Use of centrifugation-aided infection to increase virus titer

Abstract

The invention relates to a process for increasing the observed titer of a virus stock for the purpose of increasing the calculated log reduction (LRV) in virus clearance studies. A tissue culture or assay plate is seeded with an indicator cell line and titrated with a virus stock followed, by a centrifugation step for about 5 minutes to about 24 hours at a g-force ranging from about 50g to about 2400g, and at a temperature from about 4 C. to about 39 C. The resulting calculated virus titer after undergoing the centrifugation step is 10-fold higher than the virus titer would be if determined in the absence of the centrifuging step.

Claims

1. A process for increasing the observed titer of a Xenotropic murine leukemia virus (X-MuLV) stock for the purpose of increasing the calculated log reduction (LRV) in a virus clearance study comprising the steps of: a) providing a X-MuLV stock, cell culture media, indicator cell line, and an assay plate; b) adding the indicator cell line and media to the assay plate; c) adding the X-MuLV stock to the cell line and media on the assay plate; d) centrifuging the assay plate containing the cell line, media, and X-MuLV stock; e) stopping the centrifuging step; f) incubating the assay plate centrifuged in step d); and g) measuring the observed titer of the X-MuLV stock, wherein the resulting titer of the X-MuLV stock is 10-fold higher than the X-MuLV titer would be if measured in the absence of the centrifuging step (d).

2. The process of claim 1, wherein the assay plate is selected from the group consisting of a tissue culture plate, a well plate, a flask or a test tube.

3. The process of claim 1, wherein centrifuging step (d) occurs from about 5 minutes to 24 hours at g-force range from about 50g to 2400g.

4. The process of claim 1, wherein centrifuging step (d) occurs from about 30 minutes to 120 minutes at g-force range from about 500g to 1000g.

5. The process of claim 1, wherein the temperature during centrifuging step (d) is about 4 C. to 39 C.

6. The process of claim 1, wherein the temperature during centrifuging step (d) is about 21 C. to 39 C.

7. The process of claim 1, further comprising an additional incubating step, wherein the assay plate containing X-MuLV stock, media and cell line is additionally incubated prior to the centrifuging step (d).

8. The process of claim 1, wherein the X-MuLV stock is prediluted.

9. The process of claim 1, wherein the incubation temperature is about 37 C.

10. The process of claim 7, wherein the incubation temperature is about 37 C.

11. The process of claim 1, wherein the temperature during centrifuging step (d) is about 25 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graphical representation of the measured virus titer vs. centrifugation time.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(2) For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term about.

(3) Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the Invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible.

(4) Moreover, all ranges disclosed herein are to be understood to encompass all subranges subsumed therein. For example, a range of 1 to 10 includes any and all subranges between (and including) the minimum value of 1 and the maximum value of 10, that is, any and all subranges having a minimum value of equal to or greater than 1 and a maximum value of equal to or less than 10, e.g., 5.5 to 10.

(5) Before describing the present invention in further detail, a number of terms will be defined. Use of these terms does not limit the scope of the invention but only serves to facilitate the description of the invention.

(6) As used herein, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise.

(7) The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, cell culture, virology, immunology and the like which are in the skill of one in the art. These techniques are fully disclosed in current literature and reference in made specifically to Sambrook, Fritsch and Maniatis eds., Molecular Cloning, A Laboratory Manual, ed Ed., Cold Spring Harbor Laboratory Press (1989); Cells J. E. Cell Biology. A Laboratory Handbook Academic Press, Inc. (1994) and Bahnson et at, J. of Virol. Methods 54:131-143 (1995).

(8) All publications and patent applications cited in this specification are indicative of the level of skill of those skilled in the art to, which this invention pertains and are hereby incorporated by reference in their entirety.

(9) Contamination of biopharmaceutical products, e.g., antibodies, recombinant proteins, vaccines, blood derivatives, plasma, and animal products, etc., by bacteria, viruses, prions, and the like, is a serious risk that needs to be sufficiently addressed. Contamination can arise by different ways. For example, it can occur because the source material (e.g., the cells in a cell culture, the blood product, etc.) is intrinsically contaminated with viruses.

(10) The manufacturing processes of biopharmaceutical products are also susceptible to virus contamination from extrinsic sources (e.g., inadvertent introduction from use of non-sterile or improperly sterilized materials). Because of the nature of the products, manufacturers of biopharmaceutical products are highly regulated and are required to incorporate sufficient virus clearance steps into their manufacturing processes to ensure that their products are contaminant-free. Multiple virus clearance steps can be incorporated into a manufacturing process. Each of these virus clearance steps needs to be evaluated for its effectiveness and thus, validated before a biopharmaceutical Manufacturing process is approved. Virus clearance steps typically involve either virus removal steps or virus inactivation steps.

(11) Virus removal as used herein means a method in which the virus is physically removed from the sample. This is often achieved by either nanofiltration or chromatography. Nanofiltration techniques remove viruses by size exclusion. The success of chromatographic methods for removing viruses depends on the column composition and the reagents (e.g., buffers) used.

(12) Virus inactivation, as used herein means a method in which the viruses may remain in the final product, but in a non-infective (inactive) form. Many viruses contain lipid or protein coats that can be inactivated by chemical alteration. Alternatively, some viral inactivation processes denature the virus completely. Examples of virus inactivation methods include solvent and/or detergent inactivation, pasteurization (e.g., heating to high temperatures), pH inactivation (e.g., using an acidic pH), and irradiation (e.g., ultraviolet (UV) or gamma irradiation).

(13) The concentration of a contaminating virus, or the risk of virus contamination, in a manufacturing process may be extremely low, but, because viruses are by their nature infective, even one viral particle can, be sufficient to ruin an entire run in a manufacturing process. It is for this reason that special measures must be taken to determine the appropriate removal or inactivation methods in a manufacturing process. As such, the effectiveness of such virus clearance methods needs to be evaluated and validated. Spiking studies were created specifically for this purpose.

(14) Spiking studies use a scaled down model of the virus clearance step of a production-scale process to evaluating and/or validate the virus clearance steps and to evaluating and/or validate the apparatus used in the virus clearance method. Virus stocks of as high a titer as possible are desirable. This is due in part because the volume of virus spike that can be added to a test system is limited by Government Mandated Regulatory Guidelines (e.g., The U.S. Food and Drug Administration (FDA)).

(15) The titer of the virus stock therefore determines the maximum possible concentration of virus spiked into the test material. As a result, the actual capacity of the test unit to clear virus in the production process may be underestimated. Current limitations of our knowledge of virus production has resulted in methods that lack the capacity to produce virus stocks of high purity and high titer.

(16) The techniques provided herein increase the efficiency of virus titration assays, resulting in virus stocks having a measured virus titer about tenfold (10-fold) higher than the virus titer would be if measured in the absence of these techniques. This increase in measured virus titer is of great benefit for virus clearance studies because this can increase the LRVs that can be demonstrated for highly effective virus clearance operations.

(17) The Tissue Culture Infectious Dose 50% (TCID.sub.50) assay is a method for counting the number of infectious viral particles in a sample. The TCID.sub.50 is the quantity of a pathogenic agent (virus) that will produce a cytopathic effect in 50% of the cultures inoculated. The TCID.sub.50 value is proportional to, but not the same as, the number of infectious virions in a sample. Titers determined using this method are typically reported as TCID.sub.50/ml.

(18) When no virus at all is detected by the assay, a maximum possible titer for the sample is determined using a Minimal. Limit of Detection (LOD) calculation. This calculation considers the sensitivity of the assay and reports the most virus that could be in a sample without the assay detecting any. The result of these calculations is a titer reported as X, meaning that the actual titer in the sample is X or lower (to a 95% certainty). This LOD calculation depends solely on the quantity of sample tested and any predilutions made to the sample before assaying.

(19) Infectious virus titer is usually quantified by either plaque-forming unit (PFU) assay or tissue culture infectious dose 50% (TCID.sub.50) assay. Both of these techniques involve adding serial, dilutions of a virus-containing solution to a tissue culture containing susceptible host cells. The amount of virus in the original sample is then calculated by counting the number of infection events on the cells. The amount of virus detected by these assays is limited by the ability of the virus to infect the host cells and the rate at which it does so.

(20) The idea of increasing the infection of cells by retrovirus by centrifuging the cell culture is known, see for example, in Forstell, et al. (1996) J. Viral Meth 60:171. Forstell demonstrated that centrifugation during infection, or spinoculation, increased the ability of retrovirus to transduce a reporter gene into host cells.

(21) As taught herein, and previously unbeknownst to us, we have found that the centrifugation of tissue culture or assay plates (i.e., a process referred to herein as spinoculation or centrifugal inoculation of cell cultures) appears to improve the detection of retrovirus in TCID.sub.50 assay.

(22) As taught herein, and previously unbeknownst to us, when Xenotropic twine leukemia viruses (X-MuLV) are titrated onto a tissue culture or assay plate seeded with an indicator cell line are centrifuged, that centrifugation or spinoculation step of the tissue culture plates after the virus has been added to the cell lines on the plate unexpectedly increases the sensitivity of the TCID.sub.50 assay such, that the virus titer of the original stock is calculated to be approximately 10-fold higher than when the TCID.sub.50 assay is performed by standard methods. Other than the addition of the centrifugation or spinoculation step, the TCID.sub.50 assay is performed as a usual standard. TCID.sub.50 assay known in the art.

(23) The exact mechanism by which spinoculation works is unclear. The centrifugal force is insufficient, to sediment the virus onto the cells. While not wishing to be bound to any one theory, it is postulated that there may be some deformation of the cell surface that enables More efficient virus attachment. This could be especially effective for retrovirus because retrovirus is known to have a natural charge-based repulsion to the cell surface.

(24) The use of spinoculation to increase the titer of a virus stock is a technique that, up until now has not been used in areas such as virus clearance studies. We have found that when a virus stock (i.e., X-MuLV stock) is titrated by standard TCID.sub.50 methods, but with the addition of centrifugation (i.e., spinoculation) of the cell culture plates (e.g., at 1800 RPM) over increasing periods of times after addition of the virus, a gradual increase in the measured titer of the virus stock was unexpectedly discovered.

(25) The measured titer of a virus stock is dependent upon the methods and/or processes used to determine that measurement. We have found that modifying a standard TCID.sub.50 assay by the spinoculation process as taught herein provides an unexpectedly increased sensitivity to the titer of a virus stock, which has the benefit of effectively increasing the titers of existing virus stocks (e.g., X-MuLV) by 10-fold.

(26) Increasing the titers of existing virus stocks 10-fold has a direct impact upon the log reduction values (LRV) achieved when using such virus stocks for its intended purpose in virus clearance validation studies. Use of the spinoculation technique as provided herein in virus clearance studies essentially provides an extra 1 LRV for retroviruses when the clearance operation being tested results in no virus being detected in the post-unit operation material. In such cases, the LRV for a virus clearance process is based entirely on the titer of virus in the spiked material and the amount of post-unit operation material tested. Because spinoculation increases the measured titer of virus of the spiked material by 1 log, this translates directly into a 1 LRV gain in clearance (vs. titration of the spiked feed by methods traditionally used).

(27) Given that biopharmaceutical producers have hard targets that must be met for retrovirus clearance (typically a total of 12-20 LRV across the entire process), use of the spinoculation techniques as taught herein unexpectedly provides the ability to achieve an extra 1 LRV to virus filtration steps, which typically result in no virus being detected in the filtrate. In addition, the spinoculation technique taught herein can be used at every vim clearance step, and potentially add 1 LRV to each step that is effective enough to reduce virus to non-detectable levels. This could potentially add a total of 2 to 4 LRV for retrovirus to typical MAb processes.

(28) The following examples are provided for the purpose of further illustrating the present invention but are in no way to be taken as limiting. In addition, the following examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and how to practice the methods of the invention, and are not intended to limit the scope of what the inventor regards as his invention. Efforts have been made to insure accuracy with respect to numbers used (e.g. amounts, temperature, etc.), but some experimental errors and deviation should be accounted for. Unless indicated otherwise, temperature is in degree Celsius (AC), chemical reactions were performed at atmospheric pressure or transmembrane pressure, as indicated, the term ambient temperature refers to approximately 25 C. and ambient pressure refers to atmospheric pressure.

(29) The invention will be further clarified by the following examples which are intended to be exemplary of the invention.

EXAMPLES

Example 1

(30) Test materials used: A) Xenotropic murine leukemia virus (X-MuLV) 1) Strain: ATCC VR-1447 2) Prep type: VSP X-MuLV Mk1 purified 3) Lot # CP2; Production date: November 2009 4) Recorded titer: 5.94 log TCID.sub.50/ml B) Minute virus of mice (MVM) 1) Strain: ATCC VR-1346 2) Prep type: VSP MVM Mk2.1 3) Recorded titer 10.4 log TCID.sub.50/ml C) Plate preparation 1) The day prior to the experiment, 96-well plates were seeded with PG-4 cells (labeled #1 to #16) by standard protocol (each well containing 100 l media with 5.0 g polybrene/ml (final concentration will be 2.54g/ml after inoculation). 2) Also, 96-well plates were seeded with 324K cells (labeled #17 to #20) by standard protocol. D) Virus stock dilution 1) A single ten-fold dilution series was made of the X-MuLV stock virus in 50 ml tubes (10.sup.2 through 10.sup.8). (a) Label seven (7) tubes #2 through #8. (b) The following media was used for the dilutions: (1) McCoy's media supplemented with 1% FBS, 1 Penn/Strep, 1 L-Glut, 1 NEAA (as per standard X-MuLV titration protocol). (c) 49.5 ml of media was added to the #2 tube. 45 ml of media was added to tubes #3 through #8. (d) 0.5 ml virus stock was added to the #2 tube and mixed. (e) 5 ml was transferred from the #2 tube to the #3 tube, and mixed. A tenfold dilution series was continued to the #8 tube. 2) A single ten-fold dilution series of the MVM stock virus was made in 50 ml tubes (10.sup.2 through 10.sup.13). (a) Twelve (12) tubes were, labeled #2 through #13 (b) The following media was used for the dilutions: (1) DMEM media supplemented with 1% FBS, 1 Penn/Strep, 1 L-Glut, 1 NEAA (as per standard MVM titration protocol). (c) 19.8 ml of media was added to the #2 tube. 18 ml media was added to tubes #3 through #7. 3) 0.2 ml virus stock was added to the #2 tube and mixed. 4) 2 ml was transferred from the #2 tube to the #3 tube and mixed. A tenfold dilution series was continued to the #13 tube, E) Virus titration 1) The X-MuLV and MVM were titrated by standard protocols using the dilutions made above. (a) X-MuLV: titrated across plates #1 to #16 from to 10.sup.3 to 10.sup.8 (b) MVM: titrated across plates #17 to #20 from to 10.sup.8 to 10.sup.13 2) AH plates were placed in a 37 C. at 5% CO.sub.2 incubator until their turn in the centrifuge and after. F) Spinoculation 1) Plates were centrifuged for the times specified in Table 1, provided in section (e) below. (a) The 0 Min controls was simply left in the incubator. (b) Speed: 1800 RPM=670g (c) Temperature: set to 25 C. (d) Each plate was spun continuously for the complete duration of its run (i.e., no start/stopping) (e)

(31) TABLE-US-00001 TABLE 1 Centrifugation time (min) X-MuLV MVM 0 1-2 17-18 5 3-4 10 5-6 15 7-8 20 9-10 30 11-12 45 13-14 19-20 90 15-16 G) Plates were incubated at 37 C. at 5% CO.sub.2 until appropriate read time (7 days for XMLV, 10 days for MVM), then the plates were scored. H) Results provided in Tables 2 and 3

(32) TABLE-US-00002 TABLE 2 Titers for XMLV (Xenotropic murine leukemia virus) Sample Time Titer (log Std vs. Name (min) TCID.sub.50) Error time 0 Control 0 5.88 0.06 5 min Spin 5 6.44 0.13 +0.56 10 min Spin 10 6.72 0.03 +0.84 15 min Spin 15 6.75 0.00 +0.88 20 min Spin 20 7.00 0.00 +1.13 30 min Spin 30 7.00 0.06 +1.13 45 min Spin 45 7.19 0.13 +1.31 90 min Spin 90 7.34 0.09 +1.47

(33) TABLE-US-00003 TABLE 3 Titers for MVM (Minute virus of mice) Titer (log Std Sample Name TCID.sub.50) Error Control-MVM 9.53 0.22 45 min 9.56 0.06

(34) Compared to the Control, an increase of about a 1.5 log in virus titer was seen after spinning the plates containing XMLV for 90 minutes in Table 2. There was no significant difference from the Control in the MVM plates in Table 3.

(35) FIG. 1 is a graphical representation of the measured virus titer vs. centrifugation time. A X-MuLV stock was titrated by standard TCID.sub.50 methods, with the addition of centrifugation of the 96-well plates at 1800 RPM for various times after addition of the virus. As the plates were spun over increasing time periods, a gradual increase in measured virus titer was seen, with the greatest increases occurring within the first 60 minutes.

(36) The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in applications claiming priority from this or a related application. Such claims, whether directed to a different invention or to the same invention, and Whether broader, narrower, equal, or different in scope to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.