CA2084873A1

CA2084873A1 - Process and apparatus for removal of dna and viruses

Info

Publication number: CA2084873A1
Application number: CA002084873A
Authority: CA
Inventors: Stephan D. Glenn; Gregory Butchko; Edward O'connell; Paulette Smariga
Original assignee: Individual
Current assignee: Coulter Corp
Priority date: 1990-06-29
Filing date: 1991-06-20
Publication date: 1991-12-30
Also published as: EP0536297A4; EP0536297B1; JPH06507375A; DE69128249T2; DE69128249D1; ATE160294T1; EP0536297A1; AU8215091A; US5076933A; AU654669B2; WO1992000132A1

Abstract

A single filtration device (12) containing coated filter membranes (32, 38, 62, 68) and absolute pore filters (34, 36, 64, 66) is provided in which the membranes (32, 38, 62, 68) and absolute pore filters (34, 36, 64, 66) are present in two sections (30, 60) of the filter device. The filter device (12) will remove viruses, as modeled by type-C Xenotropic retrovirus, with an efficiency of at least 4.6 x 105; remove DNA from levels of 10 µg/sample to levels below 10 picograms per 500 mg sample of monoclonal antibody; and will remove at least 97 % of some bacterial endotoxins.

Description

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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 ob~ective in the preparation of pharmaceutlcal 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~
tamina-tion 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 manufac-tured 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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W092/00132 PCr/US~
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Pall et al, 4,816,162 to Rosskopf et al, and 4,~20,398 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 rnic-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 producedO
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 99O99%
per filter pass. For example, using a 0.04 micron fil-ter, applicants removed all detecta81e Reovirus (0.075 micron) from a sample containing lO virus particles per milliliter sample. An article published in the April, l99O issue of Genetic Engineering News (page 6) commen-ted on the Food and Drug Adminis-tration'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.

WO92/00132 PcT/us~ d~
~,,,..~, _3_ 2 ~ t 3 Ano-ther contaminant which can be presen-t 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 antlbody preparations of less than lO 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 mee-t 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 b~f 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 ~NA/ml. In a more dilute solution containing O.O01 ~g 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, endotoxinsare 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 ~092/00132 PC~/US9~f~ 7 ~3~ 7,~ ~_ above have also been repor-ted to remove endotoxins. How-ever, we have no-t 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 ofthe 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 filterO 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 contalning 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 manufa~turing 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 o-~ either pressure to push the solution .
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~ ~ _5 2 ~ 8 ~~ ~) 7 through the filter elements, as when adminis-tering to a patient, or vacuum to pull the solu-tion 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 an efficiency oflOat least 4.6 x 10 or approximately 99.995~, or 3xlO bacteriophage (99.99999997~) ; remove DNA from levels of 10 ~g/sample to levels below 10 picog-ra~s 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 absor-b less than 10~ of the pharmaceutical or biological phar~
maceu-tical, and most often 6% or less of such phar-maceuticals, particularly monoclonal antibodies andbovine s~rum albumin.
In an alternative embodiment of the invention, the DEAE filter membranes are replaced by absolute pore fil-ters which have been coated with DEA~, 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 QA~ 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, O.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. l;
Fiy. 3 is a perspective view of a multiple unit fil-ter apparatus embodying the invention;
Fig. 4 is an exploded view or the apparatus shown in FigO 3.

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2 ~ 7, ~ - 6~ .7 sest Mode for Carrying out the Invention Referring to Fig . 1, the invention is a filt~r 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 meansO
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 intern~l 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 elçments 32, 34, 36 and 38 in sequenti.al 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 SO, 6~ 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 suppor-ted by the in-teri.or of housing 16; and wherein sealing said interior ' ~ ' ' "
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WO92/00132 PC~/US91l~"DJ~
( -7- ~ '73 elements by joinlng 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 sai.d 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 F'IG. 3 comprislng a first DNA remo~al filter unit having a top filter housing part 14 with inlet port 18 and a base filter housing part 16 with 01..it-let port 20, and internal members flush to the interio:r walls and sequentially ln 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 seali.ng 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 o:f the support; and a second virus removal filter unit 6 having a top filter housing part 15 with inlet port l9 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 fla-t 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 frorr said inner wall; a virus filter section 60 having filter elements 62, 64, 66 and 68; and a fil-ter 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 sai.d virus removal WO92/00l3~ Pcr/ US9i/0~ 3 1 2~ 8- r filter unit 6 by connecting outlet port 20 and inlet port 19; wherein the top to bottom face contact of the elements is 28 to 24, 32 -to 28, 34 -to 32, 3~ -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 fac-t 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 thelr respective housings 16 and 17; and whereby enclosing sa:Ld 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 respecti~e filter sections; and said first DNA removal filter par-t and said second virus removal filter part being joined b~
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 6U. The filter units can be sized to handle commer-cially useful quantities of water for use in the manufac-ture or preparation of buffer s~lution, 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 manufacturiny 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 patien-t, for example, the fi.lter 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 cel-:, ~

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WC) 92/001~ PCr/US~ 451 , 9 2 ~ t; 3 ~ ~'3 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 or higher, remove DNA to level of 10 micrograms/ml to levels below 1 picogram/ml and ~ill remove about 97% of some bacterial endotoxins. In addi-tion, the filter elements of the present invention absorb 6~ or less of proteins from the solu-tion unde:r 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 obtalned 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 , '' : ' ' WO92/00l32 PCI/U~j 11 f ~, /
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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 ths 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, Ool and 0.04 micron elements were absolute pore fllters, 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.

x0 [ 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-anaIyzed in trip-licate for retrovirus. No retrovirus found in any of the three monoclonal antibody filtrates. Antibody recovery .' ,' ~' 't ' . ~ ' '' ' .," . . , ' ' WO9~/OOl32 Pcr/us~

was greater than 90~.

Example ~. Removal Of Bacterlophage 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 bacterOophage 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 concentra-tion of an 0.1 micron bacteriophage by at least 3.0 x 10 fold (99.99999997~)0 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 retrovOirus in the conditioned raw material by at least 3xlO fold. Thus, solutions con-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

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solution having an undetectable virus level. Using two of the filter devices of the present invention in series would allow -the removal of ap~ro~imately 10 - 10 viruls particles per ml [(3xlO j x (3xlO )/ 1000 =
9~10 ].

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-10 fore and after filtration showed 727 pg and 442 pg of DNA
per sample before filtration; and 5pg and pg DNA, res-pectively, after filtra-tion (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 contaminationO
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 flve showed less than 1 picogram per dose~
The results are shown in Table 1.

Table 1 SUMMARY of DNA REMOVAL from antibody products Mean DNA Concentration Product No. Before Filtration After Filtration p~ DNA/mg Mab: p~ DNA/dosepg DNA/dose 1 0.65 260 2.6 3~ 2 0.30 120 0.4 3 0.34 3.4 2.6

4 0.13 1.3 3.1 0.14 140 0 ' ' ' .

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Example 5. DNA Removal Validation In order to validate DNA rernoval for commercial pur-poses, the DNA/Virus removal filters were challenged with 500 mg samples of a pharmaceutical grade monoclonal an-tibody (B1) 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 (onl~) solutions without DNA and two buffer (only) solutions spiked with 100 micrograms of DNA were used as con-trols.
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. I'he 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 da-ta is linearly transformed by a lo~-logit transformation where the peak heights are converted to a logit (relative to a standard that will give ~ximum color development and a blank) versus the log of the picoyrams of DNA standard added. Test samples were then interpolated from the standard curve of DNA to color in-tensity. Thc, results are given in Table 2 and indicate -wo s2/00l32 ~c-r/lJs~

that a slngle 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 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 i.s 6.1 pg DNA/500 mg antibody.

Table 2 __. ...

Sample DNA Spike DNA Detected % Recovery of Mean total in sample after protein DNA detec-ted DNA spiking concentration Mean af-te.~
(Lowry)filtrati.on 15 Bl 500 mg 100 ug81 ug 98.9% 16.5 pg B1 500 mg 100 ug92 ug 98.0~ 8.6 pg 12O3pg Bl 500 mg 100 ug74 ug 96.7~ 11.7 pg B1 500 mg 0 0 96.3~ 3.3 pg Bl 500 mg - 0 0 92.8~ 3.2 pg 3O2.pg 20 Buffer lOO ug89 ug N/A16~9 pg Buffer 100 ug96 ug N/A 3.3 pg lO.lpg Buffer 0 0 N/A 9.8 pg Buffer 0 0 N/A 2.6 pg 6O2pg * total DNA in 500 mg sample of monoclonal antibody (mean ob-sexvation of samples assayed in duplicate) Exampls 6 Endotoxin Removal A lOOml solution of 50m~/ml bovine serum albumin in 10% maltose-phosphate buffer solution contaminated with DNA
and a endotoxin was filtered through a 47 mrn DNA/virus 3~ removal filtration device. The starting S~ T IT5~1TE SH!E~T

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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 de-tected in any analyzed portion of filtrate. Endotoxin levels wereo first= 30.72 EU/ml, middle= 30.72 EU/ml and last= 61O44 EU/ml. Endotoxin removal in the end sample was 96~9%~
Solution recovery was 95% (95ml) with no change in protein concentration.

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Claims

1. A process for the removal of DNA, endotoxins and viruses from aqueous buffer solutions, aqueous phar-maceutical solutions and aqueous biological phar-maceutical solutions comprising, passing one of said aqueous buffer solutions, aqueous pharmaceutical solutions and aqueous biological pharmaceutical solutions through a first DNA filter section and a second virus filter section, wherein said DNA, viruses and endotoxins are substantially removed; and collecting said aqueous buffer solutions, aqueous pharmaceutical solutions or aqueous biological pharmaceutical solutions; said first DNA filter section having:
(i) a first absolute pore filter, (ii) a first and second filter selected from one of (a) a DEAE cellulose filter membrane and (b) an absolute pore filter coated with at least one of DEAE cellulose, quaternary aminoethyl salts and quaternary aminomethyl salts, and (iii) a second absolute pore filter;
and said second virus filter section having absolute pore filters of smaller pore diameter than the absolute pore filters in the DNA filter section.

2. A process in accordance with claim 1 wherein the yield of pharmaceuticals or biological pharmaceuticals, in the filtered solutions is 90% or higher compared to the starting solution.

3. A process in accordance with claim 1 wherein the virus removal is 99.9999997% or higher when the virus is 0.100 microns or larger, and the yield of pharmaceutical or biological pharmaceutical in the filtered solution of same of 90% or higher compared to the starting solution.

4. A process in accordance with claim 1 wherein said DNA in the filtered solutions is less than 10 picog-rams per 100 ml of solution.

5. A process in accordance with claim 1 wherein the aqueous buffer solutions, aqueous pharmaceutical solutions and aqueous biological pharmaceutical solutions have a pH in the range of 3 to 9.

6. A process in accordance with claim 1 wherein the aqueous buffer solutions, aqueous pharmaceutical solutions and aqueous biological pharmaceutical solutions each have a specific salt content of less than 0.5 Molar, said specific salt being at least one selected from the group consisting of the lithium, sodium, potassium and ammonium salts of the phosphate, chloride, bromide, iodide, sulfate and acetate anions.

7. A process in accordance with claim 1 wherein said DNA in the filtered solutions is less than 1 picog-ram per 100 ml of solution.

8. A process for the removal of DNA and viruses from aqueous pharmaceutical solutions and aqueous biological pharmaceutical solutions, comprising passing either one of the solutions through a first DNA filter section and a second virus filter section to obtain a filtered aqueous pharmaceutical solution or a filtered aqueous biological pharmaceutical solution having substantially reduced DNA and virus levels, said solutions passing through:
(a) a first DNA filter section comprising a first 0.2 micron absolute pore filter, a first diethylaminoet-hyl cellulose filter, a second diethylaminoethyl cel-lulose filter and a second 0.2 micron absolute pore fil-ter, and (b) a second virus filter section comprising a first 0.1 micron absolute pore filter, a second 0.1 micron ab-solute pore filter, a first 0.04 micron absolute pore filter and a second 0.04 micron absolute pore filter; and collecting the filtered solutions.

9. A process in accordance with claim 8 wherein the yield of the pharmaceutical or biological pharmaceutical in the filtered solution is 90% or higher.

10. A process in accordance with claim 8 wherein the virus removal is 99.99999997% or higher when the virus is 0.100 microns or larger, and the yield of the pharmaceutical or biological pharmaceutical in the fil-tered solution is 90% or higher.

11. A process in accordance with claim 8 wherein the DNA in the filtered pharmaceutical or biological pharmaceutical solution is reduced to less than 10 picog-rams per 100 ml of solution.

12. A process in accordance with claim 8 wherein the pharmaceutical solution or biological pharmaceutical solution has a pH in the range of 6 to 8.

13. A process in accordance with claim 8 wherein the pharmaceutical or biological pharmaceutical has a specific salt content, excluding salts of pharmaceuticals or biological pharmaceuticals, of less than 0.5 Molar, said specific salts being at least one selected from the group consisting the lithium, sodium, potassium and am-monium salts of the phosphate, chloride, bromide, iodide, sulfate and acetate anions.

14. A process in accordance with claim 1 or 8 wherein said aqueous buffer solutions, aqueous phar-maceutical solutions and aqueous biological phar-maceutical solutions are passed through said DNA filter section and said virus filter section by a vacuum means or a pressure means.

15. A process in accordance with claim 8, wherein the DNA in the filtered pharmaceutical or biological pharmaceutical solution is reduced to less than 1 picog-ram per 100 ml of solution.

16. An improved apparatus for the removal of DNA, endotoxins and viruses from aqueous buffer solutions, aqueous pharmaceutical solutions and aqueous biological pharmaceutical solutions, said apparatus having a housing with suitable inlet/outlet means, internal gaskets and filter supports, and internal filters wherein the improvement comprises a first DNA filter section having, from inflow to outflow, a first 0.2 micron filter, a first diethylaminoethyl cellulose filter, a second diet-hylaminoethyl cellulose filter and a second 0.2 micron filter, the filters having face-to-face contact; and a second virus filter section having, from inflow to outflow, a first 0.1 micron filter, a second 0.1 micron filter, a first 0.04 micron filter and a second 0.04 mic-ron filter, said filters having face-to-face contact.

17. An improved apparatus in accordance with claim 16 wherein said 0.2, 0.1 and 0.04 filters are absolute pore filters.

18. The improved apparatus in accordance with claim 16 wherein said apparatus is capable of removing DNA to a level of less than 10 picograms per 100 ml of solution.

19. The improved apparatus of claim 16 wherein said apparatus is capable of removing 99.9999997% of virus when the virus is 0.100 microns or larger.

20. The improved apparatus of claim 16 wherein the apparatus gives a yield of pharmaceutical or biological pharmaceutical in the filtered solution of 90% or higher compared to the unfiltered solution.

21. The improved apparatus of claim 16 where the solution to be filtered is passed through said apparatus by pressure or vacuum means.

22. The improved apparatus of claim 16 wherein said apparatus is a means of administration of said aqueous buffer solution, aqueous pharmaceutical solution or aqueous biological pharmaceutical solution to a patient which means contains said DNA and virus filter sections.

23. The improved apparatus of claim 16 wherein said apparatus is a syringe filtration device containing said DNA and Virus removal filters.

24. An improved apparatus for the removal of DNA, endotoxins and viruses from aqueous buffer solutions, aqueous pharmaceutical solutions and aqueous biological pharmaceutical solutions, said apparatus having a housing with suitable inlet/outlet means, internal gaskets and filter supports, and internal filters wherein the improvement comprises a first DNA filter section having, from inflow to outflow, a first 0.2 micron filter, first and second absolute pore filters coated with at least one selected from the group consisting of DEAE cellulose, QAE, QAM and other quaternary ammonium salts, a second 0.2 micron filter, the filters having face-to-face contact; and a second virus filter section having, from inflow to outflow, a first 0.1 micron filter, a second 0.1 micron filter, a first 0.04 micron filter and a sec-ond 0.04 micron filter, said filters having face-to-face contact.

25. The improved apparatus in accordance with claim 14, wherein said apparatus is capable of removing DNA to a level of less than 1 picogram per ml of solution.

26. An improved apparatus for the removal of DNA, endotoxins and viruses from aqueous buffer solutions, aqueous pharmaceutical solutions and aqueous biological pharmaceutical solutions, said apparatus having a housing with suitable inlet/outlet means, internal gaskets and filter supports, and internal filters wherein the improvement comprises a first DNA filter section having, from inflow to outflow, a first 0.2 micron filter, a sec-ond 0.2 micron filter, the filters having face-to-face contact; and a second virus filter section having, from inflow to outflow, a first 0.1 micron filter, a second 0.1 micron filter, a first 0.04 micron filter and a sec-ond 0.04 micron filter, said filters having face-to-face contact; wherein at least one of the absolute pore fil-ters is coated with at least one of the group consisting of DEAE, QAE, QAM and other quaternary ammonium salts.

27. Apparatus for removing DNA, endotoxins and viruses from aqueous buffer solutions, aqueous phar-maceutical solutions and aqueous biological phar-maceutical solutions comprising, a housing having inlet and outlet means for such solutions, a stacked assembly of filter sections and internal gasketing and support means for said filter sections, said assembly including a DNA filter section and a virus filter section constructed and arranged to substantially remove DNA, virus and en-dotoxins from such solution passed therethrough, and means for collecting said solutions after such removal of dna, endotoxins and viruses.

28. Apparatus according to claim 27 in which said DNA filter section has (i) a first absolute pore filter, (ii) a first and second filter selected from one of (a) a DEAE cellulose filter membrane and (b) an absolute pore filter coated with at least one of DEAE cellulose, quaternary aminoethyl salts and quaernary aminomethyl salts, and (iii) a second absolute pore filter;
and said virus filter section has absolute pore filters of smaller pore diameter than the absolute pore filters in the DNA filter section.