Method for removing biopolymer aggregates and viruses from a fluid

Abstract

The present invention relates to a method for removing biopolymer aggregates and viruses from a fluid. In a first step, the biopolymer aggregates are selectively removed by filtration through a porous, polyamide-comprising shaped body having a native surface. In a second step, the biopolymer aggregate-free fluid is filtered through at least one suitable virus-retentive membrane.

Claims

1. A method for removing biopolymer aggregates and parvoviruses from a fluid, comprising the steps of: (a) filtering the fluid containing the biopolymer aggregates and parvoviruses through a porous, polyamide shaped body consisting of nylon-6, the porous, polyamide shaped body having internal and external surface regions and being configured so that all of the internal and external surfaces regions have the same chemical properties, the filtering of the fluid containing the biopolymer aggregates and parvoviruses through the porous, polyamide shaped body causing the biopolymer aggregates to be selectively depleted from the fluid by adsorption, whereas the parvoviruses permeate through the shaped body in a biopolymer aggregate-depleted fluid; and (b) filtering the biopolymer aggregate-depleted fluid from step (a) through at least one parvovirus-retentive membrane having a molecular weight cut-off of from 100 to 1000 kD, wherein the content of parvoviruses in the fluid that has passed through the at least one parvovirus-retentive membrane is reduced by at least 99.9% with respect to the content of parvoviruses prior to carrying out step (a); wherein: the biopolymer aggregates are selected from the group of the dimers, trimers or multimers of peptides, proteins, nucleic acids or mixtures thereof; the fluid comprises a human blood plasma product, a protein solution obtained from a cell culture, a protein solution obtained from extraction of plant products, or a protein solution obtained from microorganisms; the pH of the fluid during steps (a) and (b) is 9; the porous, polyamide shaped body comprises a microporous membrane; and the membrane used in step (b) comprises a material selected from the group consisting of polyethersulfone and polysulfone.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph that shows the course of filtration of Example 1.

(2) FIG. 2 shows a plot of t/V against t of the filtration shown in FIG. 1.

(3) FIG. 3 is a graph that shows the course of filtration of the Comparative Example 1.

(4) FIG. 4 shows a plot of t/V against t for the Comparative Example 1.

(5) FIG. 5 is a graph that shows the course of filtration of both Example 2 and Comparative Example 2.

(6) FIG. 6 is a graph that shows the filtration of the protein solution at pH=6 across two different membranes.

(7) FIG. 7 is a graph similar to FIG. 6, but showing filtration of the protein solution at pH=9.

(8) FIG. 8 is a graph that shows the course of filtration for Example 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(9) The present invention will now be more particularly elucidated with reference to the following nonrestricting examples.

EXAMPLES

(10) In the following examples, the filtration capacity Vmax is the asymptotic value for the filtration volume at 100% blockage of the filter, obtained as the reciprocal of the slope of the line from a plot of the quotient t/V against t in the case of filtration at constant pressure as described in Ho et al., Journal of Colloid and Interface Science 232, 389-399 (2000) (equation 1):

(11) $\begin{array}{cc}\frac{t}{V}=\frac{1}{V_{\max }}.Math.t+\frac{1}{Q_0}& \left(\mathrm{equation}1\right)\end{array}$

(12) In the equation, t refers to the filtration time, V the filtration volume, Vmax the filtration capacity and Q0 the initial flow in the case of the filtration at constant pressure.

Example 1: Filtration of a Protein Solution of a Monoclonal Antibody from the Cell Line CHO dg44 ST1-C6

(13) The monoclonal antibody in the protein solution originates from the cell line CHO dg44 ST1-C6 and was made available by means of the following method:

(14) Firstly, a cell culture in which the target protein was produced was set up. Culturing took place under usual conditions known to a person skilled in the art, using a Biostat® CultiBag RM single-use bioreactor (Sartorius Stedim Biotech GmbH) with a culture volume of 25 L and an inoculation cell density of 5×10.sup.5 cells/mL and the growth medium Pro CHO5 (Lonza BE12-766P).

(15) After attainment of the maximum cell density after 9 days, the cells were harvested and purification carried out. In a typical method, the cell suspension was firstly filtered across depth filter layers and particle filters having a pore size of 0.2 μm. The antibody was isolated from the cell-free solution in chromatographic methods, such as protein A affinity chromatography and ion-exchange chromatography.

(16) To prepare the protein solution for the filtration, the purified solution after the chromatographic steps was subjected to ultrafiltration/diafiltration. To this end, a Sartocon Slice, Hydrosart® 30 kD MWCO (3051445901E-SW) filtration cassette having a filtration area of 0.1 m.sup.2 and a molecular weight cut-off of 30 kD was used. After this step to alter the buffer composition and protein concentration, the solution of the antibody having a concentration of 10 g/L in phosphate buffer, pH 6.6 (Sigma Aldrich, catalog No. P8165), and 10 mM EDTA (Sigma Aldrich, catalog No.: E6758) was obtained.

(17) The protein solution of the monoclonal antibody was guided, in step (a), across a porous polyamide surface composed of nylon-6 in the form of a microporous membrane (pore size 0.2 μm, external area 17.5 cm2). Subsequently, the filtrate from step (a) was filtered, in step (b), across one layer of a virus-retentive membrane, Virosart® CPV Minisart (polyethersulfone membrane, pore size 0.02 μm, area 5 cm.sup.2, Sartorius Stedim Biotech GmbH), at 1 bar.

(18) FIG. 1 shows a graph of the course of the filtration of the monoclonal antibody, where the filtered volume, normalized to an area of 1 m2, is plotted on the y-axis and the filtration time in minutes is plotted on the x-axis. FIG. 2 shows a plot of t/V against t of the same filtration, from which the reciprocal of the slope of the line allows a filtration capacity V.sub.max of 365 L/m.sup.2 to be determined according to equation 1.

Comparative Example 1: Filtration of a Protein Solution of a Monoclonal Antibody from the Cell Line CHO dg44 ST1-C6

(19) The filtration of comparative example 1 was carried out analogously to example 1, with the microporous polyamide membrane having a pore size of 0.2 μm in step (a) having been replaced by a polyethersulfone membrane, Sartopore® 2 (pore size 0.1 μm, area 17.5 cm2, Sartorius Stedim Biotech GmbH). In step (b), the filtrate from step (a) was filtered normally, i.e. in dead-end filtration across one layer of a virus-retentive membrane, Virosart® CPV Minisart (area 5 cm.sup.2, Sartorius Stedim Biotech GmbH), at 1 bar.

(20) FIG. 3 shows the course of the filtration, with the filtered volume on the y-axis, normalized to an area of 1 m2, and the filtration time in minutes on the x-axis. FIG. 4 shows a plot of t/V against t of the same filtration, from which the reciprocal of the slope of the line allows a filtration capacity Vmax of 33 L/m2 to be determined according to equation 1. The filtration capacity is thus less than 10% of the filtration capacity when using the porous polyamide surface in step (a) of example 1.

Example 2: Filtration of a Solution of a Human IgG Protein

(21) For the filtration, a solution of a human IgG protein (5% strength solution, SeraCare, catalog No. HS-475-1L) diluted with phosphate buffer, pH 6.6 (Sigma Aldrich, catalog No. P8165), and EDTA (Sigma Aldrich, catalog No. E6758) to a concentration of 10 g/L protein and 10 mM EDTA was used. The solution was contacted, in step (a), with a porous polyamide surface composed of nylon-6 in the form of a microporous membrane (pore size 0.1 μm, external area 7 cm.sup.2) by filtration normal to the external surface. Subsequently, in step (b), the filtrate from step (a) was filtered normally across a double-layer Virosart® CPV Minisart virus filter (area 5 cm.sup.2, Sartorius Stedim Biotech GmbH) at 2 bar.

Comparative Example 2: Filtration of a Solution of a Human IgG Protein

(22) Comparative example 2 was carried out analogously to example 2, but, in step (a), with the porous polyamide surface from example 2 having been replaced by a polyethersulfone membrane, Sartopore® 2 (pore size 0.1 μm, area 17.5 cm2, Sartorius Stedim Biotech GmbH).

(23) FIG. 5 shows a graph of the course of the filtration of the human IgG protein of both example 2 (symbol .circle-solid.) and comparative example 2 (symbol ◯), where the filtered volume, normalized to an area of 1 m2, is plotted on the y-axis and the filtration time in minutes is plotted on the x-axis. The filtration capacity is 109 L/m2 for the filtration with use of the polyethersulfone membrane. The polyamide surface as prefilter results in a filtration capacity of 217 L/m2.

(24) The use of the polyamide surface in the first step increases the filtration capacity in the case of this medium by about a factor of 2 relative to the use of a microporous polyethersulfone membrane.

Example 3: Investigation of pH Dependence

(25) A protein solution was prepared as described in example 1, and the concentration was adjusted to 10 g/L. The pH was adjusted via changes in the buffer composition with the aid of citric acid (Sigma Aldrich, catalog No. C2404) and tris base (2-amino-2-(hydroxymethyl)propane-1,3-diol, Sigma Aldrich, catalog No. T1503) to pH=6.0 and 9.0, respectively. The two protein solutions were adjusted to a uniform conductivity of 15 mS/cm by addition of sodium chloride (Sigma Aldrich, catalog No. S5886). Both solutions were filtered across a Sartopore® 2 polyethersulfone membrane (pore size 0.1 μm) in order to exclude the effects of different pore sizes in further prefiltration steps. In step (a), the protein solutions were filtered across either a charged Sartobind® S membrane (external area 6 cm.sup.2, Sartorius Stedim Biotech GmbH) or a microporous polyamide membrane (pore size 0.1 μm, external area 6 cm.sup.2). In step (b), the solutions were filtered across one layer of a Virosart® CPV parvovirus filter (external area 2.5 cm.sup.2, Sartorius Stedim Biotech GmbH) and at a differential pressure of 2 bar.

(26) In FIG. 6, the filtration of the protein solution at pH=6 across the porous polyamide surface in the form of a microporous polyamide membrane (symbol .circle-solid.) and the filtration of the protein solution at pH=6 across the charged Sartobind® S membrane (symbol ◯) are shown together. In FIG. 7, the filtration procedures for the same protein solution at pH=9 are shown. In both figures, the filtered volume, normalized to an area of 1 m2, is plotted on the y-axis and the filtration time in minutes is plotted on the x-axis.

(27) The difference between the Sartobind® S membrane and the polyamide membrane as the first step prior to filtration across a virus-retentive membrane can be clearly seen. The series of filtration experiments implemented here illustrates that the increase in the filtration capacity of a virus-retentive membrane when using polyamide surfaces occurs to a similar extent at different pH levels of the solution, whereas the known process of using charged membranes exhibits a clear pH dependence. At pH=6, the gap, in terms of the filtration capacity of the virus filter, between the polyamide surface (V.sub.max=550 L/m.sup.2) and the charged membrane (V.sub.max=200 L/m.sup.2) is still relatively small. At pH=9, a distinctly larger gap becomes apparent, and in this case the filtration capacity of the virus filter after the charged membrane collapses greatly; with the charged membrane, only V.sub.max=8 L/m.sup.2 is achieved. By contrast, in the case of use of the polyamide surface before the virus filter, V.sub.max=260 L/m.sup.2 is achieved.

Example 4: Depletion of Biopolymer Aggregates and Viruses

(28) Example 4 shows a repeat of example 1 in a slightly modified form and demonstrates in addition the depletion of the viruses by the method according to the invention.

(29) A protein solution of a monoclonal antibody from the cell line CHO dg44 ST1-C6 was prepared as described in example 1. However, before being used as filtration medium, the protein solution was admixed with a solution of PP7 bacteriophage, a recognized model system for small, nonenveloped viruses, and so the solution contained 10 g/L protein and 4.5.Math.10.sup.7 pfu/mL (plaque forming units) of the bacteriophage. The filtration was carried out twice analogously to example 1, but at 2.0 bar differential pressure. The prefilter used was, in each case, a polyamide prefilter from example 1. As main filter, two virus filters were selected at random from a batch of 200 Virosart® CPV virus filters (No. 41 (symbol .circle-solid.) and No. 44 (symbol ◯)) and used for the filtration. At both 10 L/m.sup.2 and 20 L/m.sup.2, a sample was taken and the titer of the bacteriophages was determined in a plaque assay. The results of the titer determination are shown in table 1.

(30) TABLE-US-00001 TABLE 1 Fil- Down- No. of Fil- tered Upstream stream main trate volume Filtration titer titer filter* No. [L/m.sup.2] time [pfu/mL] [pfu/mL] Log10 ** 41 1.1 10 4 min 22 s 4.5 .Math. 10.sup.7 2.0 .Math. 10.sup.1 6.3 41 1.2 20 9 min 10 s 4.5 .Math. 10.sup.7 2.0 .Math. 10.sup.1 6.3 44 2.1 10 4 min 4 s  4.5 .Math. 10.sup.7 2.0 .Math. 10.sup.1 6.3 44 2.2 20 8 min 37 s 4.5 .Math. 10.sup.7 2.0 .Math. 10.sup.1 6.3 *Main filter = Virosart CPV virus filter ** The value of 6.3 entered in the column “Log10” indicates that the bacteriophage titer was reduced by 10.sup.6.3 pfu.

(31) The course of the filtration is shown in FIG. 8. A plot according to equation 1 gives rise to the filtration capacities of 286 L/m2 for main filter No. 41 and 130 L/m2 for main filter No. 44. Because of the low blockage according to the invention at the end point of the filtration, the extrapolation to the maximum capacity is associated with a certain margin of variation.