News Column

Patent Application Titled "Method for Removing Biopolymer Aggregates and Viruses from a Fluid" Published Online

May 20, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Thorm, Volkmar (Goettingen, DE); Hansmann, Bjoern (Goettingen, DE), filed on April 28, 2012, was made available online on May 8, 2014 (see also Sartorius Stedim Biotech GmbH).

The assignee for this patent application is Sartorius Stedim Biotech GmbH.

Reporters obtained the following quote from the background information supplied by the inventors: "The present invention relates to a method for removing biopolymer aggregates and viruses from a fluid.

"Biopolymer aggregates, for example aggregates of proteins, and viruses are frequently undesired contaminants in fluids obtained by biotechnological methods, such as protein and peptide solutions. Public health authorities prescribe the removal of said contaminants from the fluid before it is used therapeutically.

"Protein solutions, for example immunoglobulins in blood plasma, or solutions comprising recombinant proteins or natural proteins generally contain a detectable proportion of dimers and higher aggregates of said proteins. Prior to parenteral administration to patients, depletion of the aggregates is necessary. In general, an aggregate content of in total less than 1%, based on the total protein content of the solution, is striven for. The removal of said aggregates by means of size-exclusion filtration is not advantageous from a technical point of view, since the filters used become blocked during said filtration. As an alternative, methods which can remove impurities, for example aggregates of biopolymers, by means of selective affinity binding to chromatographic columns, such as ion exchangers, have been established.

"For the depletion of viruses as contaminants from protein solutions which are used therapeutically, size-exclusion filtration is known to be an effective process which can be used reliably on all virus species and under a large range of conditions. The filtration of protein solutions in order to deplete small, nonenveloped viruses is a robust step recognized by public health authorities for reducing virus concentration in protein solutions originating from biological sources. In said filtration, it is necessary to have in one production process at least two robust virus-depletion methods which act differently.

"The requirements typical for filtration procedures are applicable to the effective elimination of viruses from protein solutions: Separation selectivity should lead to a reduction in the titer, i.e. the virus concentration, by a factor of at least 103. The water permeability of the filtration membrane used for the removal of the viruses should be maximal. By contrast, the tendency toward blockage, i.e. the reduction in flow capacity owing to an obstruction by accompanying components, or the closure of filtration membrane pores, should be minimized. Owing to the simultaneously necessary passage of the therapeutic proteins as target compounds, the hydrodynamic radius of which is of a similar order of magnitude to that of the viruses to be retained, the production of suitable so-called parvovirus filters is complicated compared to microfiltration membranes for other applications. The small difference in size between proteins and viruses results in the parvovirus filters being especially sensitive with respect to blockage and, compared to microfilters, in a high demand on the purity of the solution to be filtered.

"Because of these high demands on product purity, the parvoviral filtration is carried out relatively late in the process sequence for producing recombinant proteins or blood plasma products. Typically, the filtration is preceded by chromatographic separation and purification processes. Highly relevant to the viral filtration is the requirement that no extractable constituents (leachables) or particles are released into the filtrate from the filtration media used. The release of such contaminants occurs with preference when kieselguhr-containing depth filters are used.

"U.S. Pat. No. 7,118,675 B2 discloses a method for selectively removing protein aggregates and viruses from a protein solution, wherein, in a first step, the protein solution is filtered through one or more layers of a charged or surface-modified microporous membrane in order to retain the aggregates. In a second step, the protein aggregate-free filtrate is filtered through one or more ultrafiltration membranes, whereby the virus content is reduced by at least three powers of ten. This method, which has proved effective in principle, requires, as a function of the protein solution to be processed and the aggregates to be removed, individual adjustment of the pH and the conductivity of the protein solution. These aforementioned parameters influence, on their part, the optimal selection of a suitable charged or surface-modified membrane for the first step of the method. As an alternative, in the first step, one or more layers of a depth filter can be used in addition to the microporous membranes.

"This optimization problem has already been identified by A. Brown et al. in 'Increasing Parvovirus Filter Throughput of Monoclonal Antibodies Using Ion Exchange Membrane Adsorptive Pre-Filtration', Bioseparations and Downstream Processing, Biotechnology and Bioengineering, DOI 10.1002/bit.22729, 2010 Wiley Periodicals, Inc. For the removal of biopolymer aggregates from virus-containing solutions, charged membranes can be used effectively only within a narrow pH range and at low salt concentrations because undesired competition between the binding of salt ions and the antibodies as target proteins occurs in said membranes at high salt concentrations.

"A. Brown et al. describe the use of charged membrane prefilters to increase the filtration capacity of downstream Viresolve.RTM. parvovirus filters. Here, the prefilters remove aggregates of monoclonal antibodies, the monomers of the antibody being the target product. For cation-exchanging membranes having sulfonic acid ligands (Mustang.RTM. S) as virus prefilter, the filtration capacities of a parvovirus filter were determined within a pH range of between 4 and 7 and at conductivities between 3 mS/cm and 25 mS/cm. The filtration capacities achieved, defined as V50 (filtration volume in the case of a permeability drop of 50% with respect to the start of filtration), show a strong dependence on pH and conductivity, which is additionally influenced by the monoclonal antibody used. The pH dependence is explained by the change in the net charge of the proteins and the aggregates. The dependence on conductivity is attributed to shielding of the charged groups of the prefilter by salts. In the case of the monoclonal antibody used from Chinese hamster ovary cells, an optimum (sweet spot) arises within a pH range of 5 to 6 and at a conductivity between 5 mS/cm and 12 mS/cm. The differences between the abovementioned optimized conditions of the sweet spot and other suboptimal ranges of pH and conductivity differ from one another by up to a factor of 9 with regard to V50.

"When using charged membranes for aggregate removal, these optimized conditions for the 'sweet spot' have to be ascertained on a individual basis for each protein, this optimization amounting to great effort for successful application of this method.

"EP 1 624 950 B1 discloses a method for selectively removing protein aggregates and viruses from a protein solution, wherein, in a first step, the aggregates are retained by a fibrous pad and kieselguhr, whereas, in the subsequent second step, the viruses are retained by at least one ultrafiltration membrane, whereby the virus content is reduced by at least three powers of ten. The fibrous pad can contain one or more layers of microporous membranes composed, for example, of polyamide, regenerated cellulose, poly(ether)sulfone, polyimide or PVDF, which, on their part, are filled with charged particles, known from U.S. Pat. No. 5,531,899 or U.S. Pat. No. 5,093,197, for improved aggregate retention. As charged particles, use is made of, for example, particles based on Diphonix cation-exchange resins or particles having surface silanol groups in polyolefin membranes. As an alternative, the membrane to be used as prefilter can be filled with silica gel particles.

"The use of membranes filled with charged particles results here in the aforementioned optimization problem with regard to pH and conductivity of the protein solution to be processed.

"With regard to undesired release of particles into the filtrate containing the target proteins, the use of kieselguhr in the first step of the method can be problematic.

"A further method in which a charged membrane is likewise used as prefilter for the downstream virus filter is disclosed by U.S. Pat. No. 7,465,397 B2. Viruses and protein aggregates are removed from a protein solution in at least two steps in this method, in which surface- or charge-modified polyamide membranes are used in the first filtration stage, whereas the downstream ultrafiltration module for virus removal comprises polyamide membranes which are not modified on their surface.

"US 2009/0326211 A1 discloses a method for selectively removing large microbiological particles, such as algae, fungi, amoebas, or inorganic particles from a water sample containing microorganisms, such as Legionella pneumophila or viruses. Unlike the other documents already mentioned, removal is effected here according to the principle of size-exclusion filtration and can be carried out in a similar manner largely independent of the material of the filter. However, removal according to the principle of size-exclusion filtration likewise does not solve the optimization problem already discussed for the other documents with regard to pH and conductivity of the protein solution to be processed.

"In the first step of the method according to US 2009/0326211 A1, the particles are retained by a prefilter consisting of a steel screen, glass fibers, polypropene, polyethersulfone, nylon, PVC, PTFE, polyester, polycarbonate or polyester, whereas the microorganisms pass through the prefilter. The filtrate containing the microorganisms is subsequently fed to a main filter having a smaller pore size, by means of which the microorganisms are retained. Subsequently, the microorganisms can be subjected to lysis on the main filter for analysis. The material of the main filter is selected from the group of the materials which are also used for the prefilter.

"It is an object of the present invention to provide a method which overcomes the aforementioned disadvantages of the prior art and which permits the selective removal of biopolymer aggregates and viruses from biotechnological fluids in a simple and cost-effective manner. Said method shall avoid the use of complicated-to-produce charged or surface-modified membranes as virus prefilters and of materials which can release particles into the filtrate. Furthermore, the method to be provided shall allow the reliable selective removal of said aggregates and viruses across a wide range for the pH and conductivity of the protein solution to be processed."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "The present invention provides a method for removing biopolymer aggregates and viruses from a fluid, comprising the following steps:

"(a) filtering the fluid containing the biopolymer aggregates and viruses through a porous, polyamide-comprising shaped body, the internal and external surfaces of which have the same chemical and physical properties as the shaped-body matrix which is enclosed by the surfaces, wherein the biopolymer aggregates are selectively depleted from the fluid by adsorption, whereas the viruses permeate through the shaped body; and

"(b) filtering the fluid from step (a) through at least one membrane having a molecular weight cut-off of from 100 to 1000 kD, wherein the content of viruses in the fluid is reduced by at least 99.9% with respect to the content of viruses prior to carrying out step (a).

"According to the invention, the term 'biopolymer aggregates' is not subject to any particular restriction. In a preferred embodiment of the present invention, the biopolymer aggregates are selected from the group of the dimers, trimers and multimers of peptides, proteins, nucleic acids or mixtures thereof.

"According to the invention, the term 'virus' is not subject to any restriction, i.e. the term encompasses both enveloped (e.g. MLV) and small, nonenveloped viruses (parvoviruses) such as, for example, PPV or MVM.

"According to the invention, the term 'fluid' is not subject to any restriction. In a preferred embodiment of the present invention, the fluid comprises a blood plasma product, a protein solution obtained from a cell culture, a protein solution obtained from extraction of animal or plant products, or a protein solution obtained from microorganisms.

"In step (a) of the method according to the invention, the biopolymer aggregates are selectively removed by filtration through a porous, polyamide-comprising shaped body, whereas the viruses and target compounds permeate through the shaped body. According to the invention, 'selective removal of biopolymer aggregates' is understood to mean that the biopolymer aggregate content of the fluid is reduced after the filtration with respect to the starting biopolymer aggregate content. This selective removal of the biopolymer aggregates in the first step of the method according to the invention can thus advantageously prevent blockage of the pores of virus-retentive membranes in step (b) of the method. According to the invention, the filter material in step (a) is a native surface of a polyamide in porous form. In this connection, native means that the internal and external surfaces of the polyamide-comprising shaped body have the same chemical and physical properties as the matrix (the base material).

"According to the invention, polyamide is understood to mean a polymer composed of repeating units which are covalently bonded to one another by means of amide functions. The monomer units can be aliphatic and/or aromatic and be joined to one another via ring opening or polycondensation. In a preferred embodiment of the present invention, the porous, polyamide-comprising shaped body comprises an aliphatic and/or aromatic polyamide having no more than five different repeat units (monomers). Among the aforementioned monomers, particular preference is given to .epsilon.-caprolactam, 1,6-hexanedioic acid and 1,6-hexanediamine or sebacic acid. In a particularly preferred embodiment, the porous, polyamide-comprising shaped body consists of an aliphatic and/or aromatic polyamide having no more than five different repeat units. In a further preferred embodiment, a polyamide consisting only of aliphatic monomer units is used. In a particularly preferred embodiment of the method according to the invention, the polyamide used is nylon-6 and/or nylon-6,6.

"According to the invention, the porous, polyamide-comprising shaped body itself is not subject to any restriction in terms of its form; for example, it can be present in the form of fibers, for example as nonwoven, as woven fabric, as porous membrane, as monolith, as gel, as bed of particles, or the like. According to the invention, the pore size of the porous, polyamide-comprising shaped body is not subject to any restriction. In a preferred embodiment of the present invention, the polyamide surface has a pore size smaller than 0.5 .mu.m. In a further preferred embodiment of the present invention, the polyamide-comprising shaped body comprises a microporous membrane, a monolith, a gel, or a bed of particles. In a particularly preferred embodiment, the polyamide-comprising shaped body is a microporous membrane.

"In step (b) of the method according to the invention, the fluid from step (a) is filtered through at least one virus-retentive membrane. In this connection, the membrane filter is considered to be virus-retentive if the membrane filter achieves at least 99.9% depletion of small viruses, for example PPV (plum pox virus, sharka virus), and model systems, such as the bacteriophage PP7. In a preferred embodiment of the present invention, at least 99.99% depletion of enveloped viruses is additionally achieved. In this connection, a person skilled in the art will easily select a suitable virus-retentive membrane which is known from the prior art and which meets this standard and, in addition, allows the target molecules to permeate.

"According to the invention, the membrane has a molecular weight cut-off of from 100 to 1000 kD. In this connection, the molecular weight cut-off (MWCO) of a membrane refers to the nominal molecular weight of molecules and particles of which 90% can pass through the membrane. To this end, a person skilled in the art will, in a manner known to him or her, compare the molecular weight distribution of a filtrate of the membrane to be investigated of a mixture of dextrans of varying molar mass with the unfiltrate in a gel permeation chromatography procedure, with the flow through the membrane being adjusted to 0.0001 cm/s.

"In a preferred embodiment of the present invention, the membrane used in step (b) comprises polyethersulfone, polysulfone, polypropylene, polytetrafluoroethene, polyamide, polyimide, polyvinylidene fluoride, cellulose, cellulose derivatives or mixtures thereof. In a particularly preferred embodiment of the present invention, the membrane consists of polyethersulfone, polysulfone or mixtures thereof.

"The method according to the invention can be used effectively across a wide pH range of the fluid. In a preferred embodiment of the present invention, the pH of the fluid during steps (a) and (b) is between 5 and 9.

"Owing to the use of a porous, polyamide-comprising membrane, the method according to the invention has particular advantages over the use of known chromatographic columns. By selectively removing the biopolymer aggregates in the first step of the method according to the invention, blockage of the pores of virus-retentive membranes can be advantageously avoided in step (b) of the method. It was found that, surprisingly, in the case of filtration of a fluid through a porous, polyamide-comprising shaped body having a native surface prior to filtration across a virus-retentive membrane, there was a distinct increase in the filtration capacity of the virus-retentive membrane compared to the use of prefilters composed of other materials, for example polyethersulfone, and in particular of membranes having charged surfaces or surfaces chemically or physically modified with respect to the membrane matrix. The increase in the filtration capacity of virus filters works independently of the net charge of the protein aggregates, and the ratio between volume of the protein solution and the surface area influences the filtration capacity proportionally. Furthermore, the surface effects of the polyamide can be combined with a size-exclusion effect. Advantageously, the increase in the filtration capacity is independent of the pH of the fluid in the range of pH=5 to 9, and this distinguishes the method according to the invention from the methods known to a person skilled in the art using charged or surface-modified membranes. Furthermore, the porous surface of the polyamide-comprising shaped body advantageously exhibits only a low release of organic carbon and particles compared to typical depth filter materials which are described for the removal of biopolymer aggregates and are therefore of only limited suitability in typical processes for depleting viruses from protein solutions. In addition, the porous polyamide surfaces used for the method according to the invention, for example in the form of membranes, are distinctly more conveniently available compared to classic chromatography media. Accordingly, separation by means of the method according to the invention using the same volume of medium is, advantageously, not only distinctly more selective and faster than is possible with the methods known to date, but also in addition distinctly more cost-effective.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

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

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

For more information, see this patent application: Thorm, Volkmar; Hansmann, Bjoern. Method for Removing Biopolymer Aggregates and Viruses from a Fluid. Filed April 28, 2012 and posted May 8, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=6417&p=129&f=G&l=50&d=PG01&S1=20140501.PD.&OS=PD/20140501&RS=PD/20140501

Keywords for this news article include: Viruses, Virology, Sartorius Stedim Biotech GmbH.

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Source: Life Science Weekly


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