Compositions and methods for detecting biological contaminants

Abstract

Provided herein are compositions and methods for determining whether a microbial contaminant is present in a biotherapeutic production process. In particular, artificial positive amplification control plasmids and specific quantitative PCR detection probes are provided that enable rapid and real-time detection of false positive results.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202210143904.4. Among them, the Chinese Patent Application No. 202210143904.4 is a divisional application of Chinese Patent Application No. 201680025470.0.

[0002] Cross-reference to related applications

[0003] This application claims the priority of U.S. Provisional Patent Application No. 62 / 139,321, filed on March 27, 2015, under 35 USC § 119(e), which is hereby incorporated by reference in its entirety. Technical field

[0004] The present invention generally relates to methods for manufacturing biomolecules by cell culture. The present invention more specifically but not exclusively relates to compositions and methods for detecting biological contaminants in cell cultures. Background art

[0005] Biopharmaceuticals, particularly therapeutic antibodies, are produced by mammalian cell culture. Chinese hamster ovary (CHO) cells are the most commonly used host cells. These production systems are prone to exogenous and endogenous viral infections, which pose potential safety problems for biopharmaceuticals. Therefore, virus clearance procedures and virus load measurements are used to enhance the safety of the drugs. Steps for reducing virus load include nanofiltration, virus inactivation by heat or pH maintenance, and chromatography. The virus load and the effectiveness of virus removal can be monitored by time-consuming infectivity assays or by rapid quantitative assays such as real-time PCR or quantitative polymerase chain reaction (Q-PCR).

[0006] Q-PCR requires appropriate negative and positive controls to be reliable. Nucleic acid extraction controls are added to test samples to control for proper nucleic acid extraction. If the nucleic acid extraction control is negative or outside the expected recovery range during Q-PCR, the sample is rejected. Conversely, if the nucleic acid extraction control is positive or within the expected recovery range during Q-PCR, the nucleic acid extraction from the test sample is considered reliable. Negative controls, such as buffers without samples, are usually included in Q-PCR assays. The presence of a positive signal in the negative control may indicate contamination of the Q-PCR or nucleic acid extraction reagents with viral material.

[0007] Positive amplification controls can also be included in Q-PCR virus load assays. Such positive controls can include conserved viral nucleic acid sequences amplified using primers that amplify true viral contaminants. Failure to detect the positive control in Q-PCR may indicate that the amplification procedure will not be able to detect viral contaminants if they are present in the test sample.

[0008] Using a positive amplification control that mimics the target contaminant creates its own problems. If the test sample is contaminated by even a slight amount of the positive control, given the high sensitivity of Q-PCR, the test sample may show false positives. By using a low level of the positive amplification control, performing positive control work in an isolated chamber, using the UNG / dUTP system to selectively degrade PCR products containing dUTP, using disposable containers and displacement pipettes, and thoroughly cleaning the work area and equipment, false positives can be mitigated to some extent. Regardless of which mitigation agent is used, false positive results can still occur during the PCR testing process.

[0009] In biopharmaceutical manufacturing, the risk of obtaining false positives for biological contaminants cannot be ignored and can lead to costly corrective actions. There is a great need for systems and methods to determine whether a given positive PCR result is a true positive or a false positive caused by cross - contamination from the positive control. The applicant has developed and now discloses positive control compositions, systems, and methods that allow for the real - time determination of false positive Q - PCR signals. Summary of the Invention

[0010] The applicant has solved the problem of real - time identification of whether a positive Q - PCR signal for a target contaminant in a test sample is a true positive or a false positive due to cross - contamination. The applicant has created a Positive Amplification Control (PAC) plasmid that includes a biological contaminant target sequence (i.e., the positive control sequence) and a unique artificial plasmid - specific sequence. This unique artificial plasmid - specific sequence enables the assay technician to specifically identify the plasmid in the sample. Thus, when a positive contaminant signal is detected and the absence of the artificial plasmid - specific sequence is determined, the technician can be confident that the result is a true positive result. Conversely, in the case of a false positive, the presence of the unique artificial plasmid sequence allows the technician to quickly rule out an apparently positive result as a false positive.

[0011] In some embodiments, a positive control human artificial plasmid specific sequence (“specific sequence”; also known as PACP or positive amplification control polynucleotide) is detected using a fluorescently labeled artificial oligonucleotide detection probe (“unique detection probe” or “UDP”) included in a Q-PCR reaction mixture. The unique detection probe comprises a nucleic acid polymer covalently bound to a fluorophore and a quencher. The “unique” nucleic acid polymer is designed to specifically anneal to the specific sequence and be incorporated into the amplified copy of the specific sequence during PCR. The “unique” nucleic acid polymer is designed not to recognize or anneal to any naturally occurring parvovirus under the hybridization conditions used in the target assay operation. In one embodiment, the “unique” nucleic acid polymer comprises 17 to 20 nucleotides, wherein no more than seven (7) to 10 internal consecutive nucleotides and no more than six (6) consecutive 3’ nucleotides are identical to any parvovirus sequence. In one embodiment, the “unique” nucleotide polymer comprises 17 to 20 nucleotides, wherein no more than 7 to 10 consecutive nucleotides and no more than six (6) consecutive 3’ nucleotides are identical to any parvovirus sequence shown in SEQ ID NOs: 9 and 12 - 37. When the nucleic acid polymer of the unique detection probe is not incorporated into the amplified copy, the quencher remains in close proximity to the fluorophore. If the fluorophore is excited, the quencher absorbs the emitted light and prevents the light from being detected (by FRET or contact quenching). When the nucleic acid polymer is incorporated into the amplified copy (i.e., when the specific sequence is present in the sample), the fluorophore and the quencher are released from the unique detection probe and are thus spatially separated. In this case, when the fluorophore is excited, the quencher is far enough away that it cannot effectively quench the emitted light. Thus, the emitted light can be detected. Therefore, when the specific sequence is present in the sample, as PCR progresses, the intensity of the detectable emission wavelength increases. When the specific sequence is absent, as PCR progresses, the quencher does its job and no emission wavelength is detected. In one embodiment, the fluorophore is attached at or near the 5’ end of the nucleic acid polymer, and the quencher is attached at or near the 3’ end. In an alternative embodiment, the fluorophore is attached at or near the 3’ end of the nucleic acid polymer, and the quencher is attached near or at the 5’ end.

[0012] Any fluorophore-quencher pair known now or discovered later can be used in the practice of the present invention. (See, e.g., S.A. Marras, “Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes,” Methods Mol. Biol. 2006; 335:3-16.). In some embodiments, the excitation wavelengths of the fluorophores are 495 nm, 538 nm, or 646 nm, respectively, and the emission wavelengths are 520 nm, 554 nm, or 669 nm, respectively. In some embodiments, the quencher is a dye with an absorption peak from 430 nm to 672 nm. In some embodiments, the quencher is selected from DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, and DHQ-3. In one embodiment, the excitation wavelength of the fluorophore is 495 nm, the emission wavelength is 520 nm, such as FAM, and the quencher is BHQ-1. In one embodiment, the nucleic acid polymer comprises the nucleic acid sequence of SEQ ID NO: 3 (5’-TGTCGATGGCGAATGGCTA-3’).

[0013] One aspect of the present invention is the unique detection probe itself containing a nucleic acid polymer and a linked fluorophore and quencher as described above. Other aspects of the present invention include the positive amplification control (PAC) plasmid itself containing a biological contaminant target sequence and a unique artificial plasmid-specific sequence, and using the plasmid as a positive control to evaluate the presence of the target biological contaminant in a cell culture. The PAC plasmid is used to control a successful PCR amplification reaction designed to amplify the target biological contaminant sequence. For example, separate and parallel PCR reactions are run that contain the same components as the test sample and are run under the same parameters, but contain the positive control plasmid instead of the test sample. If the sample containing the positive control plasmid produces a positive “contaminant” signal while the test sample does not, then it can be concluded that the test sample does not have the target biological contaminant. In some embodiments, the test sample is obtained from a mammalian cell culture, such as a bioreactor culture containing CHO cells engineered to produce a therapeutic protein of interest.

[0014] In one embodiment, the PAC plasmid contains (a) a target amplification polynucleotide (TAP) sequence, such as a parvovirus nucleic acid sequence or the sequence of another target contaminant, and (b) a plasmid amplification control polynucleotide (PACP) sequence. The PACP sequence (sense strand or Crick strand) is complementary to the nucleic acid polymer of a unique sequence probe (antisense strand or Watson strand).

[0015] In one embodiment, the PAC plasmid is deployed in a separate Q-PCR reaction in parallel with the Q-PCR reaction containing the test sample. The TAP sequence is designed to represent the target biological contaminant. For example, if the test sample Q-PCR reaction fails to produce a TAP amplicon and the positive control (i.e., the sample containing the PAC plasmid) Q-PCR fails to produce a TAP amplicon, the Q-PCR reaction can be considered a failure. In one embodiment, the target biological contaminant is rodent parvovirus and the TAP sequence contains rodent parvovirus sequences. In one embodiment, the TAP sequence includes all or part of the parvovirus NS1 sequence, which is conserved across several rodent parvovirus strains. See Cotmore, et al., “Replication Initiator Protein NS1 of the parvovirus Minute Virus of Mice Binds to Modular Divergent Sites Distributed throughout Duplex Viral DNA,” J. Virol. 2007 Dec;81(23):13015-13027. In some embodiments, several rodent parvovirus strains include murine parvovirus prototype strain (MVMp), murine parvovirus immunosuppressive strain (MVMi), murine parvovirus Cutter strain (MVMc), murine parvovirus 1b (MPV-1b), murine parvovirus 1a (MPV-1a), murine parvovirus 1c (MVP-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a (RPV-1a), rat parvovirus (RMV), and the University of Massachusetts strain of rat virus L (RV-Umass). See O.-W. Merten, “Virus Contaminations of Cell Cultures - A Biotechnological View,” Cytotechnology. 2002 July;39(2):91-116. In one embodiment, the TAP sequence contains the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and the complementary sequence of SEQ ID NO: 4.

[0016] In other aspects, the present invention relates to PCR mixture compositions and methods of using the PCR mixture to detect target contaminants in a test sample and to exclude false positives due to contamination of the PAC test sample.

[0017] In one embodiment, the PCR mixture specifically comprises a target contaminant-specific forward oligonucleotide primer, a target contaminant-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the above-described unique detection probe (UDP), and a target contaminant-specific reverse oligonucleotide primer. In an embodiment where the target contaminant is rodent parvovirus, the PCR mixture specifically comprises a rodent parvovirus-specific forward oligonucleotide primer, a rodent parvovirus-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the above-described unique detection probe (UDP), and a rodent parvovirus-specific reverse oligonucleotide primer. Each detection probe (i.e., the target contaminant-specific oligonucleotide detection probe, such as the rodent parvovirus-specific oligonucleotide detection probe and the artificial oligonucleotide detection probe) contains a nucleic acid sequence with one end (5' or 3') linked to a fluorophore and the other end (3' or 5' respectively) linked to a quencher.

[0018] Here, the nucleic acid sequences of the rodent parvovirus-specific forward oligonucleotide primer and the rodent parvovirus-specific oligonucleotide detection probe hybridize to the antisense strand of parvovirus. The rodent parvovirus-specific reverse oligonucleotide primer hybridizes to the sense strand of parvovirus. In one embodiment, the parvovirus sequence to which the primer and the parvovirus-specific oligonucleotide probe hybridize is a conserved rodent parvovirus sequence. In one case, the conserved parvovirus sequence is the parvovirus NS1 sequence, such as the nucleic acid sequence described in SEQ ID NO: 9. By using the conserved sequence, a single probe will effectively detect multiple rodent parvovirus strains.

[0019] In one embodiment, the artificial oligonucleotide detection probe does not hybridize to parvovirus nucleic acid or any biological contaminant sequence. The nucleic acid of the artificial oligonucleotide detection probe is synthetic and will not hybridize to any biological contaminant nucleic acid sequence under any stringency. In one embodiment, the nucleic acid of the artificial oligonucleotide detection probe (also known as the "unique" nucleic acid polymer or unique sequence) is designed not to recognize or anneal to any naturally occurring parvovirus under the hybridization conditions used in the assay of this study. In one embodiment, the "unique" nucleic acid polymer contains 17 to 20 nucleotides, where no more than seven (7) to 10 internal consecutive nucleotides and no more than six (6) consecutive 3' nucleotides are identical to any parvovirus sequence. In one embodiment, the "unique" nucleotide polymer contains 17 to 20 nucleotides, where no more than 7 to 10 consecutive nucleotides and no more than six (6) consecutive 3' nucleotides are identical to any parvovirus sequence shown in SEQ ID NOs: 9 and 12 - 37. However, the nucleic acid of the artificial oligonucleotide detection probe (i.e., the unique sequence) hybridizes to the PACP sequence of the PAC plasmid. Thus, the artificial oligonucleotide detection probe detects the PAC plasmid but does not detect parvovirus or other biological contaminant sequences.

[0020] In one embodiment, the PCR mixture can be used to determine whether the PAC plasmid is present in a test biological sample, resulting in false positive results. Here, if the test sample shows a positive Q-PCR signal for parvovirus-specific oligonucleotide probes and a negative Q-PCR signal for artificial oligonucleotide detection probes, the test sample is considered to be free of PAC contamination (i.e., true positive).

[0021] In one embodiment, the target contaminant-specific forward oligonucleotide primer comprises the sequence of SEQ ID NO: 1; the target contaminant-specific oligonucleotide detection probe nucleic acid comprises the sequence of SEQ ID NO: 2; the artificial oligonucleotide detection probe nucleic acid (i.e., UDP) comprises the sequence of SEQ ID NO: 3; and the target contaminant-specific reverse oligonucleotide primer comprises the sequence of SEQ ID NO: 4.

[0022] In other aspects, the present invention provides systems and methods for detecting biological contaminants in a test sample. Here, the test sample is a cell culture, such as an industrial-scale mammalian cell culture for the production of therapeutic proteins. Mammalian cells that can be used in the practice of the present invention include, but are not limited to, CHO cells, CHO-K1 cells, and EESYR cells (see U.S. Patent No. 7,771,997). In addition to the primers, probes, mixtures, and PAC plasmids used as described above, the systems and methods also include the use of a nucleic acid extraction control (NEC). In one embodiment, the NEC is M13K07 phage, which is included in the test sample prior to nucleic acid extraction. If the nucleic acid extraction is sufficient to detect contaminant DNA or RNA, the NEC nucleic acid (e.g., M13K07 nucleic acid) is detected by Q-PCR in the "spiked" test sample. In a specific embodiment, the Q-PCR reaction mixture contains a target contaminant-specific forward oligonucleotide primer, a target contaminant-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the aforementioned unique detection probe (UDP), a target contaminant-specific reverse oligonucleotide primer, an NEC-specific forward oligonucleotide primer, an NEC-specific oligonucleotide detection probe, and an NEC-specific reverse oligonucleotide primer. In one embodiment, the test sample is taken from a therapeutic protein production cell culture to which NEC (e.g., M13K07 phage) has been added prior to nucleic acid extraction and subsequent Q-PCR analysis.

[0023] In a specific embodiment, the target contaminant is rodent parvovirus. In this case: (1) the target contaminant-specific forward oligonucleotide primer is a rodent parvovirus-specific forward oligonucleotide primer, more specifically comprising the sequence of SEQ ID NO: 1; (2) the target contaminant-specific oligonucleotide detection probe is a rodent parvovirus-specific oligonucleotide detection probe, more specifically comprising the nucleic acid sequence of SEQ ID NO: 2; (3) the artificial oligonucleotide detection probe is a unique detection probe (UDP), more specifically comprising the nucleic acid sequence of SEQ ID NO: 3; (4) the target contaminant-specific reverse oligonucleotide primer is a rodent parvovirus-specific reverse oligonucleotide primer, more specifically comprising the sequence of SEQ ID NO: 4; (5) the NEC-specific forward oligonucleotide primer is an M13 forward oligonucleotide primer, more specifically comprising the sequence of SEQ ID NO: 5; (6) the NEC-specific oligonucleotide detection probe is an M13 detection probe, more specifically comprising the nucleic acid sequence of SEQ ID NO: 6; and (7) the NEC-specific reverse oligonucleotide primer is an M13 reverse oligonucleotide primer, more specifically comprising the nucleic acid of SEQ ID NO: 8. In one embodiment of the method, if an NEC Q-PCR signal is detected (i.e., the signal is within the expected recovery range), then the nucleic acid extraction of the test sample can be considered successful. On the other hand, if no NEC signal is detected (i.e., the signal is outside the expected recovery range), then the nucleic acid extraction of the test sample can be considered a failure, and any negative Q-PCR target contaminant signal is considered invalid (i.e., false negative). Detailed Description

[0024] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as these methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value can vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 94.4, etc.).

[0026] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Other embodiments will become apparent by referring to the following detailed description.

[0027] The following detailed description is given to provide a complete understanding of the invention described herein.

[0028] The present invention relates to improved materials and methods for detecting any biological contaminants in any cell culture producing a therapeutic protein. Specifically, the present invention relates to quantitative polymerase chain reaction (Q-PCR) materials and methods that include a positive amplification control element or step that is easily detectable to eliminate false positives.

[0029] PCR and quantitative PCR

[0030] As used herein, the phrase "polymerase chain reaction" ("PCR") refers to a method for preparing copies of nucleic acids (e.g., DNA) by using multiple cycles of denaturation (separation of template DNA strands), annealing (hybridization of single-stranded oligonucleotides to single-stranded template DNA strands), and DNA synthesis (catalysis by a DNA polymerase of the synthesis of a new DNA strand initiated from the 3' end of a hybridized oligonucleotide using the template DNA strand as a template). To effect amplification, at least two different oligonucleotide primers (simply referred to as "primers") are used in the PCR reaction. One primer, commonly referred to as the forward primer, hybridizes to the antisense strand of the template DNA and forms the 5' end of the newly synthesized sense strand. The other primer, commonly referred to as the reverse primer, hybridizes to the sense strand of the template DNA and forms the 5' end of the newly synthesized antisense strand. In each cycle, each template strand is replicated to form a new double-stranded DNA molecule, which is also referred to as an "amplicon". Thus, using a non-limiting amount of oligonucleotide primers, DNA polymerase (i.e., Taq polymerase or other thermostable DNA polymerases; see Innis et al., DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA, 85(24) Proc Natl Acad Sci U S A. 9436-40 (1988)), and nucleoside triphosphates, the number of DNA molecules (template and amplicons) doubles in each cycle. PCR is described in U.S. Patent No. 4,683,202 (issued July 28, 1987). See also PCR Primer: A Laboratory Manual (Carl W. Dieffenbach & Gabriela S. Dveksler eds., 1995).

[0031] As used herein, the term "cycle" refers to a single round of (1) DNA strand unwinding, called "denaturation", followed by (2) hybridization of oligonucleotide primers to the resulting single-stranded DNA by the rules of base pairing, a process called "annealing", and (3) polymerization starting from the 3' end of the oligonucleotide primer and moving in the 5' to 3' direction to form a new DNA strand, a process called "amplification" or "extension". Typically, polymerization uses a DNA polymerase such as Taq polymerase to catalyze the formation of phosphodiester bonds between adjacent deoxynucleotide triphosphates ("dNTPs"), placing the dNTPs along the exposed single-stranded template DNA by hydrogen bonding according to the rules of base pairing. Denaturation, annealing, and amplification are carried out at certain temperatures, based in part on the GC content of the DNA template and oligonucleotide primers and the length of the DNA strand to be replicated. The temperatures of denaturation and annealing and the ionic strength of the reaction buffer control the stringency of hybridization and the fidelity of DNA replication.

[0032] "Quantitative PCR" or "qPCR" or "Q-PCR" (also known as "real-time PCR") is a type of PCR that is capable of monitoring the formation of amplicons during the PCR cycle. Q-PCR can be used to quantify the amount of a specific template DNA in a sample. In addition to forward and reverse oligonucleotide primers, Q-PCR also introduces at least one oligonucleotide detection probe into the reaction mixture. The detection probe is a single-stranded oligonucleotide that hybridizes to the sense or antisense strand of the target template DNA somewhere between the forward primer binding site and the reverse primer binding site. During the annealing step, the oligonucleotide detection probe anneals to the single-stranded template. When polymerization occurs, the probe is cleaved and degraded by the 5' nuclease activity of the DNA polymerase. Thus, as the specific template sequence is amplified, the detection probe is degraded at an exponential rate.

[0033] Q-PCR oligonucleotide detection probes are typically constructed with a linked fluorophore (also referred to as a reporter fluorochrome, or simply "reporter") and a linked quencher. In most cases, the fluorophore is linked at or near the 5' end of the oligonucleotide, and the quencher is linked at or near the 3' end of the oligonucleotide. However, any feasible construct can be used in the practice of the present invention. When the oligonucleotide detection probe is intact, the fluorophore and quencher are in close proximity such that the quencher absorbs the light emitted by the excited fluorophore, thereby significantly reducing the detectable fluorophore emission. When the oligonucleotide detection probe is cleaved or degraded, the fluorophore and quencher are released and are thus spatially separated. The quencher is no longer close enough to quench the fluorophore emission. When more specific amplicons are formed, more oligonucleotide detection probes are cleaved, more fluorophores and quenchers are released, such that more fluorophore / quencher pairs are separated, and the fluorescence emission amplitude increases. In other words, the increased fluorophore emission signal is correlated with the amount of specific target DNA in the sample. For a review of Q-PCR, see Ian M. Mackay et al., Survey and Summary: Real-Time PCR in Virology, 30(6) Nucleic Acids Research 1292-1305 (2002).

[0034] Fluorescence quenching can occur by direct contact between the reporter and the quencher (also known as static quenching) or by fluorescence resonance energy transfer (FRET) between the reporter and the quencher when both are within each other's radius. A specific fluorophore can be excited by light of one or more specific wavelengths or a wavelength range having a maximum wavelength. This is referred to as the excitation wavelength. After the fluorophore is excited, it returns to the ground state and emits light of a longer wavelength than the excitation wavelength. This is referred to as the emission wavelength. During FRET, a second fluorophore, dye, lanthanide molecule, etc. having an absorption spectrum that matches or overlaps the emission spectrum of the fluorophore absorbs the light emitted by the fluorophore excited within the radius, thereby quenching or reducing the fluorophore emission wavelength. Contact or static quenching occurs when the reporter and the quencher form a ternary complex in the ground state of the fluorophore. The ternary complex is non-fluorescent, i.e., essentially non-excitable, and thus does not emit light at the expected emission wavelength.

[0035] For a review of static quenching and FRET, see Salvatore A.E. Marras et al., Efficiencies of Fluorescence Resonance Energy Transfer and Contact-Mediated Quenching in Oligonucleotide Probes, 30(21) Nucleic Acids Research e122, pp. 1-8 (2002). Marras et al. also discuss reporter / quencher pairs for use in applications for Q-PCR.

[0036] Nucleic Acids

[0037] As used herein, the terms "polynucleotide", "oligonucleotide", "probe", "primer", or "nucleotide primer" or "oligonucleotide primer", "template", or "template nucleic acid" or "template DNA" are used in their ordinary sense to a person of ordinary skill in the art in the field of molecular biology. For a detailed description of each of these, see, for example, PCR Primer: A Laboratory Manual (Carl W. Dieffenbach & Gabriela S. Dveksler eds., 1995).

[0038] As used herein, "amplicon" refers to a DNA product produced by amplifying a template nucleic acid sequence by PCR. As PCR proceeds and the template is amplified, the newly formed DNA amplicons serve as templates for subsequent rounds of DNA synthesis.

[0039] Cell culture

[0040] The present invention relates to an improved Q-PCR method for detecting biological contaminants in cell cultures. Cell cultures are commonly used to produce complex biomolecules for therapeutic use, such as antibodies, trap molecules, and Fc fusion proteins. These cultures should be kept free of biological contaminants. Detection of contaminants is important for determining whether a particular batch is to be discarded or repaired.

[0041] Cell cultures include media and cells typically derived from a single cell line. Here, the cell line includes cells capable of producing biotherapeutic proteins. Examples of cell lines routinely used for the production of protein biotherapeutics specifically include primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells, and chicken embryo cells. The Chinese hamster ovary (CHO) cell line or one or more of several specific CHO cell variants such as the CHO-K1 cell line is optimized for large-scale protein production. The cell line is a specialized CHO cell line optimized for enhancing the production of a protein of interest. For details regarding the cells, see U.S. Patent No. 7,771,997 (issued August 10, 2010).

[0042] "Cell culture" or "culture" refers to cells growing and multiplying outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See, for example Animal cell culture: A Practical Approach (D. Rickwood, ed., 1992). Mammalian cells can be cultured in suspension or attached to a solid matrix. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors with or without microcarriers and operating in batch, fed-batch, continuous, semi-continuous, or perfusion mode can be used for mammalian cell culture. Cell culture medium or concentrated feed medium can be added continuously or intermittently to the culture during culturing (i.e., fed-batch). For example, the culture can be fed daily, every other day, every three days, or when the concentration of a specific medium component monitored directly or indirectly falls outside the desired range.

[0043] Animal cells such as CHO cells or Cells can be cultured in small-scale cultures, such as in a 125 ml container with approximately 25 ml of culture medium, a 250 ml container with approximately 50 to 100 ml of culture medium, or a 500 ml container with approximately 100 to 200 ml of culture medium. Alternatively, the culture can be large-scale, such as a 1000 ml container with approximately 300 to 1000 ml of culture medium, a 3000 ml container with approximately 500 ml to 3000 ml of culture medium, an 8000 ml container with approximately 2000 ml to 8000 ml of culture medium, and a 15000 ml container with approximately 4000 ml to 15000 ml of culture medium. The culture used for manufacturing can contain 10,000 L or more of culture medium. Large-scale cell cultures, such as those used for the clinical manufacture of protein therapeutics, are typically maintained for days or weeks while the cells produce the desired protein. During this period, culture samples can be taken and tested for the presence of biological contaminants.

[0044] Production of therapeutic proteins

[0045] Cell cultures monitored for biological contamination can be used to produce proteins or other biomolecules of interest, such as therapeutically effective antibodies or other biopharmaceutical substances. The protein product (target protein) can in particular be an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody or a tetrabody, a Fab fragment or an F(ab’)2 fragment, an IgA antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody.

[0046] The protein of interest can be a recombinant protein containing an Fc portion and another domain (e.g., an Fc-fusion protein). The Fc-fusion protein can be a receptor Fc-fusion protein that contains one or more extracellular domains of one or more receptors coupled to the Fc portion. In some cases, the Fc portion contains a hinge region followed by the CH2 and CH3 domains of IgG. In some cases, the receptor Fc-fusion protein contains two or more different receptor chains that bind a single ligand or multiple ligands. For example, the Fc-fusion protein is a trap, such as an IL-1 trap (e.g., rilonacept, which contains the ligand-binding region of IL-1RAcP fused to the extracellular region of Il-1R1 fused to the Fc domain of hIgG1; see U.S. Patent No. 6,927,004) or a VEGF trap (e.g., aflibercept, which contains the Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1 fused to the Fc domain of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159).

[0047] The present invention is not limited to any particular type of cell or cell line for protein production. Examples of cell types suitable for protein production include mammalian cells, such as CHO-derived cells such as insect cells, avian cells, bacterial cells, and yeast cells. The cells can be stem cells or recombinant cells transformed with a vector for recombinant gene expression, or cells transfected with a virus for production of viral products. The cells can contain a recombinant heterologous polynucleotide construct encoding the protein of interest. The construct can be episomal (e.g., an extrachromosomal plasmid or fragment), or it can be physically integrated into the genome of the cell. The cells can also produce the protein of interest without encoding the protein on a heterologous polypeptide construct. In other words, the cells can naturally encode the protein of interest, such as B cells that produce antibodies. Methods and vectors for genetically engineering cells or cell lines to express a protein of interest are well known to those skilled in the art. For example, various techniques are described in Current Protocols in Molecular Biology , Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., MolecularCloning:ALaboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R.J., Large Scale Mammalian CellCulture, 1990, pp. 15 - 69. A variety of cell lines suitable for culturing growth can be obtained from the American Type Culture Collection (Manassas, Va.) and commercial suppliers.

[0048] The cells can also be primary cells, such as chicken embryo cells or primary cell lines. Examples of useful cells include BSC cells, LLC - MK cells, CV - 1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK - 1 cells, LLCPK cells, PK15 cells, LLC - RK cells, MDOK cells, BHK - 21 cells, chicken embryo cells, NS - 1 cells, MRC - 5 cells, WI - 38 cells, BHK cells, 293 cells, Per.C6 cells, and CHO cells. In various embodiments, the cell line is a CHO cell derivative, such as CHO - K1, CHO DUX B - 11, CHO DG - 44, Veggie - CHO, GS - CHO, S - CHO, CHO lec mutant line or cell line.

[0049] In one particular case, the cells are CHO cell derivatives that ectopically (heterologously) express a protein, such as cells. The protein contains an immunoglobulin heavy chain region, such as the CH1, CH2, or CH3 region. In one embodiment, the protein contains human or rodent immunoglobulin CH2 and CH3 regions. In one embodiment, the protein contains human or rodent immunoglobulin CH1, CH2, and CH3 regions. In one embodiment, the protein contains a hinge region and CH1, CH2, and CH3 regions. In specific embodiments, the protein contains an immunoglobulin heavy chain variable domain. In specific embodiments, the protein contains an immunoglobulin light chain variable domain. In specific embodiments, the protein contains an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain. In specific embodiments, the protein is an antibody, such as a human antibody, a rodent antibody, or a chimeric human / rodent antibody (e.g., human / mouse, human / rat, or human / hamster).

[0050] The protein production phase of the cell culture can be carried out at any culture scale, from single culture flasks and shake flasks or shake bags to one-liter bioreactors and to large-scale industrial bioreactors. Large-scale processes can be carried out in volumes of approximately 100 liters to 20,000 liters or greater. One or more of several methods can be used to control protein production, such as temperature shift or chemical induction. The growth phase of the cells can be carried out at a higher temperature than the production phase of protein expression and / or secretion. For example, the growth phase can be carried out at a first temperature of about 35°C to 38°C, and the production phase can be carried out at a second temperature of about 29°C to 37°C, optionally about 30°C to 36°C or about 30°C to 34°C. In addition, a chemical inducer of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetamide (HMBA), can be added either before or after the temperature shift, or simultaneously. If the inducer is added after the temperature shift, it can be added from 1 hour to 5 days after the temperature shift, for example, 1 to 2 days after the temperature shift. The production cell culture can be carried out as a continuous-feed culture system, such as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec; 17(6):1032-41), or according to a fed-batch (batch-fed) process (Huang, 2010).

[0051] Therapeutic protein products

[0052] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably throughout and refer to a molecule comprising two or more amino acid residues linked to each other by peptide bonds. Peptides, polypeptides, and proteins can also include modifications such as glycosylation, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Peptides, polypeptides, and proteins can have scientific or commercial interest, including protein-based drugs. Peptides, polypeptides, and proteins include, in particular, antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins are produced by recombinant animal cell lines using cell culture methods.

[0053] "Antibody" refers to an immunoglobulin molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds). Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed among more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, produced, or isolated by recombinant methods, such as antibodies isolated from host cells transfected to express an antibody. The term antibody also includes bispecific antibodies, which include heterotetrameric immunoglobulins that can bind to more than one epitope. Bispecific antibodies are generally described in U.S. Patent Application Publication No. 2010 / 0331527, which is incorporated herein by reference.

[0054] The "antigen-binding portion" (or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab’)2 fragments, divalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al. (1989) Nature 241:544-546), which consist of the VH domain, (vi) isolated CDRs, and (vii) scFv, which consists of the two domains VL and VH of an Fv fragment linked by a synthetic linker to form a single polypeptide chain, wherein the VL and VH regions pair to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also included under the term "antibody" (see, e.g., Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0055] An antibody or antigen-binding portion thereof can be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the preparation of tetramerized scFv molecules using streptavidin core regions (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the preparation of divalent and biotinylated scFv molecules using cysteine residues, tag peptides, and C-terminal polyhistidine tags (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab’)2 fragments, can be prepared from whole antibodies using conventional techniques, e.g., by digestion of the whole antibody with papain or pepsin. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques (see Sambrook et al., 1989).

[0056] The term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention can, for example, include amino acid residues not encoded by human germline immunoglobulin sequences in the CDRs, particularly in CDR3 (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, the term “human antibody” as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0057] The term “recombinant human antibody” as used herein is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from transgenic animals (e.g., mice) for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, produced, or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using transgenic animals for human Ig sequences), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that may not naturally occur in the human antibody germline repertoire when derived from and related to human germline VH and VL sequences.

[0058] "Fc fusion protein" contains portions or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, which are not found together in nature. The preparation of fusion proteins containing certain heterologous polypeptides fused to various portions of antibody-derived polypeptides, including the Fc domain, has been described, for example, in Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. "Receptor Fc fusion protein" contains one or more extracellular domains of a receptor coupled to an Fc portion, which in some embodiments includes a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc fusion protein contains two or more different receptor chains that bind one or more ligands. For example, the Fc fusion protein is a trap, such as an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcP ligand-binding region fused to the extracellular region of IL-1R1 fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004) or a VEGF trap (e.g., aflibercept, which contains the Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159).

[0059] Biological contaminants

[0060] As used herein, the term "biological contaminant" refers to any unwanted, undesired, harmful, or potentially harmful biological entity. Such entities specifically include prions (the etiological cause of bovine / spongiform encephalopathy), virions, viruses, mycoplasmas, other bacteria, contaminating metazoan cells, DNA, RNA, transposons, other transposable elements, yeasts, other fungi, algae, protists, and other exogenous and endogenous agents. Particular concern in the production process of biotherapeutic agents using rodent cells such as CHO cells and derivatives of CHO cells is exogenous viruses associated with the cells or the culture medium or the manufacturing raw materials. Contamination of the large-scale processed materials of cell culture and the resulting drug products poses a direct risk to patients and an indirect risk of interrupting the drug supply.

[0061] Non-exhaustive lists of exogenous and / or endogenous agents that can infect CHO cell cultures include: single-stranded (-)RNA viruses such as Cache Valley virus, influenza A / B virus, parainfluenza 1 / 2 / 3, simian virus 5, mumps virus, bovine respiratory syncytial virus, and vesicular stomatitis virus; single-stranded (+)RNA viruses such as bovine coronavirus, herpesvirus 2117, encephalomyocarditis virus, coxsackievirus B-3, Semliki Forest virus, and Sindbis virus; double-stranded RNA viruses such as bluetongue virus, epizootic hemorrhagic disease virus, and reovirus 1 / 2 / 3; single-stranded DNA viruses such as porcine circovirus 1, and particularly problematic parvoviruses, which include murine parvovirus (also known as minute virus of mice); and double-stranded DNA viruses such as adenovirus and pseudorabies virus. Among these potential exogenous agents, four viruses are primarily from large harvest samples of CHO cell cultures from various manufacturers. These viruses are reovirus type 2, Cache Valley virus, epizootic hemorrhagic disease virus, and the rodent parvovirus murine parvovirus. For a detailed review of exogenous viral contamination of CHO cell cultures, see Andreas Berting et al., Virus Susceptibility of Chinese Hamster Ovary (CHO) Cells and Detection of Viral Contaminations by Adventitious Agent Testing, 106(4) Biotechnology and Bioengineering 598-607 (2010), and Andrew Kerr & Raymond Nims, Adventitious Viruses Detected in Biopharmaceutical Bulk Harvest Samples over a 10 Year Period, 64(5) PDA Journal of Pharmaceutical Science & Technology 481-485 (2010).

[0062] Exogenous agent testing is divided into two major categories. The first category is the classical virological method using in vitro virus assays. Here, the test sample is applied to an indicator cell line, the cells are incubated and passaged for 14 to 28 days, and then endpoints such as cytopathic effect or hemagglutination reaction are measured (Berting, 2010). The second category is PCR-based assays, which measure in real time the presence of nucleic acids associated with exogenous or endogenous agents. See, for example, Zhan et al., Detection of Minute Virus of Mice Using Real Time Quantitative PCR in Assessment of Virus Clearance during the Purification of Mammalian Cell Substrate Derived Biotherapeutics, 30(4) Biologicals 259-270 (2002).

[0063] Mouse minute virus (also known as MMV, murine minute virus, or MVM) poses a particular problem for the manufacture of biotherapeutics. The US Food and Drug Administration (FDA) and European Medicines both require specific testing for MVM. This virus is a member of the Parvoviridae family (parvoviruses) and is common in mice. It is excreted in urine and feces and persists robustly in the environment. It can be easily introduced into the biotherapeutic manufacturing process. See Moody et al., Mouse Minute Virus (MMV) Contamination–A Case Study: Detection, Root Cause Determination, and Corrective Actions, 65(6) PDA Journal of Pharmaceutical Science and Technology 580-288 (2011). Other rodent parvoviruses can have a negative impact on cell culture-based biopharmaceutical production. In addition to the MVM prototype strain, these rodent parvoviruses specifically include the MVM immunosuppressive strain and cutter strain, murine parvovirus 1a (MPV-1a), MPV-1b, MPV-1c, hamster parvovirus, Toolan's parvovirus (parvovirus H-1), Kilham rat virus, rat parvovirus 1a, rat parvovirus, and the Umass strain of rat virus L. See S.F. Cotmore & P. Tattersal, The Autonomously Replicating Parvoviruses of Vertebrates, 33 Advances in Virus Research 91-174 (1987), and Jacoby et al., Rodent Parvovirus Infections, 46(4) Lab Anim Sci. 370-80 (1996).

[0064] These parvoviruses share a conserved nucleic acid sequence called NS-1 (NS1), which encodes a large non-structural protein involved in viral genome amplification. The conserved NS1 nucleotide sequence from MVM is shown in SEQ ID NO: 9. Nucleotides 875-956 of this sequence are at least 97% conserved among a wide range of rodent parvovirus NS1 sequences and thus serve as a good target sequence for PCR-based testing of exogenous and endogenous agents. PCR-based testing for rodent parvoviruses (as well as other exogenous and endogenous agents) can be performed at the raw materials, pre-harvest media, various points along the overall process of purification of the biotherapeutic molecule, and the formulation and packaging stages. Detection of contaminants may require remediation steps such as treating contaminated materials, replacing raw materials, and decontaminating the facility.

[0065] In addition to testing for exogenous agents, endogenous agents, and other biological contaminants during manufacture, which enables corrective and preventive actions (CAPA), special manufacturing and batch processing steps (i.e., unit operations) can be employed to eliminate, reduce, or inactivate viral contaminants. Chemical inactivation, virus retention filtration, and chromatography have been shown to be effective in reducing herpesviruses, retroviruses, and parvoviruses in harvested or partially purified cell culture fluids. The most commonly used chemical inactivation step is low pH treatment, which is thought by those skilled in the art to be due to the denaturation of viral envelope proteins. Protein A, hydroxyapatite, cation exchange, and anion exchange chromatography steps have been shown to remove viruses to some extent. See, for example, Miesegaes et al., Analysis of Viral Clearance Unit Operations for Monoclonal Antibodies, 106(2) Biotechnology and Bioengineering 238-246 (2010), and Liu et al., Recovery and Purification Process Development for Monoclonal Antibody production, 2(5) mAbs 480-499 (2010).

[0066] Q-PCR test: positive and negative controls

[0067] Testing for biological contaminants should be appropriately controlled to ensure accuracy, reliability, and credibility. As used herein, a "negative control" contains most or all of the experimental reagents and conditions, but does not contain the test sample. The test sample can be replaced with a buffer or mock medium known not to contain the biological contaminant of interest. In addition, as used herein, a "negative control" should produce a negative result for the biological contaminant. If the negative control produces a positive biological contaminant result, then those skilled in the art or scientists may conclude that a positive result for a parallel test sample may not accurately reflect whether the test sample contains the biological contaminant.

[0068] As used herein, one or more "positive controls" are used to evaluate whether the experimental conditions are sufficient to operably detect biological contaminants. Positive controls can be used at any step along the experimental process to ensure that each step is functioning and to determine at which step of the process a failure occurs.

[0069] In some embodiments, a positive control is used in the nucleic acid extraction step to evaluate whether the expected extraction of any biocontaminant nucleic acid is efficient enough to detect the biocontaminant. This positive control is referred to as a "nucleic acid extraction control" or "NEC". In some cases, the NEC is chosen to mimic the target biocontaminant in the form of a protein-nucleic acid structure. If the NEC is extracted in a manner sufficient to be detected, then the technician can assume that the target biocontaminant nucleic acid is also extracted in a manner sufficient to be detected. In one embodiment, the NEC is a single-stranded DNA phage, not entirely unlike parvovirus.

[0070] In a specific embodiment, the NEC is the M13 phage, which consists of circular single-stranded DNA of approximately 6,407 nucleotides. In a more specific embodiment, the NEC is the M13K07 strain, which is a commonly available molecular biology reagent for cloning and other laboratory purposes. See van Wezenbeek et al., Nucleotide Sequence of the Filamentous Bacteriophage M13 DNA Genome: Comparison with Phage fd, 11(1-2) Gene 129-148 (1980). The nucleotide sequence of the M13K07 phage is shown in SEQ ID NO: 8. Although the M13 phage or the M13K07 phage strain can be used as the NEC in a specific embodiment, the present invention is in no way limited to using this particular agent as the NEC. One of ordinary skill in the art can substitute another agent as the NEC in the practice of the present invention without departing from the scope of the present invention (e.g., the MS2 phage for reverse transcriptase-PCR assays; see Kothapalli et al., Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template, 109(2) J. Virol. Methods 203-207 (2003)).

[0071] In some embodiments of the present invention, a positive control is used in the PCR amplification step to evaluate whether the PCR reagents (including primers) and the PCR step are sufficient to detect the template nucleic acid of biological contaminants. This positive control is referred to as the "plasmid amplification control" or "PAC". In some cases, the PAC is selected or designed to match the target biological contaminant nucleic acid sequence and exactly match the forward and reverse oligonucleotide primers of the test sample. In a specific embodiment, the PAC additionally contains a "unique sequence" not found in the target biological contaminant nucleic acid. In a more specific embodiment, the unique sequence does not exist in nature. A "unique" nucleic acid polymer is designed to specifically anneal to this unique sequence and be incorporated into the amplified copies of the unique sequence during PCR. The "unique" nucleic acid polymer is designed not to recognize or anneal to any naturally occurring parvovirus under the hybridization conditions used in the target assay operation. In one embodiment, the "unique" nucleic acid polymer contains 17 to 20 nucleotides, where no more than seven (7) to 10 internal consecutive nucleotides and no more than six (6) consecutive 3' nucleotides are identical to any parvovirus sequence. In one embodiment, the "unique" nucleotide polymer contains 17 to 20 nucleotides, where no more than 7 to 10 consecutive nucleotides and no more than six (6) consecutive 3' nucleotides are identical to any parvovirus sequence shown in SEQ ID NO: 9 and 12 - 37.

[0072] This unique sequence enables those skilled in the art to distinguish between the true target biological contaminant sequence and cross - contamination of the test Q - PCR reaction with the PAC plasmid.

[0073] In one embodiment, the PAC (as a plasmid, also known as the PAC plasmid) is included in the same but separate Q - PCR reaction to control PCR amplification. The PAC reaction uses exactly the same oligonucleotide primers and probes as those used in the test sample Q - PCR reaction to accurately reflect the amplification of the actual target biological contaminant nucleic acid. The test sample Q - PCR reaction and the separate PAC plasmid Q - PCR reaction include a target detection probe and a unique sequence probe. If the PAC reaction functions properly, a positive target signal and a positive unique sequence signal are expected. Obtaining a positive target signal and a positive unique sequence signal in the reaction containing the PAC plasmid indicates that whenever the target biological contaminant nucleic acid is present, the test sample Q - PCR reagents and conditions can adequately generate a positive target signal in the test sample.

[0074] Thus, the PAC serves as a control for proper PCR amplification. If a negative target signal is obtained in the test sample, but the PAC shows a positive target signal, one skilled in the art can conclude that no detectable target contaminant DNA is present in the test sample. Conversely, if a positive target sequence signal and a positive specific sequence signal are obtained in the test sample, one skilled in the art can assume that the PAC plasmid has cross-contaminated the test sample, and thus the positive target sequence signal may be a false positive.

[0075] Detailed description of several implementation plans

[0076] In some embodiments, an improved positive control system (i.e., compositions and methods) is provided for detecting biological contaminants by using polymerase chain reaction, more specifically Q-PCR.

[0077] In one aspect, the present invention provides a unique sequence probe (USP) for detecting a positive amplification control plasmid (PAC plasmid). The USP contains an artificial nucleotide sequence, a fluorophore, and a quencher that are capable of hybridizing to a unique artificial plasmid specific sequence (also referred to as a unique sequence or PACP specific sequence, or UAPS). In a specific embodiment, the artificial nucleotide sequence capable of hybridizing to the UAPS comprises the nucleic acid sequence of SEQ ID NO: 3 (5'-TGTCGATGGCGAATGGCTA-3'), which is antisense to the sense strand sequence of the UAPS (e.g., i.e., SEQ ID NO: 10 - 5'-TAGCCATTCGCC ATCGACA-3').

[0078] As described above, the Q-PCR detection probe contains a fluorophore and a quencher. Depending on the fluorophore / quencher pair, quenching can occur by contact quenching or FRET. Here, the fluorophore and the quencher are covalently linked to the USP oligonucleotide. In one embodiment, the fluorophore has an excitation wavelength of 495 nm to 680 nm and an emission wavelength of 515 nm to 710 nm. In some embodiments, the excitation wavelengths of the fluorophore are 495 nm, 538 nm, or 646 nm, respectively, and the emission wavelengths are 520 nm, 554 nm, or 669 nm, respectively. In some embodiments, the quencher is a dye with an absorption peak of 430 nm to 672 nm. Examples of useful quenchers include Dabcyl, Iowa Black Iowa Black and In a specific embodiment, the fluorophore is fluorescein amidite (FAM; described in U.S. Patent No. 5,583,236, issued December 10, 1996), which has an absorbance maximum of approximately 495 nm and an emission maximum of approximately 520 nm, and the quencher is Black Hole ( Biosearch Technologies, Inc., Petaluma, CA), which absorbs at 480 nm to 580 nm. Quenching occurs by FRET and contact quenching. Generally but not always, the quencher is linked to the 3'-hydroxyl group of the oligonucleotide by an ether bond, while the fluorophore is linked to the 5'-phosphate group of the oligonucleotide by an ester bond.

[0079] On the other hand, the present invention provides a mixture of Q-PCR reagents comprising a plurality of oligonucleotides and probes, which can be used to detect biological contaminants in cell cultures, raw materials, partially purified and purified biomolecules, etc. In one embodiment, the biological contaminant is a DNA virus, more specifically a parvovirus, and more specifically a rodent parvovirus, such as MVM. The parvovirus contains an NS1 gene having a nucleic acid sequence that is at least 88% identical to any of the sequences listed in Table 1. In another embodiment, the parvovirus contains an NS1 gene having a nucleic acid sequence that is at least 97% identical to the sequence shown in SEQ ID NO: 9 (i.e., the mouse minute virus (MVM) NS1 gene). In another embodiment, the parvovirus contains an NS1 gene comprising the consensus sequence of SEQ ID NO: 37.

[0080] Table 1: Parvoviridae NS1 Sequences

[0081]

[0082] In a specific embodiment, the mixture contains (1) a rodent parvovirus-specific forward oligonucleotide primer; (2) a rodent parvovirus-specific oligonucleotide detection probe; (3) an artificial oligonucleotide detection probe, such as USP; and (4) a rodent parvovirus-specific reverse oligonucleotide primer. This mixture can be used in test samples and positive control samples. In a more specific embodiment, the oligonucleotide primer and the parvovirus-specific oligonucleotide detection probe hybridize to the NS1 sequence, such as the NS1 sequence shown in SEQ ID NO: 9. In a more specific embodiment, (1) the forward primer contains the sequence of SEQ ID NO: 1; (2) the parvovirus-specific oligonucleotide detection probe contains the sequence of SEQ ID NO: 2; (3) the artificial probe is USP and contains the sequence of SEQ ID NO: 3; and (4) the reverse primer contains the sequence of SEQ ID NO: 4.

[0083] As described above, USP contains a fluorophore and a quencher such that amplicons containing this sequence (i.e., PAC DNA) can be detected in real time as Q-PCR progresses. In a similar manner, the parvovirus-specific detection probe contains an oligonucleotide with a covalently linked fluorophore and quencher. In practice, the emission wavelength of the fluorophore of the parvovirus detection probe should be different from that of USP to distinguish a true parvovirus positive signal from a false positive due to PAC plasmid or other PACP contaminating the test sample (e.g., PACP-contaminated amplicons). Thus, in an embodiment where the USP fluorophore is FAM, the fluorophore linked to the parvovirus detection probe oligonucleotide should have an emission wavelength other than approximately 520 nm. In a specific embodiment, the fluorophore linked to the rodent parvovirus-specific oligonucleotide detection probe is a dye (Life Technologies, Inc., Carlsbad, CA) that has a maximum absorbance at 538 nm and a maximum emission at approximately 554 nm. Here, the quencher can be a FRET quencher or a contact quencher. In one embodiment, the quencher is a minor groove binding non-fluorescent quencher (MGBNFQ) (see Sylvain et al., Rapid Screening for HLA-B27 by a TaqMan-PCR Assay Using Sequence-Specific Primers and a Minor Groove Binder Probe, a Novel Type of TaqMan TM Probe, 287(1-2) Journal of Immunological Methods 179-186 (2004)).

[0084] The mixture of the above primers and probes is used to test and distinguish the positive amplification control construct from true parvovirus contaminants. In some embodiments, the mixture of primers further comprises a set of primers and probes for detecting a nucleic acid extraction control (NEC). In a specific embodiment, the NEC is M13 bacteriophage, such as the M13K07 strain (SEQ ID NO: 8). Thus, in some embodiments, in addition to the parvovirus primers and probes and the USP probe, the mixture of primers and probes contains (5) an M13-specific forward oligonucleotide primer; (6) an M13-specific oligonucleotide detection probe; and (7) an M13-specific reverse oligonucleotide primer.

[0085] In some embodiments, the M13 primers and probes hybridize to the sequence of SEQ ID NO: 8. In a specific embodiment, the M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 5; the M13-specific oligonucleotide detection probe comprises the nucleic acid sequence of SEQ ID NO: 6; the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 7. As in the case of the non-overlapping fluorophore emission spectra of the parvovirus probe and the USP, the NEC probe contains a fluorophore that emits light in a non-overlapping spectrum. In a specific embodiment, the fluorophore linked to the M13-specific oligonucleotide detection probe is Cy5, which is a cyanine dye having an absorbance maximum of about 650 nm and a maximum emission maximum of 670 nm (see Southwick et al., Cyanine Dye Labeling Reagents: Carboxymethylindocyanine Succinimidyl Esters, 11 Cytometry, 418 - 430 (1990)). Here, the quencher can operate by FRET or contact quenching. In one embodiment, the quencher is Black Hole ( Biosearch Technologies, Inc., Petaluma, CA), and quenches in the range of about 550 nm to about 650 nm.

[0086] On the other hand, the present invention provides a method for detecting biological contaminants during the production of biomolecules. Biological contaminants more specifically include parvoviruses, more particularly rodent parvoviruses, and most particularly those parvoviruses having at least 97% identity with the MVM NS1 gene. In some embodiments, the NS1 gene comprises the sequence of SEQ ID NO: 9. In one embodiment, the biomolecule production method is a mammalian cell culture method for preparing antibodies, trap molecules, or other therapeutic antibodies. A test sample is taken from the cell culture (or bulk components) and nucleic acid is extracted. In some cases, the test sample is added to a nucleic acid extraction control (NEC), such as M13 (e.g., SEQ ID NO: 8), to serve as a control for proper nucleic acid extraction prior to Q-PCR. The method comprises the steps of: (1) mixing (a) a nucleic acid sample extracted from the test sample, (b) oligonucleotide primers and probes (as described above), and (c) a DNA polymerase, preferably a thermostable DNA polymerase with 5' exonuclease activity such as Taq polymerase; (2) subjecting the mixture to polymerase chain reaction (PCR); and (3) monitoring the generation of various amplicons by the fluorescence emission amplitude.

[0087] The formation of specific amplicons is related to the presence and amount of various template nucleic acids in the test sample. Specific amplicons include (1) target amplified polynucleotides (TAPs), such as rodent parvovirus sequences (e.g., those biological contaminants containing the NS1 sequence), (2) nucleic acid extraction controls (NECs), such as M13 (e.g., M13K07) polynucleotides (NECPs), and (3) plasmid amplification control polynucleotides (PACPs), such as uniquely human artificial plasmid specific sequences (UAPs). If TAP and NECP are produced and PACP is not produced, it can be concluded that the test sample contains biological contaminants and does not contain a cross-contaminated positive amplification control plasmid. However, if TAP and PACP (i.e., UAP) are produced in the test sample Q-PCR reaction, it can be concluded that the test sample is cross-contaminated with the PAC plasmid and the TAP result may be a false positive.

[0088] In a specific embodiment, where the primers and probes comprise (1) a rodent parvovirus-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO: 1, (2) a rodent parvovirus-specific oligonucleotide detection probe, a VIC fluorophore, and an MGBNFQ quencher, (3) an artificial oligonucleotide detection probe, such as one comprising the sequence of SEQ ID NO: 3 and labeled with 6-FAM and Labeled USP, (4) rodent parvovirus - specific reverse oligonucleotide primers containing the sequence of SEQ ID NO:4, (5) M13 - specific forward oligonucleotide primers containing the sequence of SEQ ID NO:5, (6) M13 - specific oligonucleotide detection probe, which contains SEQ ID NO:6 and is labeled with Cy5 and label, and (7) M13 - specific reverse oligonucleotide primers containing the sequence of SEQ ID NO:7, the production of TAP is monitored at about 533 nm to about 580 nm, the production of PACP is monitored at about 465 nm to about 510 nm, and the production of NECP is monitored at about 618 nm to about 660 nm.

[0089] In one embodiment, 96 to 72 hours before harvesting the culture, the test sample is taken from a production CHO cell culture, for example, a production culture of cells transformed with a nucleic acid encoding a protein of interest. If TAP and NECP are detected in the test sample, but PACP is not detected in the test sample, a confirmation test can be performed on a second test sample obtained from the same production cell culture 48 hours before harvesting. If TAP and NECP are again detected in the second test sample, but PACP is not detected in the test sample, the cell culture is considered contaminated and may not be further processed. Alternatively, the second test may not be performed, but the cell culture can be considered contaminated and the culture is not further processed.

[0090] In one embodiment, nucleic acids are extracted from a test sample obtained from a production cell culture. Here, 1 ml of the test sample is subjected to cell lysis, proteolysis, and heat denaturation, then the sample is combined with a nucleic acid extraction control (NEC) sample, and then nucleic acids are extracted from the sample. In one embodiment, an automated nucleic acid extraction system (e.g., Instrument) (Qiagen, Inc., Valencia, CA) is used to extract nucleic acids from the test sample (or NEC - spiked test sample) (see Lee et al., Comparative evaluation of the QIAGEN SP system and bioMérieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory, 52(4) J. Clin. Virol. 339 - 43 (2011)).

[0091] In one embodiment, uracil-N-glycosylase (UNG) is added to the Q-PCR reaction mixture prior to performing PCR to selectively degrade contaminating amplicons (see Taggart et al., Use of heat labile UNG in an RT-PCR assay for enterovirus detection, 105(1) J. Virol. Methods. 57-65 (2002)). The reaction mixture is incubated at 50 °C for at least 2 minutes, more specifically for 2 minutes or 5 minutes in some cases.

[0092] In a specific embodiment, after optional UNG treatment, the reaction mixture is incubated at 95 °C for 2 minutes and then subjected to 8 cycles: (1) denaturation at 95 °C for 10 seconds, then (2) annealing and extension for 30 seconds such that the first annealing temperature is 70 °C and then the annealing temperature is decreased by 1 °C per cycle, with the eighth annealing at 62 °C. After the initial 8 cycles, 40 cycles of DNA amplification are performed, which include the steps of: (1) denaturation at 95 °C for 10 seconds, then (2) annealing and extension at 62 °C for 30 seconds. In a specific embodiment, the rate of temperature change from the denaturation temperature to the annealing temperature is about 4.4 °C / second, and from the annealing temperature to the denaturation temperature is about 2.2 °C / second.

[0093] In some embodiments, a method for detecting biological contaminants in a production cell culture medium or its product includes performing an external positive amplification control (PAC) assay run separately from the test sample assay, and performing an external negative control assay run separately from the test sample assay and the PAC assay. If the negative control or the positive control fails, the results obtained from the test sample assay are rejected.

[0094] In one embodiment, the external positive control includes the steps of: (1) specifically mixing in the absence of a test sample (a) a positive amplification control (PAC) plasmid which, in a specific embodiment, contains SEQ ID NO: 11, (b) a rodent parvovirus-specific forward oligonucleotide primer containing the sequence of SEQ ID NO: 1, (c) a rodent parvovirus-specific oligonucleotide detection probe containing the sequence of SEQ ID NO: 2 labeled with VIC and MGBNFQ, (d) an artificial oligonucleotide detection probe, such as one containing 6-FAM and USP of the sequence of SEQ ID NO: 3 that is labeled, (e) a rodent parvovirus-specific reverse oligonucleotide primer comprising the sequence of SEQ ID NO: 4, and (f) a DNA polymerase, preferably a thermostable DNA polymerase with 5'-exonuclease activity such as Taq polymerase; (2) subjecting the positive control mixture to a positive control polymerase chain reaction (PCR); (3) monitoring the production of (a) target amplified polynucleotide (TAP), (b) nucleic acid extraction control amplified polynucleotide (NECP), and (c) plasmid amplification control polynucleotide (PACP) during the PCR. Monitor the production of TAP at about 533 nm to about 580 nm, the production of PACP at about 465 nm to about 510 nm, and the production of NECP at about 618 nm to about 660 nm.

[0095] The positive amplification control PCR reaction is carried out in the same manner as the test sample PCR reaction (as described above). If TAP and PACP are produced in the positive control reaction, it can be concluded that the PCR amplification procedure is operating properly. If TAP is not produced in the positive amplification control reaction, any negative TAP in the test sample can be discounted as a failed PCR reaction. In one embodiment, NEC (i.e., for example, M13K07) is included in the positive amplification control. A properly functioning control should also show a positive NECP signal (see Table 1).

[0096] In one embodiment, the external negative control comprises the steps of: (1) specifically mixing, in the absence of a test sample and without a PAC plasmid, (a) a blank, which can be a buffer that mimics the test sample buffer system or simply water, (b) a rodent parvovirus-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO: 1, (c) a rodent parvovirus-specific oligonucleotide detection probe comprising the sequence of SEQ ID NO: 2 labeled with VIC and MGBNFQ, (d) an artificial oligonucleotide detection probe, such as one comprising 6-FAM and USP of the sequence of SEQ ID NO: 3 that is labeled, (e) a rodent parvovirus-specific reverse oligonucleotide primer comprising the sequence of SEQ ID NO: 4, and (f) a DNA polymerase, preferably a thermostable DNA polymerase with 5'-exonuclease activity such as Taq polymerase; (2) subjecting the positive control mixture to a positive control polymerase chain reaction (PCR); (3) monitoring the production of (a) target amplified polynucleotide (TAP), (b) nucleic acid extraction control amplified polynucleotide (NECP), and (c) plasmid amplification control polynucleotide (PACP) during the PCR. Monitor the production of TAP at about 533 nm to about 580 nm and the production of PACP at about 465 nm to about 510 nm.

[0097] The negative control PCR reaction is carried out in the same manner as the test sample PCR reaction (as described above). If TAP and PACP are produced in the negative control reaction, it can be concluded that the PCR reagent is contaminated with the PAC plasmid. If TAP is produced in the negative control reaction but PACP is not produced, it can be concluded that the PCR reagent is contaminated with parvovirus. In both cases, the test sample results are discarded. However, if the production of TAP and PACP in the negative control reaction is negative, the production of TAP and PACP in the positive amplification control is positive, and the production of TAP (or optionally NECP) in the test sample reaction is positive and the production of PACP is negative, then the technician can conclude that the test sample is contaminated (see Tables 2 and 3).

[0098] In other aspects, the present invention provides a mixture of a positive amplification control plasmid (PAC plasmid) and a positive control reagent comprising the positive control plasmid. In one embodiment, the PAC plasmid comprises (1) a parvovirus nucleic acid sequence, (2) an M13K07 nucleic acid sequence, and (3) an artificial nucleic acid sequence unique to the plasmid (also referred to as the "UAPS" or "unique" sequence). In a specific embodiment, the parvovirus nucleic acid sequence comprises the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4; the M13K07 nucleic acid sequence comprises the sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the unique sequence comprises the antisense sequence of SEQ ID NO: 3. In a more specific embodiment, the nucleotide sequence of the PAC plasmid consists of the sequence shown in SEQ ID NO: 11.

[0099] In some embodiments, the mixture of the positive control reagent particularly includes the above-mentioned PAC plasmid, a rodent parvovirus-specific forward oligonucleotide primer, a rodent parvovirus-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe (i.e., USP), a rodent parvovirus-specific reverse oligonucleotide primer, an M13-specific forward oligonucleotide primer, an M13-specific oligonucleotide detection probe, an M13-specific reverse oligonucleotide primer, and a buffer. The mixture optionally contains dNTP and Taq polymerase.

[0100] Table 2: Assay Control and Test Session Status

[0101]

[0102] Table 3: Test Session Status

[0103] Sample description Parvovirus UAP Signal M13K07 Is the test sample suitable? Negative test samples 0 0 + yes False negative test samples 0 0 0 no Positive test samples + 0 + yes False positive test samples + + + yes

[0104] In a specific embodiment, the rodent parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 1, the rodent parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binder quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO: 2, the USP comprises a VIC fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO: 3, the rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 4, the M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 5, the M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO: 6, and the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 7.

[0105] Example 1: Oligonucleotides and nucleic acid reagents

[0106] Rodent parvovirus, M13K07, and artificial specific oligonucleotides (Oligos) were obtained from various suppliers in different scales and in various forms, which are described in Table 4. All oligonucleotides were assigned a three-year shelf life upon receipt from the supplier.

[0107] The oligonucleotides were reconstituted in water to a concentration of 100 μM to produce a master stock solution. Before setting up the qPCR reaction, the master stock solution was further diluted to produce a 10X stock solution. Table 5 describes the qPCR oligonucleotides (primers and probes) at 10X and 1X concentrations.

[0108] Table 4: Primers and Probes for Parvovirus, M13, and Specific Artificial Sequences

[0109]

[0110]

[0111] Table 5: Oligonucleotide Concentrations

[0112]

[0113] Example 2: Positive Amplification Control Plasmid

[0114] The parvovirus-M13 positive amplification control (PAC) plasmid was prepared in the pUC57-Kan plasmid (GeneWiz, Inc., South Plainfield, NJ). This PAC plasmid consists of the sequence shown in SEQ ID NO: 11. The parvovirus-M13 PAC plasmid contains 2.1×10 8Copy / ng, calculated using the following formula: [Quantity = (Amount * Number / Mole) / (bp * ng / g * g / Mole of bp)]; where Amount = ng, Number / Mole = 6.022×10 23 , bp = 4372, ng / g = 1×10 9 , g / Mole of bp = 650. Calculate the concentration of the plasmid and express it in ng / μL, and serially dilute it to a 10X concentration of 10 2 Copy / μL. Prepare 50 μL aliquots from the 10 2 Copy / μL dilution and store in 2 mL sterile screw-cap tubes. All aliquots are stored at ≤ -60 °C.

[0115] Example 3: Preparation of M13k07 phage

[0116] Use MEM as a diluent to serially dilute M13K07 phage tenfold to obtain M13K07 at a titer of 100 pfu / μL. Prepare 200 μL aliquots and store at ≤ -60 °C, and assign a three-year shelf life.

[0117] Example 4: Preparation of Q-PCR Reagents

[0118] The rodent parvovirus real-time PCR assay is fully automated PCR method, which consists of automated DNA purification using (Qiagen), followed by nucleic acid amplification and real-time PCR product detection on a 480 instrument (Roche Diagnostic). To assess the presence of PCR inhibitors, each test article is automatically spiked with phage M13K07 as an internal control (IC). Rodent parvovirus primer oligonucleotides are designed to hybridize within a highly conserved region of the rodent parvovirus genome (NS-1 region) to ensure broad-range detection. The assay is performed in duplex (i.e., two targets) format, with rodent parvovirus and M13K07 primers generating PCR products of approximately 110 and 97 bp, respectively. The PCR products are detected in real-time by cleavage of two probes labeled with different reporter dyes: the VIC fluorophore for rodent parvovirus and the Cy5 fluorophore for M13K07 phage. A positive amplification control (PAC) plasmid at a concentration of 100 copies / μL is used. This plasmid contains a unique (Flag) sequence (USP) that differentiates it from wild-type parvovirus using a specific probe labeled with the fluorophore FAM.

[0119] Aliquot the Master Mix into 2 mL tubes in an amount at least three times the number of test articles including controls according to Table 6. Store the tubes at 2 - 8 °C. Prepare a negative amplification control (NAC) vial by adding 50 μL of water to a 2 mL tube.

[0120] Table 6: Reaction mixture

[0121]

[0122] Example 5: Sample preparation for testing

[0123] Since all samples have the potential to be contaminated with or contain exogenous agents, aseptic practices are followed for all sample pretreatment steps. The following steps are performed inside a clean biosafety cabinet. The test article (sample) is obtained from the cell culture producing the antibody or capture molecule and frozen or used directly. The test article (sample) is obtained from the cell culture producing the antibody or capture molecule and frozen or used directly.

[0124] Aliquot 1000 μL of PBS into appropriate 2 mL tubes to be used as negative extraction controls (NEC). When PCR is used as the endpoint of the cell culture step, the positive and negative controls of the cell culture are used as positive and negative extraction controls, respectively.

[0125] Each test article is thawed at room temperature. Transfer the sample in the 60 mL bag to a 50 mL - Falcon tube and then aliquot. Pipette 1000 μL of each sample into a 2 mL tube for pretreatment. Add 400 μL of lysis buffer ( Lysis Buffer L13, Invitrogen, Cat#CS11202 or equivalent, Carlsbad, CA) to each tube and vortex for at least 10 seconds. Then add 20 μL of Proteinase K (≥10 mg / mL, ≥800 units / mL in 40% glycerol (v / v) containing 1 mM calcium acetate in 10 mM Tris - HCl, pH 7.5; SAFC, Cat#P4850 - 5ML, SigmaAldrich, St. Louis, MO) to each sample and vortex for at least 10 seconds. Then incubate the samples at 65 °C for 30 minutes. After incubation, vortex the samples and centrifuge at 17,000×g for 10 minutes.

[0126] Meanwhile, thaw the positive amplification control (PAC; at least 50 μL of 10 2 copies / μL plasmid) in a 2 mL tube and the M13K07 (10 2 PFU / mL) internal control (IC; at least 150 μL / 12 samples).

[0127] Example 6: Nucleic acid extraction

[0128] At The extraction protocol programmed on the instrument (Qiagen, Valencia, CA) requires an internal control (IC). The instrument automatically adds 120 μL of recombinant IC to each sample. For every 12 samples, the instrument requires 1.8 mL of IC (i.e., 1 vial). The IC vial is prepared by adding 1650 μL of AVE buffer (RNase-free water containing 0.04% sodium azide) to 150 μL of M13K07 IC.

[0129] Load each prepared sample into the Sample Carrier of the instrument inside the biological safety cabinet (BSC). Load the IC vials into a separate (dedicated) Sample Carrier inside the BSC at a ratio of one IC vial per 12 samples. Close all drawers and run the instrument according to the manufacturer's recommended protocol (see QIAsymphony DNA Handbook, 09 / 2010, available at http: / / www.algimed.by / download / EN-QIAsymphony-DNA-Handbool.pdf).

[0130] Use the Reagent Prep Cartridge containing all the reagents required for extraction from the DSP Virus / Pathogen Kit (see DSP Virus / Pathogen Kit Handbook, April 2013, available at https: / / www.qiagen.com / us / resources / download.aspx?id=f8bc0b3c-0aff-46ee-8807-5ed145f9e969&lang=en). The Reagent Prep Cartridge contains Proteinase K with a shelf life of approximately 2 weeks.

[0131] Perform nucleic acid extraction in a 96-well reaction plate to facilitate integration with Q-PCR. After the automated nucleic acid extraction program is completed and passes the status check, cool the reaction plate and seal it with 480 sealing foil (Roche, Branchburg, NJ). Place the 96-well plate in a plate spinner balanced with a suitable weight (e.g., another 96-well plate) and spin for 30 to 60 seconds. Check for bubbles in the wells and repeat spinning if necessary.

[0132] Example 7: Q-PCR

[0133] Use either the TaqMan Triplex and / or Veriquest Triplex program in Q-PCR was performed on a 480 instrument (Roche, Branchburg, NJ). The TaqMan Triplex protocol used TaqMan Fast Advance Custom Master Mix without the reference dye (ROX). Three fluorescence channels (FAM, VIC, and CY5) were selected, and the UNG step time was 2 minutes. The reaction parameters used are outlined in Table 7.

[0134] When using a Master Mix that also has no ROX, a 5-minute UNG step time was used. The reaction parameters used are outlined in Table 8.

[0135] Table 7: Rodent parvovirus PCR TaqMan Triplex program steps

[0136]

[0137] The crossing point (Cp) fluorescence signals of each of the M13 internal control, rodent parvovirus, and positive amplification control plasmid were determined by one or both of two algorithms. The first algorithm is the Automated Second Derivative method. This method requires no user input and generally results in higher consistency, so it is considered the preferred method. The second algorithm is the Fit Points method. This method allows the user to set a threshold line in the case of discrete background. The point at which the log-linear curve crosses this threshold line becomes the crossing point. The following filter combs were used to monitor the fluorescence signals: 533 - 580 nm (VIC signal) for rodent parvovirus (subset "Sample-Parvo"); 618 - 660 nm (Cy5 signal) for the internal control M13K07 (subset "Sample-M13"); and 465 - 510 nm (FAM signal) for the positive amplification control plasmid (subset "Sample-FAM"). In the case of ambiguous fluorescence signals, Fit Points analysis was used to determine the fluorescence background level. An acceptable background fluorescence signal was considered to be ≤ 1 unit on the amplification curve scale. Any fluorescence signal ≤ 1 unit was considered within the acceptable background and was therefore negative.

[0138] Table 8: Rodent parvovirus PCR VeriQuest Triplex program steps

[0139]

[0140] Example 8: Conditions for a valid test session

[0141] For a test session to be considered valid, the following conditions must be met. The fluorescence signal of the negative amplification control (NAC, i.e., water) must be negative in all three channels. The fluorescence signal of the negative extraction control (NEC, i.e., PBS) or the cell culture negative control vial must be negative in the [533 - 580] channel (i.e., rodent parvovirus probe VIC signal), negative in the [465 - 510] channel (i.e., positive amplification control [PAC] antisense Flag probe - FAM signal), and positive in the [618 - 660] channel (i.e., M13K07 probe - CY5 signal). The fluorescence signal of PAC must be positive in all three channels. The M13K07 Cp value in the NEC is used as a reference for evaluating the presence of inhibitory substances in the sample.

[0142] If PCR is used as the endpoint for the test article from the cell culture step with a rodent parvovirus positive control, the fluorescence signal of the positive virus control must be positive in the [533 - 580] channel (i.e., rodent parvovirus probe VIC signal), positive in the [618 - 660] channel (i.e., M13K07 probe - CY5 signal), and negative in the [465 - 510] channel (i.e., PAC antisense Flag probe - FAM signal). The M13K07 Cp value in the cell culture positive control sample is expected to be within ±4 cycles of the NEC - M13 Cp value.

[0143] For all control reactions containing the PAC plasmid, the fluorescence signal must be positive in all three channels.

[0144] Example 9: Conditions for invalid test sessions

[0145] The assay is considered invalid when one or more of the following conditions are met: (1) a very low amplification curve of PAC (<1 unit on the fluorescence scale), (2) a determinant error (machine, software, or human error) is confirmed, (3) the NAC is positive for any of the three channels, (4) the NEC is positive for amplification in the [533 - 580] VIC channel, positive for amplification in the [465 - 510] FAM channel, or negative for amplification in the [618 - 660] Cy5 channel, (5) the amplification signal of PAC is negative for any of the three channels. Whenever the assay is determined to be invalid, an investigation and retesting of the test sample are performed.

[0146] Example 10: Conditions for negative sample results in a valid test

[0147] For a valid negative parvovirus test result, all of the following conditions must be met. Within the expected Cp value range of NEC-M13 Cp ± 4 cycles, in the M13[618-660] channel, the M13K07 DNA amplification signal of the sample must be positive, indicating the absence of PCR inhibitors. In the parvovirus[533-580] channel, the parvovirus DNA amplification signal of the sample must be negative. Fluorescence signals below 1 unit on the fluorescence scale, regardless of Cp value, are considered within the acceptable fluorescence background level and are reported as negative for parvovirus DNA amplification. In the antisense Flag[465-510] channel, the PAC amplification signal of the sample must be negative. Note that fluorescence signals below 1 unit on the fluorescence scale (automatically generated by the instrument), regardless of Cp value, are considered within the acceptable fluorescence background level and are reported as negative for PAC-plasmid DNA amplification.

[0148] Example 11: "No Sample" Condition in a Valid Test Session

[0149] When one or more of the following conditions are met, a "no sample" result is generated. Whenever the M13K07 DNA amplification signal in the Cy5 channel[618-660] of the sample is negative, the parvovirus DNA amplification signal in the parvovirus channel[533-580] is negative, and the PAC amplification in the antisense Flag FAM channel[465-510] is negative, the sample or PCR reagents are investigated according to the standard operating procedure. This condition indicates the presence of PCR inhibitors or failure of proper nucleic acid extraction. The sample can be diluted (1:2, 1:5, 1:10) to overcome the inhibition. Cp values of fluorescence signals in the M13 channel[618 660] outside the range (NEC M13 Cp ± 4 cycles) indicate partial inhibition of PCR or an error in phage spiking and require retesting. Sample dilution (1:2, 1:5, 1:10) can be considered to overcome any inhibition of PCR. Any evidence of determinant error or unexpectedly very low fluorescence signal (less than 1 unit on the fluorescence scale) observed in the M13 channel[618-660] (which does not allow a conclusive assessment of sample suitability) is considered a "no sample result" and indicates failure during amplification or DNA extraction. This requires retesting.

[0150] Example 12: Conditions for Initial Output Specification (IOOS) Sample Results in a Valid Test Session

[0151] When the parvovirus DNA amplification signal in (i) the parvovirus channel [533 - 580] (i.e., the VIC signal of the rodent parvovirus probe) of the sample is positive, with its fluorescence signal on the fluorescence scale being higher than 1 unit (for at least one of the two replicate wells) and (ii) the PAC amplification in the antisense Flag FAM channel [465 - 510] is negative (indicating no cross - contamination from PAC), the sample is considered iOOS. Therefore, the technician must (i) initiate the GLIF (General Laboratory Investigation Form), (ii) notify the department that will submit the sample for testing to QC Virology, (iii) initiate the NOE, (iv) save the amplification tube (frozen at - 20 °C) for further research (such as Flag sequence screening or sequencing), and (v) repeat the assay and retest the sample.

[0152] Example 13: Repeat and retest plan

[0153] Whenever the assay is invalid or the sample result is considered "no result", a repeat test is initiated. The retest is performed to confirm the iOOS event (i.e., the DNA amplification signal in the parvovirus channel [533 - 580] of the first sample result is positive). The retest or repeat test must be performed using fresh reagent aliquots (i.e., master mix reagent, extraction kit).

[0154] If the fluorescence of NAC is positive in any channel; repeat the entire test session starting from the amplification (PCR) step using freshly prepared master mix with the already purified DNA sample. If the fluorescence of NEC is negative in the M13 channel [618 - 660], the entire test session will be repeated starting from the DNA extraction step. If the fluorescence of NEC is positive in the parvovirus channel [533 - 580] or the antisense Flag channel [465 - 510], the entire test session will be repeated starting from the DNA extraction step. If the fluorescence of PAC is negative in any channel, repeat the test session starting from the amplification (PCR step) using freshly prepared master mix with the same already purified DNA sample.

[0155] For those samples with a "no sample" result, repeat the test session for the affected samples starting from the DNA extraction step. As part of the investigation and problem - solving process, before DNA extraction, the sample can be diluted (1:2, 1:5, or 1:10) (in addition to the undiluted sample) to demonstrate the presence of inhibitors.

[0156] The retesting to confirm the initial positive result (iOOS) is carried out as follows using the following four separate aliquots: two aliquots from the original sampling event (e.g., one day after the final feed) and two aliquots from a different sampling event (e.g., two days after the final feed if possible) or different sample bags. If any additional testing of the four aliquots results in a positive signal without evidence of a definitive error (proven by investigation), the batch is considered not to meet the requirement of the absence of rodent parvovirus viral genomic material. For such a positive Q-PCR result, an infectivity assay must be performed for final disposition. CHO-K1 cells are used as the indicator cell line to determine the infectivity status of the detected nucleic acid.

[0157] Whenever the retest result is negative, confirmatory testing is required at a different sampling event (e.g., three days after the final feed) to confirm the absence of rodent parvovirus genomic material.

[0158] Example 14: Retesting Plan for Other Sample Types with IOOS

[0159] Other sample types, including for example untreated bulk materials, cells at the end of production, cells at the in vitro lifespan limit, and soybean materials with an initial positive result (iOOS), are retested using four separate aliquots as follows:

[0160] Retest two aliquots from the original sample container (i.e., e.g., a bag) and two aliquots from a different sample container according to the standard operating procedure. Inoculate one sample aliquot of the test article from the original sample container into CHO-K1 indicator cells. Harvest the inoculated indicator cells CHO-K1 after culturing for 1 - 3 days according to the standard operating procedure to determine the infectivity status of the detected nucleic acid. A standard curve can be used for quantifying the re-extracted nucleic acid.

[0161] Whenever any additional PCR testing of the four aliquots is positive without evidence of a definitive error (proven by investigation), the infectivity assay determines the final disposition of the article. After confirming the initial OOS, sequencing of the nucleic acid and transmission electron microscopy (TEM) can be considered to identify the microorganism and exclude any laboratory errors.

[0162] An infectivity assay using CHO-K1 as the indicator cell for the suspected contaminated material is required to determine the infectivity status of the detected nucleic acid. Whenever the investigation fails to support the possibility of parvovirus contamination and all additional PCR testing of the four aliquots of the test article and the CHOK1 culture is negative, the batch is considered to meet the requirement of the absence of infectious rodent parvovirus.

[0163] Example 15: Parvovirus Detection during Recombinant Protein Production

[0164] The Q-PCR procedure described above was performed as an in-process control in the critical process of testing cell culture medium (i.e., untreated bulk material from a production bioreactor containing CHO cell derivatives that contain heterologous heavy and light chain constructs of antibodies). Each heterologous monoclonal antibody (mAb) binds to a different target or epitope. QC virology scientists performed Good Manufacturing Practice (GMP) testing in a large-scale bioprocess production facility. Sixteen (16) of these tests are listed in Table 9. In each case, the test session was valid, the assay system suitability criteria were met, and no false positive detections were observed. In tests #13 and #14, false negative detections did occur, indicating the presence of PCR inhibitors or nucleic acid extraction failure.

[0165] Table 9: Rodent Parvovirus PCR Tests

[0166]

[0167]

[0168] UPB = Untreated Bulk Material; EA = Early Alert; Rodent Parvo PCR = the Q-PCR procedure described above.

[0169] In summary, the present invention provides the following embodiments:

[0170] 1. A composition comprising an artificial nucleotide sequence, a fluorophore, and a quencher, wherein the artificial nucleotide sequence comprises no more than five consecutive nucleic acids at its 3'-end and is identical to the sequence of any one of SEQ ID NOs: 9 and 12 - 37.

[0171] 2. The composition of embodiment 1, wherein the fluorophore is selected from fluorophores having any excitation wavelength from 495 nm to 680 nm and any emission wavelength from 515 nm to 710 nm.

[0172] 3. The composition of embodiment 2, wherein the quencher is selected from dyes having any absorption peak from 430 nm to 672 nm.

[0173] 4. The composition of embodiment 1, wherein the fluorophore has an excitation wavelength of 495 nm and an emission wavelength of 520 nm.

[0174] 5. The composition of embodiment 4, wherein the fluorophore is FAM.

[0175] 6. The composition of embodiment 5, wherein the quencher is BHQ-1.

[0176] 7. The composition of embodiment 1, wherein the fluorophore is attached to the 5' end of the artificial nucleotide sequence and the quencher is attached to the 3' end of the artificial nucleotide sequence.

[0177] 8. The composition of embodiment 1, wherein the artificial nucleotide sequence comprises the nucleic acid sequence of SEQ ID NO: 3 (5'-TGTCGATGGCGAATGGCTA-3').

[0178] 9. A composition comprising:

[0179] a. A rodent parvovirus-specific forward oligonucleotide primer;

[0180] b. A rodent parvovirus-specific oligonucleotide detection probe;

[0181] c. An artificial oligonucleotide detection probe;

[0182] d. A rodent parvovirus-specific reverse oligonucleotide primer;

[0183] e. An M13-specific forward oligonucleotide primer;

[0184] f. An M13-specific oligonucleotide detection probe; and

[0185] g. An M13-specific reverse oligonucleotide primer.

[0186] 10. The composition of embodiment 9, wherein the oligonucleotide sequences of a, b, and d each comprise the NS-1 sequence of a rodent parvovirus, and the rodent parvovirus is selected from mouse parvovirus prototype strain (MVMp), mouse parvovirus immunosuppressive strain (MVMi), mouse parvovirus Cutter strain (MVMc); mouse parvovirus 1b (MPV-1b), mouse parvovirus 1a (MPV-1a), mouse parvovirus 1c (MPV-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a, rat parvovirus, and the Umass strain of rat virus L (RV-Umass).

[0187] 11. The composition of embodiment 9, wherein the artificial oligonucleotide detection probe comprises a nucleotide sequence having no more than five consecutive nucleic acids at its 3' end, which is identical to the sequence of any one of SEQ ID NOs: 9 and 12-37.

[0188] 12. The composition of embodiment 10, wherein the oligonucleotide sequences of a, b, and d hybridize to SEQ ID NO: 37.

[0189] 13. The composition of embodiment 9, wherein each detection probe of b, c, and f comprises a fluorophore and a quencher such that the fluorophores of each detection probe emit light at different wavelengths.

[0190] 14. The composition of embodiment 13, wherein the fluorophore of each probe is selected from fluorophores having any excitation wavelength from 495 nm to 680 nm and any emission wavelength from 515 nm to 710 nm.

[0191] 15. The composition of embodiment 14, wherein the quencher is selected from dyes having any absorption peak from 430 nm to 672 nm.

[0192] 16. The composition of embodiment 13, wherein the artificial oligonucleotide detection probe (c) comprises the fluorophore FAM and the quencher BHQ-1; the rodent parvovirus-specific oligonucleotide detection probe (b) comprises the fluorophore VIC and the minor groove binding non-fluorescent quencher (MGBNFQ); and the M13-specific oligonucleotide detection probe comprises the fluorophore Cy5 and the quencher BHQ2.

[0193] 17. The composition of embodiment 12, wherein the rodent parvovirus-specific forward oligonucleotide primer (a) comprises the nucleic acid sequence of SEQ ID NO: 1; the rodent parvovirus-specific reverse oligonucleotide primer (d) comprises the nucleic acid sequence of SEQ ID NO: 4; and the rodent parvovirus-specific oligonucleotide detection probe (b) comprises the nucleic acid sequence of SEQ ID NO: 2.

[0194] 18. The composition of embodiment 11, wherein the artificial oligonucleotide detection probe (c) comprises the nucleic acid sequence of SEQ ID NO: 3.

[0195] 19. The composition of embodiment 9, wherein the M13-specific forward oligonucleotide primer (e) comprises the nucleic acid sequence of SEQ ID NO: 5; the M13-specific oligonucleotide detection probe (f) comprises the nucleic acid sequence of SEQ ID NO: 6; and the M13-specific reverse oligonucleotide primer (g) comprises the nucleic acid sequence of SEQ ID NO: 7.

[0196] 20. A method for detecting a biological contaminant in a test sample, comprising the steps of:

[0197] a. Mixing components to prepare a reaction mixture, wherein the components comprise (i) a nucleic acid sample derived from the test sample, (ii) oligonucleotides, and (iii) a DNA polymerase;

[0198] b. Subjecting the reaction mixture to polymerase chain reaction (PCR);

[0199] c. Monitoring the production of (i) target amplified polynucleotide (TAP), (ii) nucleic acid extraction control amplified polynucleotide (NACP), and (iii) plasmid amplification control polynucleotide (PACP) during PCR; and

[0200] d. Comparing the production of TAP with the production of NACP and PACP,

[0201] wherein the presence of TAP, the presence of NACP, and the absence of PACP produced during PCR indicate that the test sample contains a biological contaminant and does not contain a positive amplification control plasmid.

[0202] 21. The method of embodiment 20, wherein the oligonucleotide comprises (a) a rodent parvovirus-specific forward oligonucleotide primer, (b) a rodent parvovirus-specific oligonucleotide detection probe, (c) an artificial oligonucleotide detection probe, (d) a rodent parvovirus-specific reverse oligonucleotide primer, (e) an M13-specific forward oligonucleotide primer, (f) an M13-specific oligonucleotide detection probe, (g) an M13-specific reverse oligonucleotide primer.

[0203] 22. The method of embodiment 20, wherein the test sample is obtained from a mammalian cell culture or a purified portion thereof.

[0204] 23. The method of embodiment 20, wherein the test sample is spiked with M13K07 phage.

[0205] 24. The method of embodiment 20, wherein the nucleic acid sample is obtained by subjecting about 1 mL of the test sample to lysis, proteolysis, and heat denaturation, then combining the sample with an extraction control sample, and then extracting nucleic acid from the sample.

[0206] 25. The method of embodiment 24, wherein the extraction control sample is M13K07 phage.

[0207] 26. The method of embodiment 25, wherein (a) the rodent parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 1; (b) the rodent parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binding non-fluorescent quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO: 2; (c) the artificial oligonucleotide detection probe comprises a FAM fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO: 3; (d) the rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 4; (e) the M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 5; (f) the M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO: 6; and the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 7.

[0208] 27. The method of embodiment 20, wherein the composition comprises uracil-N-glycosylase (UNG).

[0209] 28. The method of embodiment 20, which comprises the step of incubating the reaction mixture at 50 °C for at least 2 minutes.

[0210] 29. The method of embodiment 28, wherein the PCR step (b) comprises the following steps: (i) incubating the reaction mixture at 95 °C for 2 minutes; then (ii) eight (8) cycles: (1) denaturing at 95 °C for 10 seconds, then (2) annealing for 30 seconds, wherein the annealing temperature for the first cycle of the 8 cycles is 70 °C, and the annealing temperature is reduced by 1 °C for each cycle, and the annealing temperature for the last cycle of the 8 cycles is 62 °C; then (iii) at least 40 cycles of DNA amplification, comprising the following steps: (1) denaturing at 95 °C for 10 seconds, then (2) annealing at 62 °C for 30 seconds, wherein the rate of temperature change from the denaturing temperature to the annealing temperature is about 4.4 °C / second, and from the annealing temperature to the denaturing temperature is about 2.2 °C / second.

[0211] 30. The method of embodiment 26, wherein (a) the production of TAP is monitored by measuring the fluorescence at 533 - 580 nm in each amplification cycle; (b) the production of NACP is monitored by measuring the fluorescence at 618 - 660 nm in each amplification cycle; and (c) the production of PACP is monitored by measuring the fluorescence at 465 - 510 nm in each amplification cycle.

[0212] 31. The method of embodiment 20, comprising the steps of: comparing the result obtained by the method of operating on the test sample with an external positive control and an external negative control, and if the negative control or the positive control fails, the result obtained by the method of operating on the test sample is rejected.

[0213] 32. The method of embodiment 31, wherein the external positive control comprises the steps of:

[0214] a. Mixing positive control components to prepare a positive control mixture, wherein the positive control components comprise (i) a positive amplification control (PAC) plasmid, (ii) a positive control oligonucleotide mixture, and (iii) no test sample;

[0215] b. Subjecting the positive control mixture to a positive control polymerase chain reaction (PCR); and

[0216] c. Monitoring the production of (i) a target amplified polynucleotide (TAP), (ii) a nucleic acid extraction control amplified polynucleotide (NACP), and (iii) a plasmid amplification control polynucleotide (PACP) during the PCR;

[0217] wherein the presence of TAP, NACP, and PACP produced during the PCR indicates normal operation of the PCR, and the absence of any one or more of TAP, NACP, or PACP indicates that the positive control has failed.

[0218] 33. The method of embodiment 32, wherein the PAC plasmid comprises (i) a parvovirus nucleic acid sequence, (ii) an M13K07 nucleic acid sequence, and (iii) an artificial nucleic acid sequence unique to the plasmid.

[0219] 34. The method of embodiment 33, wherein (i) the parvovirus nucleic acid sequence comprises the sequence of SEQ ID NO: 37, (ii) the M13K07 nucleic acid sequence comprises the sequence of SEQ ID NO: 8, and (iii) the artificial nucleic acid sequence unique to the plasmid comprises the sequence of SEQ ID NO: 10.

[0220] 35. The method of embodiment 34, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO: 11.

[0221] 36. The method of embodiment 33, wherein the positive control oligonucleotide mixture comprises:

[0222] a rodent parvovirus-specific forward oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO: 1;

[0223] A rodent parvovirus-specific oligonucleotide detection probe comprising a VIC fluorophore, an MGBNFQ quencher, and a nucleic acid sequence of SEQ ID NO: 2;

[0224] An artificial oligonucleotide detection probe comprising a FAM fluorophore, a BHQ quencher, and a nucleic acid sequence of SEQ ID NO: 3;

[0225] A rodent parvovirus-specific reverse oligonucleotide primer comprising a nucleic acid sequence of SEQ ID NO: 4;

[0226] An M13-specific forward oligonucleotide primer comprising a nucleic acid sequence of SEQ ID NO: 5;

[0227] An M13-specific oligonucleotide detection probe comprising a Cy5 fluorophore, a BHQ-2 quencher, and a nucleic acid sequence of SEQ ID NO: 6; and

[0228] An M13-specific reverse oligonucleotide primer comprising a nucleic acid sequence of SEQ ID NO: 7.

[0229] 37. The method of embodiment 36, wherein the positive control PCR comprises the steps of:

[0230] a. Incubating the positive control mixture at 95 °C for 2 minutes; then

[0231] b. 8 cycles: (i) denaturing at 95 °C for 10 seconds, then (ii) annealing for 30 seconds, such that the annealing temperature of the first cycle of the 8 cycles is 70 °C, the annealing temperature decreases by 1 °C for each cycle, and the annealing temperature of the last cycle of the 8 cycles is 62 °C; then

[0232] c. 40 cycles of DNA amplification, comprising the steps of: (i) denaturing at 95 °C for 10 seconds, then (ii) annealing at 62 °C for 30 seconds.

[0233] 38. The method of embodiment 36, wherein the production of TAP in the positive control PCR is monitored by measuring fluorescence at 533 - 580 nm in each amplification cycle, the production of NACP in the positive control PCR is monitored by measuring fluorescence at 618 - 660 nm in each amplification cycle, and the production of PACP in the positive control PCR is monitored by measuring fluorescence at 465 - 510 nm in each amplification cycle.

[0234] 39. A composition comprising:

[0235] a. A positive amplification control (PAC) plasmid;

[0236] b. A rodent parvovirus-specific forward oligonucleotide primer;

[0237] c. Rodent parvovirus-specific oligonucleotide detection probe;

[0238] d. Artificial oligonucleotide detection probe;

[0239] e. Rodent parvovirus-specific reverse oligonucleotide primer;

[0240] f. M13-specific forward oligonucleotide primer;

[0241] g. M13-specific oligonucleotide detection probe;

[0242] h. M13-specific reverse oligonucleotide primer; and

[0243] i. Buffer.

[0244] 40. The composition of embodiment 39, wherein the PAC plasmid comprises (i) a parvovirus nucleic acid sequence, (ii) an M13K07 nucleic acid sequence, and (iii) an artificial nucleic acid sequence unique to the plasmid.

[0245] 41. The composition of embodiment 40, wherein the parvovirus nucleic acid sequence comprises the sequence of SEQ ID NO: 37, the M13K07 nucleic acid sequence comprises the sequence of SEQ ID NO: 8, and the unique nucleic acid sequence comprises the sequence of SEQ ID NO: 10.

[0246] 42. The composition of embodiment 41, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO: 11.

[0247] 43. The composition of embodiment 42, wherein

[0248] The rodent parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 1;

[0249] The rodent parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binder quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO: 2;

[0250] The artificial oligonucleotide detection probe comprises a VIC fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO: 3;

[0251] The rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 4;

[0252] The M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 5;

[0253] The M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO: 6; and

[0254] The M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 7.

Claims

1. A positive amplification control (PAC) plasmid comprising: A unique artificial plasmid-specific sequence (UAPS), A target amplified polynucleotide (TAP) sequence, wherein the TAP sequence comprises all or part of a parvovirus NS-1 sequence, and A nucleic acid extraction control (NEC) nucleotide sequence, wherein the NEC nucleotide sequence comprises an M13 phage nucleotide sequence.

2. The PAC plasmid according to claim 1, wherein the UAPS comprises a sequence of 17 to 20 nucleotides, and wherein no more than 7 to 10 consecutive internal nucleotides and no more than 6 consecutive 3'-nucleotides of the UAPS are identical to any parvovirus sequence.

3. The PAC plasmid according to claim 1, wherein the UAPS does not recognize any naturally occurring parvovirus or does not anneal to any naturally occurring parvovirus.

4. The PAC plasmid according to claim 1, wherein the UAPS comprises SEQ ID NO:

10.

5. The PAC plasmid according to claim 1, wherein the parvovirus NS-1 sequence is at least 88% identical to any one of SEQ ID NOs: 12 - 37 or 99% identical to SEQ ID NO:

9.

6. The PAC plasmid according to claim 1, wherein the parvovirus NS-1 sequence comprises SEQ ID NO: 9 or comprises any one of SEQ ID NOs: 12 - 37.

7. The PAC plasmid according to claim 1, wherein the parvovirus NS-1 sequence comprises SEQ ID NO:

37.

8. The PAC plasmid according to claim 1, wherein the M13 phage nucleotide sequence is the M13K07 nucleotide sequence.

9. The PAC plasmid according to claim 8, wherein the M13K07 nucleotide sequence comprises SEQ ID NO:

8.

10. The PAC plasmid according to claim 1, further comprising a binding site that hybridizes to one or more oligonucleotide primers.

11. The PAC plasmid according to claim 10, wherein the one or more oligonucleotide primers are selected from a forward oligonucleotide primer, a reverse oligonucleotide primer, and combinations thereof.

12. The PAC plasmid according to claim 10, wherein the one or more oligonucleotide primers include a parvovirus oligonucleotide primer.

13. The PAC plasmid according to claim 1, further comprising a binding site that hybridizes to an M13 oligonucleotide primer.

14. The PAC plasmid according to claim 13, wherein the M13 oligonucleotide primer is selected from a forward oligonucleotide primer, a reverse oligonucleotide primer, and combinations thereof.

15. The PAC plasmid according to claim 1, wherein the parvovirus NS-1 sequence is selected from: murine parvovirus prototype strain (MVMp), murine parvovirus immunosuppressive strain (MVMi), murine parvovirus Cutter strain (MVMc); murine parvovirus 1b (MPV-1b), murine parvovirus 1a (MPV-1a), murine parvovirus 1c (MPV-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a, rat parvovirus and the Umass strain of rat virus L (RV-Umass).

16. The PAC plasmid according to claim 1, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO:

11.

17. A composition comprising a unique detection probe nucleotide sequence (UDP), a fluorophore, and a quencher, wherein the UDP comprises the nucleic acid sequence of SEQ ID NO:

3.

18. The composition according to claim 17, wherein the UDP does not anneal to any naturally occurring parvovirus in a Q-PCR assay.

19. The composition according to claim 17, wherein the fluorophore is selected from a group of fluorophores having an excitation wavelength anywhere from 495 nm to 680 nm and an emission wavelength anywhere from 515 nm to 710 nm, and the ranges of the excitation wavelength and the emission wavelength both include the endpoint values.

20. The composition according to claim 17, wherein the fluorophore has an excitation wavelength of 495 nm and an emission wavelength of 520 nm.

21. The composition according to claim 17, wherein the fluorophore is fluorescein amidite (FAM).

22. The composition according to claim 17, wherein the quencher is selected from a group of dyes having an absorption peak anywhere from 430 nm to 672 nm, and the range includes the endpoint values.

23. The composition according to claim 17, wherein the quencher is Black Hole Quencher-1 (BHQ-1).

24. The composition according to claim 17, wherein the fluorophore is linked to the 5'-end of the UDP, and the quencher is linked to the 3'-end of the UDP.

25. The composition according to claim 17, wherein the fluorophore is linked to the 3'-end of the UDP, and the quencher is linked to the 5'-end of the UDP.

26. The composition according to claim 17, wherein the UDP hybridizes to the unique artificial plasmid specific sequence (UAPS) of a positive amplification control (PAC) plasmid.

27. The composition according to claim 26, wherein the PAC plasmid comprises: i) a target amplified polynucleotide (TAP) sequence, wherein the TAP sequence comprises all or part of the parvovirus NS-1 sequence, and ii) a nucleic acid extraction control (NEC) nucleotide sequence, wherein the NEC nucleotide sequence comprises the M13 phage nucleotide sequence.

28. The composition according to claim 27, wherein the parvovirus NS-1 sequence comprises SEQ ID NO: 9 or comprises any one of SEQ ID NOs: 12-37.

29. The composition according to claim 27, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO:

11.

30. The composition according to claim 27, wherein the PAC plasmid comprises a binding site that hybridizes to an M13 oligonucleotide primer.

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