PROCESS AND APPARATUS FOR REMOVAL OF DNA AND VIRUSES
Technical Field
The present invention relates to a process for removing DNA and viruses from physiological fluids and medicant solutions administered to humans and animals, and an apparatus for performing said process. More par¬ ticularly, the invention is especially effective for removing DNA, viruses and endotoxins from biological pharmaceutical solutions and biological media, for example, DNA, viruses and endotoxins from a monoclonal antibody solution, buffer solutions or a solution of bovine serum albumin. Background Art
One objective in the preparation of pharmaceutical solutions, buffer solutions, life support solutions, saline solutions and other such solutions which are to be administered to animals and humans is that they be as free as possible from substances which might cause an ad¬ verse reaction in the host. While a goal of zero con¬ tamination by substances such as DNA, viruses and en- dotoxins is always sought, in actual practice very minute amounts of such substances are sometimes present. The Food and Drug Administration (FDA) has sets standards for such substances which cannot be exceeded. Manufacturers, ever mindful that a batch of medicant may be rejected if the level of such substances is too high, continually seek new methods to ensure that their products do not exceed FDA standards. Consequently, in all phases of the manufacturing process, manufacturers seek to ensure the purity of the reagents used in the manufacture as well as the final product. Many of the medicants and other prod¬ ucts mentioned above are either sold as aqueous solutions or are manufactured in aqueous medium. Consequently, the manufacturers seek to ensure that the water they use is free of DNA, viruses and endotoxins. One technology that such manufacturers often use is ultrafiltration. United States Patent Nos. 4,431,545 to
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Pall et al, 4,816,162 to Rosskopf et al, and 4,420,398 to Castino, describe dual-module filtration to remove pat¬ hological and/or toxic substances from various fluids including water, blood and plasma. Patent No. 4,431,545 utilizes dual filters, one of which has a negative zeta potential and one of which has a positive zeta potential, to filter out positively and negatively charged parti¬ cles. Neutral particles are removed in accordance with the pore size ratings of the filters which are 0.01 mic- rons or larger as disclosed. Patent No. 4,816,162 de¬ scribes an apparatus that removes immunoglogins, albumin and lipoproteins from blood, blood plasma or serum, but does not describe the removal of DNA or viruses. The fil¬ ter in this patent is designed for use in circulating and purifying blood during surgery. Patent No. 4,420,398 de¬ scribes a filtration method for separating cell produced antiviral substances, including monoclonal antibodies, from the reaction "broth" in which they are produced. This patent does not indicate whether the resulting species are free of viruses, endotoxins and DNA which may cause a reaction within a patient.
It is known in the prior art that multiple fil¬ tration with a 0.04 micron absolute pore size filter will remove viruses of 0.075 micron size, but not smaller viruses. For example, filtration of calf serum contain¬ ing MS 2 phage (0.024 micron) through 0.04 micron will not remove the virus. In those circumstances where virus can be removed, removal rate is typically 99.9 to 99.99% per filter pass. For example, using a 0.04 micron fil- ter, applicants removed all detectable Reovirus (0.075
8 micron) from a sample containing 10 virus particles per milliliter sample. An article published in the April, 1990 issue of Genetic Engineering News (page 6) commented on the Food and Drug Administration's (FDA) increasing emphasis on viral removal protocols with regard to the preparation of biological pharmaceuticals and the efforts being made by filter manufacturers to achieve higher de¬ grees of virus removal.
Another contaminant which can be' present in biological pharmaceuticals such as monoclonal antibodies is DNA. It is generally felt in the industry that the FDA seeks to achieve a DNA level in monoclonal antibody preparations of less than 10 picograms of DNA per dose of monoclonal antibody.
Manufacturers of biological pharmaceuticals such as monoclonal antibodies are required to establish Quality Assurance (QA) procedures to which verify that their products meet standards. In the procedures used to show compliance with the standards, it is necessary that the DNA in a sample be concentrated or solid phased (collected in solid form) from a solution of the biological pharmaceutical. It is known that DNA can be concentrated, solid phased or removed from solution by the use of diethylaminoethyl cellulose (DEAE) filter membranes. A manufacturer's literature (Schleicher & Schuell) indicates that DEAE filters will solid phase more than 90% of E. coli DNA from a solution containing 0.2 g DNA/ml. In a more dilute solution containing 0.001 jug DNA (1 nanogram) more than 80% will be solid phased. The DEAE filters work by binding a protein such as DNA to the filter. However, a major limitation arises in the use of DEAE filters with some monoclonal antibody solutions. For example, it has been found that DNA measurements of monoclonal antibody containing buffer solution having components such as maltose can result in cause false high or low DNA values. In order to assure that the DNA assay values are accurate, these false read- ings must be eliminated.
Lastly, in addition to viruses and DNA, endotoxins are important contaminating substances in biological pharmaceuticals. While some manufacturers offer column packing materials which are useful in removing endotoxins from protein solutions such as solutions of monoclonal antibodies, such packing materials often result in low product yields after passage of the protein solution through the column. The DEAE filter membranes described
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above have also been reported to remove endotoxins. How¬ ever, we have not found the membranes to be effective in removing endotoxins from all sources. In some instances removal is high, whereas in others it is low. This vari- ation is believed to be due to structural variation of the endotoxins themselves in the various samples. The variations in the endotoxins are, in turn, believed de¬ pendent on the source of the endotoxin itself and on the chemical treatment it has been subjected to. Having done a careful study of the extant art, we have developed a single filtration device capable of removing virus, DNA and at least some endotoxins to lower levels than pre¬ viously achieved. Disclosure of the Invention A single filtration device containing DEAE coated filter membranes and absolute pore filters is provided in which the membranes and absolute pore filters are present in two sections of the filter device. The first section of the device is the DNA filter section comprising a first 0.2 micron filter, a first DEAE filter, a second DEAE filter and a second 0.2 micron filter. The second section is the virus filter section comprising a first 0.1 micron filter, a second 0.1 micron filter, a first 0.04 micron filter and a second 0.04 micron filter. The filter sections can be housed in a single filter device or, alternatively, the sections can be housed in separate housings provided that in use the housing containing the DNA filter section precedes the housing containing the virus filter section and that the two are connected. In order to achieve higher levels of filtration than that afforded by a single device, multiple devices can be com¬ bined in series. The device may be used on a large scale at the point of manufacturing or packaging a phar¬ maceutical solution, or it can be used on a small scale at the point of administration to a patient. In either case, the DNA and viruses are removed by passing the pharmaceutical solution through the DNA and virus filters by the use of either pressure to push the solution
through the filter elements, as when administering to a patient, or vacuum to pull the solution through the fil¬ ter elements as in some manufacturing procedures.
The apparatus embodying the invention will remove viruses, as modeled by type-C Xenotropic retrovirus, with
5 an efficiency of at least 4.6 x 10 or approximately
99.995%, or 3x10 bacteriophage (99.99999997%) ; remove DNA from levels of 10 /g/sample to levels below 10 picog¬ rams per 500 mg sample of monoclonal antibody and preferably below 1 picogram per sample (100 ml of water or solution); and will remove at least 97% of some bac¬ terial endotoxins. Further, these filters units absorb less than 10% of the pharmaceutical or biological phar¬ maceutical, and most often 6% or less of such phar- maceuticals, particularly monoclonal antibodies and bovine serum albumin.
In an alternative embodiment of the invention, the DEAE filter membranes are replaced by absolute pore fil¬ ters which have been coated with DEAE, QAE (quaternary aminoethyl salts), QAM (quaternary aminomethyl salts) or other like quaternary salts. For example, the first and second DEAE filters can be replaced by 0.04 micron fil¬ ters coated with QAE or QAM.
In an alternative embodiment of the invention, an improved apparatus wherein DEAE functional groups, QAE, QAM or other quaternary amine functional groups are bonded directly directly to one or more of the 0.2, 0.1 and 0.04 micron absolute pore size filters, said functionalized absolute pore filters thereby replacing the DEAE cellulose filters.
Brief Description of the Drawings
Fig. 1 is a perspective view of single unit of fil¬ ter apparatus embodying the invention;
Fig. 2 is an exploded view of the apparatus shown in Fig. 1;
Fig. 3 is a perspective view of a multiple unit fil¬ ter apparatus embodying the invention;
Fig. 4 is an exploded view of the apparatus shown in Fig. 3.
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Best Mode for Carrying out the Invention
Referring to Fig. 1, the invention is a filter device 12 comprising a two-piece filter housing part hav¬ ing a top part 14 with inlet port 18, a base part 16 with outlet port 20 and a series of internal elements (not shown) with said top part and base part being joined to¬ gether in a leakproof manner; for example, by screwing the two parts together, by ball and socket attachment or other such means. Figure 2 is an exploded view of apparatus of the in¬ vention. The apparatus comprises the visible external members 14, 16, 18 and 20 as described above and internal elements, said internal elements being a first flat fil¬ ter support 24 having a plurality of channels 26 extend- ing through the thickness of the support; a first sealing member 28 extending a lateral distance inward from the inner wall of the filter housing; a first filter section 30 having filter elements 32, 34, 36 and 38 in sequential facial contact from one to the other throughout; a filter support 40 with a flat top face 42 in contact with the bottom face of filter element 38, a plurality of channels 26 extending through the thickness of the support and a plurality of rigid legs 44 at the outer edge of the bot¬ tom face of said support; a second flat filter support 46 having a plurality of channels 26 extending through the thickness of the support and whose top face 48 is in con¬ tact with legs 44; a second sealing member 50; a second filter section 60 having filter elements 62, 64, 66 and 68 in sequential facial contact from one to the other throughout; a third flat filter support 70 having a plurality of channels 26 extending through the thickness of said support; and wherein the top to bottom face con¬ tact of the element is 28 to 24, 32 to 28, 34 to 32, 36 to 34, 38 to 36, 40 to 38, 50 to 46, 62 to 50, 64 to 62, 66 to 64, 80 to 66 and 70 to 68; and the top of face of element 24 is supported by the interior of top housing 14 and the bottom fact of element 72 is supported by the in¬ terior of housing 16; and wherein sealing said interior
elements by joining said top and base-housing causes a pressure to be exerted on said sealing members 28 and 50 causing said sealing members to seal to the walls of said housing thereby preventing flow around filter sections 30 and 60, and forcing said flow to occur only through said filter sections.
Referring to Fig. 3, a second embodiment of the in¬ vention is a two section filter device 12 having a first DNA removal filter unit 4 and a second virus removal unit 6 joined by a connecting means 80.
Fig. 4 is an exploded view of the two unit filter device as shown in FIG. 3 comprising a first DNA removal filter unit having a top filter housing part 14 with inlet port 18 and a base filter housing part 16 with out- let port 20, and internal members flush to the interior walls and sequentially in facial contact with each other; said internal members being a first flat filter support 24 having a plurality of channels 26 extending through the thickness of the support; a sealing member 28 in con- tact with the inner side walls of said housing and ex¬ tending a lateral distance inward from the inner wall; a DNA filter section 30 having filter elements 32, 34, 36 and 38; a second flat filter support 72 having a plurality of channels extending through the thickness of the support; and a second virus removal filter unit 6 having a top filter housing part 15 with inlet port 19 and a base filter housing part 17 with outlet port 21 and internal members which are sequentially in facial contact with each other; said internal members being a first flat filter support 46 having a plurality of channels extend¬ ing through the thickness of said support; a first seal¬ ing member 50 in contact with the inner side walls of said housing and extending a lateral distance inward from said inner wall; a virus filter section 60 having filter elements 62, 64, 66 and 68; and a filter support member 62 having a plurality of channels 26 extending through the thickness of said support; and a connecting member 80 joining said DNA filter unit 4 and said virus removal
filter unit 6 by connecting outlet port 20 and inlet port 19; wherein the top to JDottom face contact of the elements is 28 to 24, 32 to 28, 34 to 32, 36 to 34, 38 to 36, 72 to 38, 50 to 46, 62 to 50, 64 to 62, 66 to 64, 68 to 66, and 70 to 68; and top fact of elements 24 and 46 is supported by the interior of their respective housings 14 and 15 and the bottom face of elements 70 and 72 is supported by the interior of their respective housings 16 and 17; and whereby enclosing said interior elements by joining respective top and base housings parts causes a pressure to be exerted on said sealing members thereby preventing flow around filter section 30 and 60, and forcing said flow to occur only through said respective filter sections; and said first DNA removal filter part and said second virus removal filter part being joined by connecting means 80 attached to parts 19 and 20.
The filter units of as described above can be in any size and shape -round, square, rectangular- possible, subject only to limitation of the availability of size and shape of the filter material for filter sections 30 and 60. The filter units can be sized to handle commer¬ cially useful quantities of water for use in the manufac¬ ture or preparation of buffer solution, pharmaceuticals, and pharmaceuticals solutions and the like. The filter can be used at any point in a manufacturing processes where a new aqueous material is added and is especially useful in removing DNA, viruses and endotoxins in the packaging step at the end of the manufacturing process. In addition, the filter system of the present invention can be used in conjunction with a device for administer¬ ing a physiological or a pharmaceutical solution to a patient; for example, the filter system can be built into or placed into a hypodermic syringe. In all instances of use, the solution being filtered passes through the DNA removal filter section and then passes through the virus removal filter section
The filter elements of the filter apparatus de¬ scribed above are a combination of diethylaminoethyl eel-
lulose and absolute pore filters. These filters, when used in the apparatus of this invention, will remove on
0.1 micron type-C retrovirus with an efficiency of 4.6 x
5 10 or higher, remove DNA to level of 10 micrograms/ml to levels below 1 picogram/ml and will remove about 97% of some bacterial endotoxins. In addition, the filter elements of the present invention absorb 6% or less of proteins from the solution under treatment: for example, monoclonal antibody or bovine serum albumin solution. In the preferred embodiment of the invention elements 32 and 38 are 0.2 micron absolute pore filters; elements 34 and 36 are DEAE coated filters such as, for example, Schleicher & Schuell's NA45 filters; elements 62 and 64 are 0.1 micron absolute filters; and elements 66 and 68 are 0.04 micron absolute pore filters.
In the preferred embodiment of the invention, infec¬ tious virus particles of about 0.108 micron size can be removed with an efficiency of at least 99.99% per passage through the filtration apparatus. Higher efficiencies can be obtained by using two or more of the filter ap- parati in series.
The preferred filter apparatus of the invention pro¬ vides for a synergistic effect upon use of the filter elements as specified. The smallest absolute pore filter of the invention is 0.04 microns. Manufacturer's litera¬ ture for the DEAE filters state that the pore size is 0.45 microns. However, as stated above and shown in the examples below, virus as small as 0.018 micron (the minimum virus particle size) can be removed. While the exact nature of the synergistic effect is not known, complete removal of virus 55% smaller than the smallest pore size filter element was not anticipated.
In a process utilizing the device of this invention, the water, aqueous buffer solutions and pharmaceutical solutions, including biological pharmaceutical solutions, have a pH in the range of 3 to 9. Further, these solutions have a specific salt content of less than 0.5 Molar, said specific salts being one or more selected
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from the group consisting of the lithium, sodium, potas¬ sium or ammonium salts of the phosphate, chloride, bromide, iodide, sulfate and acetate anions. When utilizing the device of this invention, solutions are first passed through the DNA removal section prior to passage through the virus removal section.
The following examples are given to illustrate the utility of the present invention and are not to be construed as limiting the scope of the invention.
Example 1. Virus Removal
The internal elements of the filter unit of the in¬ vention were assembled using eight filter element in the sequence 0.2 micron, DEAE, DEAE, 0.2 micron, 0.1 micron, 0.1 micron, 0.04 micron and 0.04 micron. The 0.2, 0.1 and 0.04 micron elements were absolute pore filters, and the DEAE elements were NA 45 filters (Schleicher & Schuell). The units were sealed in autoclavable syringes and were autoclaved or gas sterilized using standard pro¬ cedures. The sterilized syringes containing the filter elements were sent to Microbiological Associates, Inc., Life Sciences Center, 9900 Blackwell Road, Rockville, Maryland 20850 for evaluation with monoclonal antibody solutions spiked with mouse xenotropic retrovirus of similar size to type C retrovirus (0.1 micron v 0.104 micron respectively). Each syringe filter device was evaluated against one sample of retrovirus spiked mono¬ clonal antibody. By S+L- assay, the samples contained 4.37 x 10 , 5.6 x 10 and 4.1 x 10 FFU/ml.
l l [ FFU/ml = (mean number of foci/dish x volume/dish dilution
After passage of the test samples through the syringe filter units, the filtrates were re-analyzed in trip¬ licate for retrovirus. No retrovirus found in any of the three monoclonal antibody filtrates. Antibody recovery
was greater than 90%.
Example 2. Removal Of Bacteriophage By DNA/ Virus
Removal Filters. The maximum concentration of xenotropic retrovirus attainable is about 10 FFU/ml. In order to validate the DNA/Virus removal filters of this invention for higher virus particle removal efficiencies, bacteriophage T4 (approximately 0.1 micron) was chosen as a second model virus. The assay for bacteriophage T4 concentration was the formation of plaques (PFU) on a lawn of Escherichia coli B (ATCC 11303). The bacteriophage T4 was grown to maximum concentration (9.9 X 10 PFU/ml ) and the un¬ diluted bacteriophage solution was divided into three aliquots. Each aliquot was filtered through a separate DNA/Virus removal filter device. The concentration of bacteriophage T4 in the filtrate was assayed by dilution and plating on dishes of E. coli. None of the three fil¬ trates contained viable virus. The assay has an uncer¬ tainty of 3.3 FFU. These results indicate that the DNA/Virus removal filter device of the present invention is capable of reducing the concentration of an 0.1 micron bacteriophage by at least 3.0 x 10 fold (99.99999997%). Similar results should be obtainable with viruses of similar size, approximately 0.1 micron, such as type C retrovirus. Type C retrovirus has been found to be a contaminant in the conditioned raw material for mono¬ clonal antibody pharmaceutical. To the inventors' know¬ ledge, no single pass through any filter as previously achieved this level of virus removal. Using the filter device of the present invention should reduce the con¬ centration of type C retrovirus in the conditioned raw
10 material by at least 3x10 fold. Thus, solutions con-
7 taining nominal virus counts on the order of 10 should be able to be filtered to an undetectable virus level with a 1000 fold safety margin. In those cases where the virus load of a solution is higher, over 10 , the solution can be filtered two or more times to obtain a
solution having an undetectable virus level. Using two of the filter devices of the present invention in series
Example 3. DNA Removal From Spiked Antibody Solutions
Monoclonal antibody solutions containing 400 mg of antibody each and DNA were filtered through the DNA/virus removal filter unit of the invention. DNA analysis be- fore and after filtration showed 727 pg and 442 pg of DNA per sample before filtration; and 5pg and pg DNA, res¬ pectively, after filtration (99.3% and 99.8% removal).
Example 4. DNA Removal From Commercial Antibody Solutions Analysis of commercial monoclonal antibody solutions indicated that there is significant DNA contamination. The analysis was performed using an assay kit from FMC Bio Products, Rockland, Maine (FMC assay) for the detec¬ tion of DNA solid-phased on Nylon 66 membranes. Five lots of DNA containing monoclonal antibody solution were analyzed for DNA before and after filtration through a filter device of the invention: All filtered solutions had less than 10 picograms of DNA per dose of antibody and two of the five showed less than 1 picogram per dose. The results are shown in Table 1.
Example 5. DNA Removal Validation
In order to validate DNA removal for commercial pur¬ poses, the DNA/Virus removal filters were challenged with 500 mg samples of a pharmaceutical grade monoclonal an- tibody (Bl) in buffer spiked with 100 micrograms of hyb- ridoma produced DNA. The DNA used in the validation was purified from the same cell culture medium used to pro¬ duce monoclonal antibodies and was as similar as possible to the DNA actually encounted in the production of the antibody. Three antibody solutions were spiked with the DNA. Two unspiked antibody solutions, two buffer (only) solutions without DNA and two buffer (only) solutions spiked with 100 micrograms of DNA were used as controls. The actual level of DNA in the spiked solutions was determined by means of a fluorescent DNA assay technique. The spiked antibody solutions were found to have actual DNA levels of 81, 92 and 74 micrograms per sample. The spiked buffer solutions were found to have actual DNA levels of 89 and 96 micrograms per sample. All solutions samples were equal volume.
Each of the test solutions (9 solutions total ) was filter through a separate 25mm DNA/Virus removal filter device. The residual DNA in each filtrate was con¬ centrated, solid phased and quantified in duplicate using standard FMC DNA assay techniques. The quantity of DNA in each assay was determined from a standard curve of purified hybridoma DNA run in the same assay. For the standard curve, the color intensities of the sample bands, measured by the instrument's reflection den- sitometer, are measured as peak heights in centimeters.
The standard curve data is linearly transformed by a log- logit transformation where the peak heights are converted to a logit (relative to a standard that will give maximum color development and a blank) versus the log of the picograms of DNA standard added. Test samples were then interpolated from the standard curve of DNA to color in¬ tensity. The results are given in Table 2 and indicate
that a single pass through the DNA/Virus removal filter is capable of reducing the DNA levels by about 10 fold to approximately 10 picograms DNA per 500 mg of monoc¬ lonal antibody (mean = 12.3 pg DNA/500mg antibody). The 5 mean value for an equal volume of unspiked buffer (only) is 6.2 pg. Therefore, the mean net DNA detected in the filtered, spiked antibody solution is 6.1 pg DNA/500 mg antibody.
Table 2
0 Sample DNA Spike DNA Detected % Recovery of Mean total in sample after protein DNA detected DNA spiking concentration Mean after
(Lowry) filtration
0
* total DNA in 500 mg sample of monoclonal antibody (mean ob- 25 servation of samples assayed in duplicate)
Example 6 Endotoxin Removal
A 100ml solution of 50mg/ml bovine serum albumin in 10% maltose-phosphate buffer solution contaminated with DNA and a endotoxin was filtered through a 47 mm DNA/virus - - removal filtration device. The starting
SUBSTITUTESHEET
solution contained 248 pg/ml DNA and 1966 endotoxin units ml (EU/ml).
First, middle and end 20ml portions of the filtrate were collected and analyzed. No DNA was detected in any analyzed portion of filtrate. Endotoxin levels were: first= 30.72 EU/ml, middle= 30.72 EU/ml and last= 61.44 EU/ml. Endotoxin removal in the end sample was 96.9%. Solution recovery was 95% (95ml) with no change in protein concentration.