 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 module in the direction of the product stream is passed through at least one guard filter.

9. The method according to claim 8 adapted for a process for continuous, germ-reduced production and/or processing of an antibody-drug-conjugate, wherein the process is carried out in a closed and modular manner.

Description

FIGURES

[0099] FIG. 1 shows a schematic drawing of a unit for ultrafiltration and purification (1) as described herein. In detail said unit for ultrafiltration and purification (1) comprises one capillary ultrafiltration module (2)here a Gambro Polyflux 2H with capillaries that allow molecules from 1-10.0000 Da to pass one pump (4) that conveys the product stream (3)in this example the product stream comprises antibody drug conjugates as well as unbound toxophoresinto the capillaries, one positive displacement pump (5) that conveys the product stream from the capillaries of the ultrafiltration module (2), two pumps (6,7) that pass a washing fluidin this case a diafiltration buffer (9)over the outside of the capillaries and one activated carbon depth filter (8), through which all fluid leaving the at least one capillary ultrafiltration module in the direction of the product streami.e. the retentate (13)is passed. The term retentate refers to both the product stream leaving the unit for ultrafiltration and purification and the product stream leaving the activated carbon depth filter. The diafiltration buffer leaving the capillary ultrafiltration module (2) contains the unbound toxophores, i.e. constitutes the permeate (10) in this example. In this specific setting, the product stream is entering the unit for ultrafiltration and purification (1) at a rate of 10 ml/min. This is also the rate at which the product stream passes the active carbon depth filter (8). The diafiltration buffer (9) enters and leaves the capillary ultrafiltration module (2) at a rate of 30 ml/min. Thus, the driving forces that cause the separation of the product stream and the toxophores in this case are the particle size allowed to pass by the filtration membranes in the capillaries of the capillary ultrafiltration module, the difference in flow rate between the product stream and the diafiltration buffer that cause a pressure, as well as the differences in gradient e.g. of the between the product stream and the diafiltration buffer.

[0100] FIG. 2 corresponds to FIG. 1, but in addition to the details of FIG. 1 a point is depicted where a

[0101] sample (11) is taken before the product stream enters the unit for ultrafiltration and purification. A second sample (12) is taken before the retentate (13) enters the activated carbon depth filter (8) and a final sample (14) is taken after the retentate (13) has passed the activated carbon depth filter (8).

[0102] FIG. 3 Shows a schematic diagram of the toxophore concentration in parts per billion (ppb) before the product stream (feed) (3) enters the unit for ultrafiltration and purification, before the product stream (retentate) (13) enters the activated carbon depth filter (8) and after the product stream (13) leaves the activated carbon depth filter. It is depicted that the toxophore concentration of the product stream, which is already below the critical concentration, before the product stream enters the activated carbon depth filter is further reduced by the activated carbon depth filter.

[0103] FIG. 4 The set-up is as described above for FIG. 2, but the product stream was spiked with an additional load of toxophore (15) prior to entering the active carbon depth filter in order to demonstrate that the activated carbon depth filter can act as safeguard to ensure that the concentration of the toxophore is below the critical threshold once the product stream leaves the unit for ultrafiltration and purification.

EXAMPLES

[0104] 1) Exemplary Operation Mode Using a Product Stream Comprising Toxophores

[0105] In order to demonstrate that the unit for ultrafiltration and purification as described herein reliably and effectively separates toxophores from a product stream comprising toxophores and antibody-toxophore conjugates the following experiments are carried out. A unit for ultrafiltration and purification as depicted in and described with reference to FIG. 2 is operated with a flow rate of the product stream of 5 ml/min and a flow rate of the wash buffer of 15 ml/min. A sample (11) is taken before the product stream enters the unit for ultrafiltration and purification. A second sample (12) is taken before the retentate enters the activated carbon depth filter (8) and a final sample (14) is taken after the retentate has passed the activated carbon depth filter (8). As shown in FIG. 3 the toxophore concentration is above the critical threshold in the sample (11) taken before the product stream enters the unit for ultrafiltration and purification. As shown in FIG. 3 the toxophore concentration is below the critical threshold in the sample (14) taken after the product stream has passed the activated carbon depth filter.

[0106] 2 Exemplary Operation Mode Using a Marker Molecule Instead of a Toxophore

[0107] Free toxophores are potentially hazardous substances. Thus, the following model experiment was carried out using the Sunset Yellow dye instead of toxophores thereby avoiding any risk that might be associated with using toxophores. The Sunset Yellow Dye hence mimicks the presence of free toxophores.

[0108] 2.1 General Set Up Using a Marker Molecule

[0109] In detail, a product stream comprising monoclonal antibodies in a citric buffer/sodium chloride buffer at pH 5.5 and a conductivity 8.5 mS/cm was spiked with the marker molecule Sunset Yellow (CAS 2783-94-0, Sigma Aldrich 465-223-25). The monoclonal antibody concentration was 12.85 g/L the sunset yellow concentration was 913 mg/L.

[0110] The experimental set-up of the counter current diafiltration was the same as depicted in FIG. 1 except that not guard filter (8) was present. The hollow fiber ultrafiltration membrane module (Revaclear 300, Gambro) was connected to four peristaltic pumps Masterflex Easy Load II. The product stream comprising monoclonal antibodies entered the lumen side at the bottom of the membrane module. Product stream (=feed stream) and retentate stream (i.e. the product stream after leaving the unit for ultrafiltration and purification described herein) were set to the same flow rate of 10 mL/min. The diafiltration buffer was pumped in to the top shell side of the membrane module at a flow rate of 60 mL/min and was pumped out of the module at approximately the same rate in a way that the pressure at the inlet of the shell side is controlled to 100 mbar.

[0111] Pendotech pressure sensors were used at the feed inlet and the diafiltration buffer inlet. As a diafiltration buffer 20 mM L Histidin, 50 mM Arginin*HCl, 50 mM Glycin, pH 4.8 and 5.5 mS/cm was used

[0112] A central Siemens PCS7 control system was used to control the peristaltic pumps and the pressures.

[0113] The results (not shown) demonstrated that the counter current diafiltration under normal circumstances reliably cleared the marker molecule from the retentate stream.

[0114] 2.2. Operation Mode Using Guard Filter

[0115] In a second experiment a monoclonal antibody solution was again spiked with Sunset Yellow to a monoclonal antibody concentration of 11.7 g/L and a SunSet yellow concentration of 4.5 mg/L.

[0116] The resulting solution was filtered through a guard filterhere a Millistak charcoal depth filter, Type MCR4023CL3 (filtration area 23 cm.sup.2, hold-up 22 mL)in order to demonstrate that in the very unlikely case that the above described diafiltration (2.1) would not clear all marker molecules (i.e. free toxophores) from the process stream the guard filter reliably binds the marker molecules (i.e. free toxophores). A Masterflex Easy Load II pump was used as process pump Prior to processing the guard filter was primed with diafiltration buffed (described above) at a flow rate of 6 mL/min until no more air was visible in the vent outlet. The vent outlet was then closed and the filter was flushed with 650 mL diafiltration buffer at 23 mL/min.

[0117] Samples of 40 mL were taken at the outlet of the filter.

TABLE-US-00001 Sample No mAB (g/L) SunSet Yellow Feed 11.7 4.5 1 2.3 0 2 10.6 0 3 11.3 0 4 11.4 0.3 5 11.5 0.2

[0118] Approximately 12 L of product stream comprising the monoclonal antibody and the Sunset Yellow were processed in this manner. The monoclonal antibody concentration was measured in the product stream (=feed stream) as it entered the unit for ultrafiltration and purification and in the product stream when it left the unit for ultrafiltration and purification (i.e. as retentate stream) using UV 280 nm measurement (DropSense 16, Trinian). The retentate concentration was slightly increased with 14.45 g/L which can be caused by a slight slower retentate pump.

[0119] The SunSet Yellow concentration was measured using a UVVIS spectrometer NanoDrop 2000c at 481 nm using a 10 mm cuvette. The retentate concentration was measured to be 0.3 mg/L.

[0120] In other words, the charcoal depth filter reliably binds the marker molecules (free toxophores) and hence is suited as guard filter for the continuous purification and diafiltration of a product stream comprising free toxophores.

[0121] Overall this enables a continuous and even safer process for the removal of toxophores from a product stream.