EA048105B1 - COMPOSITION FOR DETECTING BIOLOGICAL POLLUTANT - Google Patents

Description

Cross-reference to related applications

This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Patent Application No. 62/139,321, filed March 27, 2015, which application is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

The invention relates generally to a process for producing biological molecules using cell culture. More particularly, the invention relates, but is not exclusively related to, compositions and methods for detecting biological contaminants in cell culture.

State of the art in the field of invention

Biopharmaceuticals, especially therapeutic antibodies, are produced in mammalian cell culture. Chinese hamster ovary (CHO) cells are the most commonly used host cells. These production systems are prone to infection by imported or endogenous viruses, which poses a potential safety issue for biopharmaceuticals. Therefore, virus removal and viral load measurement procedures are used to promote drug safety. Steps used to reduce viral load include nanofiltration, heat inactivation of the virus, or pH and chromatography. Viral load and virus removal efficiency can be monitored using time-consuming infectivity assays or rapid quantitative assays such as real-time polymerase chain reaction (PCR) or quantitative polymerase chain reaction (Q-PCR).

Q-PCR requires appropriate negative and positive controls for reliability. Nucleic acid extraction control agents are added to test samples to monitor the quality of nucleic acid extraction. If the nucleic acid extraction control was negative or outside the expected Q-PCR repair range, then the sample was rejected. Conversely, if the nucleic acid extraction control was positive or within the expected Q-PCR repair range, nucleic acid extraction from the test sample was confirmed. A negative control, such as buffer without sample, was commonly included in the Q-PCR assay. 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.

A positive amplification control may also be included in the Q-PCR viral load assay. Such a positive control may include conserved nucleic acid sequences that are amplified using primers that amplify naturally occurring viral contaminants. Failure to detect a positive control by Q-PCR may indicate that the amplification procedure may be unable to detect the viral contaminant if it is present in the test sample.

The use of a positive amplification control that mimics the target contaminant creates its own problems. If the test sample is contaminated with even a small amount of the positive control, given the extreme sensitivity of Q-PCR, the test sample may exhibit a false positive [reaction]. False positive reactions can be mitigated to some extent by using a low level of positive amplification control, by having a separate work area for the positive control, by using the UNG/dUTP system to selectively degrade dUTP-containing PCR products, by using disposable containers and piston pipettes, and by thoroughly cleaning the work area and equipment. Regardless of how meticulously these precautions are used, false positive results do occur in PCR testing.

In biopharmaceutical manufacturing, the risk of obtaining a false positive reaction to a biological contaminant is quite significant and can lead to costly actions to eliminate it. There is a great need for systems and methods for determining whether a given positive PCR result is truly positive or a false positive due to cross-contamination of a positive control. Applicants have developed and describe positive control compositions and methods that allow for real-time detection of false positive Q-PCR signals.

The essence of the invention

The applicants have solved the problem of identifying in real time 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 sequence of a biological target contaminant (i.e., the positive control sequence) and an original artificial sequence specific for the plasmid. This original artificial sequence specific for the plasmid enables the analyst to specifically identify the plasmid when it is present in the sample. Thus, when a positive signal for the contaminant is detected and the artificial sequence specific for the plasmid is not present, the analyst can

- 1 048105 be sure that this is a true positive result. On the contrary, in the case of a false positive [reaction], the presence of the original artificial plasmid sequence allows the specialist to quickly evaluate the apparent positive result as a false positive.

In some embodiments, an original artificial plasmid-specific positive control sequence (original sequence or PACP, or positive amplification control polynucleotide) is detected by a fluorescently labeled artificial oligonucleotide detection probe (original detection probe or UDP) included in the Q-PCR reaction mixture. The original detection probe comprises a nucleic acid polymer covalently linked to a fluorophore and a quencher. The original nucleic acid polymer is designed to specifically bind to a unique sequence and to be incorporated into amplicon copies of the original sequence during PCR. The original nucleic acid polymer is designed to not recognize or bind to any naturally occurring parvovirus under the hybridization conditions used to analyze the sample. In one embodiment, the original nucleic acid polymer comprises 17 to 20 nucleotides in which 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 original nucleic acid polymer comprises 17 to 20 nucleotides in which no more than 7 to 10 consecutive nucleotides and no more than six (6) consecutive 3' nucleotides are identical to any parvovirus sequence set forth in SEQ ID NOs: 9 and 12-37. When the original detection probe nucleic acid polymer is not included in the amplicon copies, the quencher remains in close proximity to the fluorophore. When the fluorophore is excited, the quencher absorbs the emitted light and prevents its detection (by fluorescence resonance energy transfer (FRET) or contact quenching). When the nucleic acid polymer is incorporated into the amplicon copies (i.e., when the original sequence is present in the sample), the fluorophore and quencher are removed from the original detection probe and are thus spatially isolated. In this case, when the fluorophore is excited, the quencher is sufficiently distant from it that it cannot effectively quench the emitted light. The emitted light can thus be detected. Thus, when the original sequence is present in the sample, the wavelength of the detectable emission increases in intensity as the PCR progresses. When the original sequence is absent, the quencher performs its function and no emission is detected as the PCR progresses. In one embodiment, the fluorophore is attached at or near the 5' end of the nucleic acid polymer and the quencher 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 at or near the 5' end.

Any fluorophore-quencher pair, known or later discovered, can be used in the practice of the present invention. (See, for example, S. A. Marras, Selection of fluorofore and quencher pairs for fluorescent nucleic acid hybridization probes, Methods Mol. Biol. 2006; 335:3-16). In some embodiments, the fluorophore has an excitation wavelength of 495 nm, 538 nm, or 646 nm and an emission wavelength of 520 nm, 554 nm, or 669 nm, respectively. In some embodiments, the quencher is a dye with an absorption peak at 430-672 nm. In some embodiments, the quencher is selected from the group consisting of 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 fluorophore has an excitation wavelength of 495 nm and an emission wavelength of 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').

One of the objects of the present invention is an original detection probe itself, as described above, comprising a nucleic acid polymer and an attached fluorophore and a quencher. Another object of the present invention includes a positive amplification control (PAC) plasmid itself, which contains a sequence of a target biological contaminant, and an original artificial plasmid-specific sequence, and the use of such a plasmid as a positive control for detecting the presence of a target biological contaminant in a cell culture. The PAC plasmid is used to control the success of a PCR amplification reaction carried out to amplify sequences of a target biological contaminant. For example, a separate and parallel PCR reaction is carried out, which contains identical components and is carried out under identical parameters as the test sample, but contains a positive control plasmid instead of the test sample. If a sample containing a positive control plasmid gives a positive signal for a contaminant, while the test sample does not, then it should be concluded that the test sample is devoid of 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.

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In one embodiment, the plasmid comprises (a) a target amplification polynucleotide (TAP) sequence, such as, for example, a parvovirus nucleic acid sequence or another target contaminant sequence, and (b) an amplification control polynucleotide plasmid (PACPP) sequence. The PACPP sequence (sense or Crick chain) is complementary to the nucleic acid polymer of the original probe sequence (antisense or Watson chain).

In one embodiment, the PAC plasmid is used in a separate Q-PCR reaction in parallel with a Q-PCR reaction containing a test sample. The TAP sequence is designed to represent a target biological contaminant. For example, if the test sample of a Q-PCR reaction fails to produce TAP amplicons and the positive control (i.e., the sample containing the PAC plasmid) Q-PCR fails to produce TAP amplicons, then it can be assumed that the Q-PCR reaction is not running. In one embodiment, the target biological contaminant is a rodent parvovirus and the TAP sequence comprises a rodent parvovirus sequence. In one embodiment, the TAP sequence comprises all or a portion of a parvovirus NS1 sequence that is conserved among multiple strains of rodent parvovirus. 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, the plurality of rodent parvovirus strains include small mouse virus prototype (MVMp), small mouse virus immunosuppressant (MVMi), small mouse virus Cutter (MVMc) strain, mouse parvovirus 1b (MPV-1b), mouse parvovirus 1a (MPV-1a), mouse parvovirus 1c (MVP-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a (RPV-1a), small rat virus (RMV), and the University of Massachusetts strain of rat virus L (RVUmass). 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 comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and the complement of SEQ ID NO: 4.

Other objects of the present invention are directed to a PCR cocktail composition and a method of using the PCR cocktail to detect a target contaminant in a test sample and to evaluate a false positive [signal] resulting from contamination of the test sample with a PCR.

In one embodiment, the PCR cocktail comprises, among other things, a forward oligonucleotide primer specific for a target pollutant, a specific oligonucleotide probe detecting the target pollutant, an artificial oligonucleotide detection probe such as the original detection probe (UDP) described above, and a reverse oligonucleotide primer to the target pollutant. In an embodiment in which the target pollutant is rodent parvovirus, the PCR cocktail comprises, among other things, a specific forward oligonucleotide primer to rodent parvovirus, a specific oligonucleotide detection probe for rodent parvovirus, an artificial oligonucleotide detection probe such as the original detection probe (UDP) described above, and a specific reverse oligonucleotide primer to rodent parvovirus. Each detection probe (i.e., a target contaminant specific oligonucleotide detection probe, such as a rodent parvovirus specific oligonucleotide detection probe and an artificial oligonucleotide detection probe) comprises a nucleic acid sequence linked to a fluorophore at one end (either 5' or 3') and a quencher at the other end (either 3' or 5', respectively).

In this case, the rodent parvovirus specific forward oligonucleotide primer and the nucleic acid sequence of the rodent parvovirus specific oligonucleotide detection probe hybridize with the antisense strand of the parvovirus. The rodent parvovirus specific oligonucleotide reverse primer hybridizes with the sense strand of the parvovirus. In one embodiment, the parvovirus sequence to which the primers and the parvovirus specific oligonucleotide probe hybridize is a conserved sequence of the rodent parvovirus. In one case, the conserved sequence of the parvovirus is a parvovirus NS1 sequence, such as, for example, the nucleic acid sequence described in SEQ ID NO: 9. Using the conserved sequence, the same probe is effective in detecting multiple strains of the rodent parvovirus.

In one embodiment, the artificial oligonucleotide detection probe does not hybridize to a parvovirus nucleic acid or any biological contaminant sequence. The nucleic acid of the artificial oligonucleotide detection probe is synthetic and does not hybridize with any stringency to any biological contaminant nucleic acid sequence. In one embodiment, the nucleic acid of the artificial oligonucleotide detection probe (otherwise referred to as the original nucleic acid polymer or original sequence) is designed not to recognize or bind to any naturally occurring parvovirus under the hybridization conditions used in the assay work for that subject. In one embodiment, the original

- 3 048 105 the original nucleic acid polymer comprises 17 to 20 nucleotides, wherein no more than seven (7) 10 internal contiguous nucleotides and no more than six (6) contiguous 3' nucleotides are identical to any parvovirus sequence. In one embodiment, the original nucleotide polymer comprises 17-20 nucleotides, wherein no more than 7-10 contiguous nucleotides and no more than six (6) contiguous 3' nucleotides are identical to any parvovirus sequence set forth in SEQ ID NOs: 9 and 12-37. However, the nucleic acid of the artificial oligonucleotide detection probe (i.e., the original sequence) hybridizes to the PAC sequence of the PAC plasmid. Thus, the artificial oligonucleotide detection probe detects the PAC plasmid, but not the parvovirus or other biological contaminant sequence.

In one embodiment, a PCR cocktail may be used to determine whether a PAC plasmid is present in a test biological sample, giving a false positive result. Wherein, if the test sample exhibits a positive Q-PCR signal to a parvovirus-specific oligonucleotide probe and a negative Q-PCR signal to an artificial oligonucleotide detection probe, then the test sample may be assumed to be free of PAC contamination (i.e., a true positive).

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

Other aspects of the present invention provide a system and method for detecting a biological contaminant in a test sample. In this case, the test sample is a cell culture, such as, for example, an industrial scale mammalian cell culture for the production of a therapeutic protein. Mammalian cells useful 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). The system and method include, in addition to the primers, probes, cocktails, and PAC plasmid used as described above, the use of a nucleic acid extraction control (NEC). In one embodiment, the NEC is phage M13K07, which is incorporated into the test sample prior to nucleic acid extraction. If the nucleic acid extraction is adequate for the purposes of detecting the DNA or RNA contaminant, then the NEC nucleic acid (e.g., M13K07 nucleic acid) is detected via Q-PCR in the test sample with an internal standard. In a particular embodiment, the additive to the Q-PCR reaction comprises a specific forward oligonucleotide primer for the target contaminant, a specific oligonucleotide detection probe for the target contaminant, an artificial oligonucleotide detection probe such as the original detection probe (UDP) described above, a specific reverse oligonucleotide primer for the target contaminant, a specific forward oligonucleotide primer for NEC, a specific oligonucleotide detection probe for NEC, and a specific reverse oligonucleotide primer for NEC. In one embodiment, the test sample is taken from a cell culture production of a therapeutic protein that has been equipped with a NEC (e.g., phage M13K07) prior to nucleic acid extraction and subsequent analysis by Q-PCR.

In a specific embodiment, the target pollutant is a rodent parvovirus, in which case (1) the target pollutant specific forward oligonucleotide primer is a rodent parvovirus specific forward oligonucleotide primer, more particularly comprising the sequence of SEQ ID NO: 1; (2) the target pollutant specific oligonucleotide detection probe is a rodent parvovirus specific oligonucleotide detection probe, more particularly comprising the nucleic acid sequence of SEQ ID NO: 2; (3) the artificial oligonucleotide detection probe is an original detection probe (UDP), more particularly 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 particularly comprising the nucleic acid sequence of SEQ ID NO: 4; (5) the NEC-specific forward oligonucleotide primer is an M13 forward oligonucleotide primer, more particularly comprising the nucleic acid sequence of SEQ ID NO: 5; (6) the NEC-specific oligonucleotide detection probe is a M13 detection probe, more particularly comprising the nucleic acid sequence of SEQ ID NO: 6; and (7) the NEC-specific reverse oligonucleotide primer is an M13-specific reverse oligonucleotide primer, more specifically comprising the nucleic acid of SEQ ID NO: 8. In one embodiment of the method, if a Q-PCR signal for NEC is detected (i.e., the signal is within the expected repair range), it can be assumed that the extraction of the nucleic acid from the test sample is successful. On the other hand, if a signal is detected for

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NEC (i.e., the signal is outside the expected repair range), then it can be assumed that the nucleic acid extraction from the test sample was unsuccessful and a negative Q-PCR signal for the target contaminant is considered erroneous (i.e., false negative).

Detailed description of the invention

Before the present invention is described, it is to be understood that the present invention is not limited to the particular methods and experimental conditions described, since such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the term about, when used with respect to a specific numerical value referred to, means that the value may 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 in between (e.g., 99, 1, 99.2, 99.3, 99.4, etc.).

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 described herein. All publications mentioned herein are incorporated by reference in their entireties. Other embodiments will become apparent from a review of the following detailed description.

In order that the invention described herein may be fully understood, the following detailed description is set forth.

The present invention relates to improved materials and methods for detecting any biological contaminant in any cell culture that produces 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 a step that is easily detected to eliminate false positives.

PCR and quantitative PCR

As used herein, the phrase polymerase chain reaction (PCR) refers to a process for producing copies of a nucleic acid (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), and DNA synthesis (DNA polymerase catalyzes the synthesis of a new DNA strand starting from the 3' end of a hybridized oligonucleotide, using the template DNA strand as a template). In order for amplification to occur, at least two different oligonucleotide primers (referred to as primers for short) are used in a 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. Another 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 copied to form a new double-stranded DNA molecule, also known as an amplicon. Thus, with an unlimited number of oligonucleotide primers, DNA polymerase (i.e., Taq polymerase or other thermostable DNA polymerase; 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 USA. 9436-40 (1988)), and nucleotide triphosphates, the number of DNA molecules (templates 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).

As used herein, the term cycle refers to a single cycle of (1) melting of DNA, referred to as denaturation, followed by (2) hybridization of oligonucleotide primers to form single-stranded DNA by the rules of complementary base pairing, a process referred to as annealing, and (3) polymerization of a new DNA strand starting at the 3' end of the oligonucleotide primer and moving in a 5'-to-3'-end direction, [i.e.] a process referred to as amplification or elongation. Typically, the polymerization uses a DNA polymerase enzyme, such as Taq polymerase, which catalyzes the formation of phosphodiester bonds between adjacent deoxynucleotide triphosphates (dNTPs), which are attached to the replicated single-stranded template DNA by hydrogen bonds according to the rules of complementary base pairing. Denaturation, annealing, and amplification are performed at specific temperatures based in part on the GC content of the template DNA and oligonucleotide primers and the length of the DNA strand being copied. The temperature of denaturation and annealing, as well as the ionic strength of the reaction buffer, controls the fidelity of hybridization and the accuracy of DNA copying.

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Quantitative PCR or qPCR or Q-PCR (also known as real-time PCR) is a type of PCR that allows for the monitoring of amplicons during a cyclic PCR process. Q-PCR can be used to quantify specific template DNA in a sample. Q-PCR includes at least one oligonucleotide detection probe in the reaction mixture in addition to a forward oligonucleotide primer and a reverse oligonucleotide primer. 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 DNA polymerase. Thus, when amplification of a specific template sequence occurs, the detection probes degrade at an exponential rate.

An oligonucleotide detection probe for Q-PCR is typically constructed with a fluorophore (also known as a reporter fluorescent dye or simply a reporter) attached and a quencher attached. In most cases, the fluorophore is attached to or near the 5' end of the oligonucleotide and the quencher is attached to or near the 3' end of the oligonucleotide. However, any [other] designs may be used in the practice of the present invention. When the oligonucleotide detection probe is intact, the fluorophore and quencher are positioned close to each other such that the quencher absorbs light emitted by the excited fluorophore, thereby substantially reducing the detectable emission of the fluorophore. When the oligonucleotide detection probe is cleaved or degraded, the fluorophore and quencher are released and, thus, sequestered in space. The quencher is not close enough to suppress fluorophore emission. As specific amplicons form, more oligonucleotide detection probes are cleaved, releasing more fluorophore and quencher, so that more fluorophore/quencher pairs are isolated from each other, and the amplitude of fluorescence emission increases. In other words, the increase in fluorophore emission signal correlates 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).

Fluorescence quenching can occur by direct contact between the reporter and quencher (also known as static quenching) or by fluorescence resonance energy transfer (FRET) between the reporter and quencher when both the reporter and quencher are within the Förster radius of each. A specific fluorophore can be excited by one or more specific wavelengths of light or a range of wavelengths that have a maximum. This is called the excitation wavelength. Once the fluorophore is excited, it returns to its baseline level and emits light at a wavelength longer than the excitation wavelength. This is called the emission wavelength. During FRET, a second fluorophore, dye, lanthanide group molecule, or similar molecule that has an absorption spectrum that matches or overlaps the emission spectrum of the fluorophore, absorbs the light emitted by the excited fluorophore within the Forster radius, thereby causing quenching or reduction in the emission wavelength of the fluorophore. Contact or static quenching occurs when the reporter and quencher form a ternary complex at the resting level of the fluorophore. This ternary complex is nonfluorescent, i.e., it is essentially unexcitable and therefore does not emit light at the expected emission wavelengths.

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 the choice of reporter/quencher pairs for use in Q-PCR.

Nucleic acids

The terms polynucleotide, oligonucleotide, probe, primer or nucleotide primer or oligonucleotide template primer or template nucleic acid or template DNA are used in this document in their usual meaning to those skilled in the art of molecular biology. For detailed explanations of each of these, see, for example, PCR Primer: A Laboratory Manual (Carl W. Dieffenbach & Gabriela S. Dveksler eds., 1995).

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

Cell cultures

The present invention is directed to an improved Q-PCR method for detecting biological contaminants in cell culture. Cell cultures are often used to produce complex biological molecules for therapeutic use, such as antibodies, decoy molecules, and Fc-containing fusion proteins. These cultures must remain free of biological contaminants. Detection of contaminants is important for determining whether a particular batch should be

- 6 048105 rejected or subject to cleaning.

Cell cultures include a culture medium and cells, typically derived from a single cell line. In this case, the cell line contains cells capable of producing a biotherapeutic protein. Examples of cell lines that are commonly used to produce protein biotherapeutics include, but are not limited to, 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, PerGY cells) and chicken embryo cells. The Chinese hamster ovary (CHO) cell line or one or more of several specific variants of CHO cells, such as the CHO-K1 cell line, are optimized for large-scale protein production. The EESYR® cell line is a specialized CHO cell line optimized for increased production of a protein of interest. For a detailed description of EESYR® cells, see U.S. Patent No. 7,771,997 (issued August 10, 2010).

Cell culture or culture refers to the growth and reproduction of cells outside a multicellular organism or tissue.

Suitable culture conditions for mammalian cells are known to those skilled in the art. See, for example, Animal Cell Culture: A Practical Approach (D. Rickwood, ed., 1992). Mammalian cells may be cultured in suspension or attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, operated in static, fed-batch, continuous, semi-continuous, or perfusion modes are available for mammalian cell culture. Cell culture medium or concentrated growth medium may be added to the culture continuously or at intervals during the culture period (i.e., fed-batch). For example, a culture may be fed once a day, every other day, every third day, or may be fed when the concentration of a specific component of the medium, which is controlled directly or indirectly, falls below a desired range [level].

Animal cells such as CHO cells or EESYR@ cells can be cultured in small scale cultures such as 125 ml containers containing about 25 ml of medium, 250 ml containers containing about 50-100 ml of medium, 500 ml containers containing about 100-200 ml of medium.

Alternatively, cultures may be large scale, such as, for example, 1000 ml containers containing about 300-1000 ml of medium, 3000 ml containers containing about 500-3000 ml of medium, 8000 ml containers containing about 2000-8000 ml of medium, and 15000 ml containers containing about 4000-15000 ml of medium. Production cultures may contain 10,000 L of medium or more. Large scale cell cultures, such as for the clinical production of protein therapeutics, are typically maintained for days or weeks while the cells produce the desired protein(s). During this time, samples of the culture may be taken and tested for the presence of biological contaminants.

Production of therapeutic proteins

The cell culture that is tested for biological contamination can be used to produce a protein or other biological molecule of interest, such as a therapeutically effective antibody or other biopharmaceutical drug substance. The protein product (protein of interest) can be, among others, an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antibody fragment that binds an antigen, 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.

The protein of interest may be a recombinant protein that contains an Fc residue and another domain (e.g., an Fc-containing fusion protein). The Fc-containing fusion protein may be an Fc-containing fusion protein receptor that contains one or more of the one or more extracellular domain(s) of the receptor associated with an Fc residue. In some cases, the Fc residue contains a hinge region followed by the CH2 and CH3 domains of IgG. In some cases, the Fc-containing fusion protein receptor contains two or more different receptor chains that bind either a single ligand or multiple ligands. For example, an Fc-containing fusion protein is a decoy, such as, for example, an IL-1 decoy (e.g., rilonacept, which contains the ligand binding region of IL-1RAcP fused to the extracellular region of I1-1R1 fused to the Fc domain of hIgG1, see U.S. Patent No. 6,927,004, or a VEGF decoy (e.g.,

- 7,048,105 aflibercept, which comprises domain 2 of the VEGF receptor Ig Fltl fused to domain 3 of the VEGF receptor Ig Fiki fused to the Fc domain of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159).

The present invention is not limited to any particular cell type or cell line for protein production. Examples of cell types suitable for protein production include mammalian cells, such as CHO-derived cells such as EESYR@, insect cells, avian cells, bacterial cells, and yeast cells. The cells may be stem cells or recombinant cells transformed with a vector for expressing a recombinant gene, or cells transfected with a virus for producing viral products. The cells may contain a recombinant heterologous polynucleotide construct that encodes a protein of interest. Such a construct may be episomal (such as an extrachromosomal plasmid or fragment) or it may be physically integrated into the genome of the cell. The cells may also produce a protein of interest. The cells may contain a recombinant heterologous polynucleotide construct that encodes a protein of interest. Such a construct may be episomal (such as an extrachromosomal plasmid or fragment) or it may be physically integrated into the genome of the cell. The cells may also produce a protein of interest without having encoding such a protein on the heterologous polypeptide construct. That is, the cell may naturally encode the protein of interest, such as B cells producing an antibody. Methods and vectors for genetically engineering cells or cell lines to express a protein of interest are well known to those of ordinary skill in the art. For example, various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R. J., Large Scale Mammalian Cell Culture 1990, pp. 15-69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and from commercial suppliers.

The cells may also be primary cells, such as chick embryo cells or primary cell lines. Examples of suitable cells include BSC cells, LLCMK 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, BHK21 cells, chick embryo cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 293 cells, PerO6 cells, and CHO cells. In various embodiments, the cell lines are derivatives of CHO cells, such as the mutant CHO-K1, CHO DUX B-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, CHO lec lines, or the EESYR@ cell line.

In one specific scenario, the cells are derivatives of CHO cells, such as EESYR@ cells, that ectopically (heterologously) express a protein. Such a protein comprises a heavy chain immunoglobulin region, such as a CH1, CH2, or CH3 region. In one embodiment, the protein comprises a CH2 and CH3 region of human or rodent immunoglobulins. In one embodiment, the protein comprises a CH1, CH2, and CH3 region of human or rodent immunoglobulin. In one embodiment, the protein comprises a hinge region and a CH1, CH2, and CH3 region. In a specific embodiment, the protein comprises an immunoglobulin heavy chain variable domain. In a specific embodiment, the protein comprises an immunoglobulin light chain variable domain. In a specific embodiment, the protein comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain. In a specific embodiment, the protein is an antibody, such as a human antibody, a rodent antibody, or a human/rodent chimeric antibody (e.g., human/mouse, human/rat, or human/hamster).

The protein production phase of cell culture may be carried out at any scale of culture from individual flasks and shake flasks or WAVE Biotech disposable bioreactors to 1 liter bioreactors and large scale industrial bioreactors. Large scale processing may be carried out at volumes of about 100-20,000 liters or more. One or more of several means may be used to control protein production, such as temperature shift or chemical induction. The cell growth phase may occur at a higher temperature than the production phase during which the protein is expressed and/or secreted. For example, the growth phase may occur initially at a temperature of about 35-38°C and the production phase may occur at a different temperature, such as about 29-37°C, or if desired, from about 30°C to 36°C or from about 30°C to 34°C. In addition, chemical inducers of protein production such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetamide (HMBA) may be added before, simultaneously with, or after the temperature shift. If inducers are added after the temperature shift, they may be added one hour to five days after the temperature shift, such as one to two days after the temperature shift.

Cell culture production can be carried out as a continuously fed-batch culture system, as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec;17(6):1032-41) or as a fed-batch culture system, as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec;17(6):1032-41).

- 8 048105 periodic process (Huang, 2010).

Therapeutic protein products

As used herein, peptide, polypeptide and protein are used interchangeably throughout and refer to a molecule comprising two or more amino acid residues joined together by a peptide bond. Peptides, polypeptides and proteins may also include modifications such as glycosylation, lipid addition, sulfation, γ-carboxylation of glutamic acid residues, alkylation, hydroxylation and ADP-ribosylation. Peptides, polypeptides and proteins may be of scientific or commercial interest, including protein-based drugs. Peptides, polypeptides and proteins include, but are not limited to, antibodies and chimeric or fusion proteins. Peptides, polypeptides and proteins are produced by recombinant animal cell lines using cell culture techniques.

An antibody is an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, joined by disulfide bridges. 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 a single domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRSs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term antibody includes reference to both glycosylated and nonglycosylated immunoglobulins of any isotype or subclass. The term antibody includes antibody molecules manufactured, expressed, generated, or isolated by recombinant means, such as isolating antibodies from a host cell transfected to express the antibody. The term antibody also includes a bispecific antibody, which includes a heterotetrameric immunoglobulin 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.

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

The antibody or its antigen-binding portion may 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 use of a streptavidin active site to form a tetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of a cysteine residue, a marker peptide, and a C-terminal polyhistidine tag to form bivalent and biotinylated scFv molecules (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 such as papain or pepsin digestion of whole antibodies. Further, antibodies, antibody portions, and immunoadhesion molecules can be produced using standard recombinant DNA techniques (see Sambrook et al., 1989).

The term "human antibody" is intended to include antibodies having variable and constant regions derived from human fetal immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human fetal immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), such as in the CDRs and in particular CDR3.

However, the term "human antibody" as used herein does not include antibodies in which CDR sequences derived from embryos of other mammalian species, such as mice, are grafted onto human framework sequences.

The term recombinant human antibody, as used herein, is intended to include all human antibodies that are manufactured, expressed,

- 9 048105 are created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial library of human antibodies, antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies made, expressed, created, or isolated by any means that involves splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human embryonic immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subject to in vitro mutagenesis (or, when animal transgenes for human Ig sequences are used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, because they are derived from or related to human germline VH and VL sequences, cannot naturally exist among the human germline antibody repertoire in vivo.

Fc-containing fusion proteins contain part or all of two or more proteins, one of which is the Fc region of an immunoglobulin molecule, that do not occur together in nature. The preparation of fusion proteins containing certain heterologous polypeptides fused to different portions of antibody-derived polypeptides (including the Fc domain) is 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. The receptor for Fc-containing fusion proteins consists of one or more extracellular receptor domains linked to an Fc fragment, which in some embodiments comprises a hinge region followed by CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc-containing fusion protein comprises two or more different receptor chains that bind one or more ligands. For example, an Fc-containing fusion protein is a decoy, such as, for example, an IL-1 decoy (e.g., rilonacept, which comprises the ligand binding region of IL-1RAcP fused to the extracellular region of IL-1R1 fused to Fc hIgG1; see U.S. Patent No. 6,927,004), or a VEGF decoy (e.g., aflibercept, which comprises domain 2 of the Ig VEGF receptor F1t1 fused to domain 3 of the Ig VEGF receptor F1k1 fused to Fc hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159).

Biological pollutants

As used in this document, the term biocontaminant means any unfavorable, undesirable, injurious, or potentially hazardous biological entity. Such entities include, but are not limited to, prions (the etiologic cause of transmissible bovine spongiform encephalopathy), virions, viruses, mycoplasma, other bacteria contaminating metazoan cells, DNA, RNA, transposons, other transmissible elements, yeast, other fungi, algae, protists, and other adventitious and endogenous agents. Of particular concern for biotherapeutic production processes that use rodent cells (such as CHO cells and CHO cell derivatives) is the introduction of viruses associated with the cells or the manufacturing environment or raw materials. Contamination of bulk cell culture process materials and the resulting drug products poses a direct risk to** patients as well as an indirect risk due to interruption of drug supply.

A partial list of imported and/or endogenous agents that can infect CHO cell cultures includes: 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, vesivirus 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 the particularly problematic parvoviruses, which include small mouse virus (also known as small murine virus)***; and double-stranded DNA viruses such as adenoviruses and pseudorabies virus. Of these potential importing agents, four viruses predominate in large CHO cell culture sample collections from various manufacturers. These viruses are reovirus type 2, Cache Valley virus, epizootic hemorrhagic disease virus, and rodent parvovirus small mouse virus. For a detailed review of adventitious 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).

Testing for imported agents falls into two main categories. The first is the classical virological approach using in vitro virus assay. In this case, the test

- 10 048105 the sample to be titrated is applied to an indicator cell line, the cells are incubated and passaged for 14-28 days, and then an outcome such as cytopathic effect or hemagglutination is measured at the end (Berting, 2010). The second [approach] is a PCR-based assay that measures in real time the presence of nucleic acids associated with imported or endogenous agents. For example, see 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 Bioterapeutics, 30(4) Biologicals 259-270 (2002).

Mouse minute virus (or MMV, mouse minute virus, or MVM) poses a particular challenge to biotherapeutic manufacturing. Both the U.S. Food and Drug Administration (FDA) and European health authorities require specific testing for MVM. This virus is a member of the Parvoviridae family (parvoviruses) and is common to mice. It is excreted in urine or feces and is persistent and survives in the environment. It can easily enter biotherapeutic manufacturing processes. See Moody et al. Mouse Minute Virus (MME) 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 may adversely affect cell culture-based biopharmaceutical manufacturing. In addition to the original MVM strain, such rodent parvoviruses include, among others, the immunosuppressive MVM strain and the truncated strain, murine parvovirus 1a (MPV-1a), MPV1b, MPV-1c, hamster parvovirus, Tulane parvovirus (H-1 parvovirus), Kilham rat virus, rat parvovirus 1a, small rat virus, 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).

These parvoviruses share a conserved nucleic acid sequence termed NS-1 (NS1), which encodes a large nonstructural protein involved in viral genome amplification. The conserved nucleotide sequence of NS1 from MVM is shown in SEQ ID NO: 9. Nucleotides 875-956 of this sequence are at least 97% conserved among a broad repertoire of rodent parvovirus NS1 sequences and therefore serve as a good target sequence for PCR-based testing of adventitious and endogenous agents. PCR-based testing for rodent parvoviruses (and other adventitious and endogenous agents) can be performed on raw materials, pre-harvest culture media, at various points in the purification process of biotherapeutic molecules in large batches, and during formulation and packaging. Detection of a contaminant may require remedial steps such as destruction of contaminated material, replacement of raw materials, and cleaning of equipment.

In addition to testing for adventitious agents, endogenous agents, and other biological contaminants that necessitate corrective and preventive actions (CAPAs) during manufacture, special manufacturing and bulk process steps (i.e., equipment operation) can be used to eliminate, reduce, or inactivate viral contaminants. Chemical inactivation, virus-retaining filtration, and chromatography have all been shown to be effective in reducing herpesvirus, retrovirus, and parvovirus in cell culture harvests or partially purified cell culture fluid. The chemical inactivation step is most often used with low pH treatment, which those of ordinary skill in the art believe is due to denaturation of viral envelope proteins. Protein A, hydroxyapatite, cation exchange, and anion exchange chromatography steps have all been shown to remove virus 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).

Q-PCR testing: positive and negative controls

Tests for biological contaminants must be adequately controlled to ensure accuracy, reliability, and validity. As used herein, a negative control includes most or all of the experimental reagents and conditions other than the test sample. The test sample may be replaced by a buffer or a culture medium known to be free of the biological contaminant of interest. Further, as used herein, a negative control must yield negative results for the biological contaminant. If a negative control yields a positive result for the biological contaminant, then an experienced technician or investigator would expect that a positive result from a parallel test sample may not accurately reflect whether the test sample contains the biological contaminant.

As used in this document, one or more positive controls are used to assess whether the experimental conditions are adequately controlled for the detection of a biological contaminant. Positive controls can be used at any stage of the experimental process to ensure that each step is working and to determine whether

- 11 048105 at what stage the process is disrupted.

In some embodiments, a positive control is used in the nucleic acid extraction step to assess whether the extraction of nucleic acid of a biological contaminant is efficient enough to detect the biological contaminant. Such a positive control is referred to as a nucleic acid extraction control or NEC. In some cases, the NEC is selected to mimic the target biological contaminant in terms of protein-nucleic acid structure. If the NEC is extracted sufficiently to be detected, then the skilled artisan can assume that the target biological nucleic acid has also been extracted sufficiently to be detected. In one embodiment, the NEC is a single-stranded DNA phage similar to parvoviruses.

In a specific embodiment, the NEC is bacteriophage M13, which consists of a circular single-stranded DNA of about 6407 nucleotides. In a more specific embodiment, the NEC is strain M13K07, which is a readily available molecular biology reagent used for cloning and other laboratory purposes. See van Wezenbeek et al., Nucleotide Sequences of the Filamentous Bacteriophage M13 DNA Genome: Comparison with Phage fd, 11 (1-2) Gene 129-148 (1980). The nucleotide sequence of phage M13K07 is set forth in SEQ ID NO: 8. Although bacteriophage M13 or bacteriophage strain M13K07 can be used as NEC in specific embodiments, the present invention is in no way limited to the use of particular reagents as NEC. Those of ordinary skill in the art may use other reagents as NEC in the practice of the present invention without departing from the scope of the present invention (e.g., phage MS2 for reverse transcriptase PCR assay; 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)).

In some embodiments of the present invention, a positive control is used in the PCR amplification step to assess whether the PCR reagents (including primers) and the PCR step are (were) sufficient to detect the template nucleic acids of the biological contaminant. Such a positive control is referred to as a plasmid amplification control or PAC. In some cases, the PAC is selected or designed to match the biological contaminant, the target nucleic acid sequence, and especially to match the forward and reverse oligonucleotide primers of the test sample. In a specific embodiment, the PAC further comprises an original sequence that is not present in the target nucleic acid of the biological contaminant. In a more specific embodiment, the original sequence is not found in nature. The polymer of the original nucleic acid is designed to specifically bind (anneal) to the original sequence and be incorporated into amplicon copies of the original sequence during PCR. The original nucleic acid polymer is designed such that it does not recognize or anneal to any naturally occurring parvovirus under the hybridization conditions used in conducting the subject assay. In one embodiment, the original nucleic acid polymer comprises 17 to 20 nucleotides, wherein no more than seven (7) to 10 internal contiguous nucleotides and no more than six (6) contiguous 3' nucleotides are identical to any parvovirus sequence. In one embodiment, the original nucleotide polymer comprises 17 to 20 nucleotides, wherein no more than 7 to 10 contiguous nucleotides and no more than six (6) contiguous 3' nucleotides are identical to any parvovirus sequence set forth in SEQ ID NOs: 9 and 12-37.

This original sequence enables experienced operators to distinguish between the sequence of a true biological target contaminant and cross-contamination of a Q-PCR test reaction with a PAC plasmid.

In one embodiment, the PAC (in the form of a plasmid, otherwise the PAC plasmid) is included in an identical but separate Q-PCR reaction to control for PCR amplification. The PAC reaction uses the same oligonucleotide primers and probes as are used with the test sample in the QPCR reaction to accurately reflect the nucleic acid amplification of the actual biological contaminant target. The test sample Q-PCR reaction and the separate PAC plasmid Q-PCR reaction include a probe detecting the target and a probe for the original sequence. The PAC reaction, if running normally, would be expected to give a positive signal for the target and a positive signal for the original sequence. Obtaining a positive signal for the target and a positive signal for the original sequence in a reaction containing the PAC plasmid indicates that the reagents and Q-PCR conditions of the test sample work adequately to produce a positive signal for the target in the test sample whenever the nucleic acid of the target biological contaminant is present.

Therefore, the RAS acts as a control for the correctness of the PCR amplification. If a negative signal for the target is obtained in the test sample, but the RAS shows a positive signal for the target, then an experienced operator can assume that the test sample is missing the detector.

- 12 048105 titrated [amount] of DNA of the target contaminant DNA. Conversely, if a positive signal for the target sequence and a positive signal for the original sequence are obtained in the test sample, then the specialist may suspect that the PAC plasmid has cross-contaminated the test sample and, therefore, the positive signal for the target sequence may be a false positive.

Detailed description of several embodiments

In several embodiments, an improved positive control system (i.e., compositions and methods) for detecting biological contaminants using polymerase chain reaction, more particularly Q-PCR, is provided.

One aspect of the present invention provides an original sequence probe (USP) for detecting a positive control for plasmid amplification (PAC plasmid). The USP comprises an artificial nucleotide sequence capable of hybridizing to an original artificial plasmid-specific sequence (the same as the original sequence or original PAC sequence, or UAPS), a fluorophore, and a quencher. In a particular embodiment, the artificial nucleotide sequence capable of hybridizing to UAPS comprises the nucleic acid sequence of SEQ ID NO: 3 (5'TGTCGATGGCGAATGGCTA-3'), which is antisense to the sense strand of the UAPS sequence (e.g., SEQ ID NO: 10-5'-TAGCCATTCGCCATCGACA-3').

As described above, the Q-PCR detection probe comprises a fluorophore and a quencher. Quenching may occur by contact quenching or by FRET, depending on the fluorophore/quencher pair. In this case, the fluorophore and quencher are covalently attached to the USP oligonucleotide. In one embodiment, the fluorophore has an excitation wavelength of 495-680 nm and an emission wavelength of 515-710 nm. In some embodiments, the fluorophore has excitation wavelengths of 495 nm, 538 nm, or 646 nm and emission wavelengths of 520 nm, 554 nm, or 669 nm, respectively. In some embodiments, the quencher is a dye with an absorption peak of 430-672 nm. Examples of suitable extinguishers include DDQ®-I, Dabcyl, Eclipse®, Iowa Black FQ®, BHQ®-1, QSY®-7, BHQ®-2, DDQ®-II, Iowa Black RQ®, QSY®-21 and DHQ®-3. 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 absorption maximum at about 495 nm and an emission maximum at about 520 nm, and the quencher is Black Hole Quencher®-1 (BHQ®-1, Biosearch Technologies, Inc., Petaluma, CA), which absorbs at 480-580 nm. BHQ®-1 causes quenching via FRET and contact quenching. Generally, but not always, the quencher is attached via an ester linkage to the 3'-hydroxyl group of the oligonucleotide, and the fluorophore is attached via an ester linkage to the 5'-phosphate group of the oligonucleotide.

Another aspect of the present invention provides a Q-PCR reagent mixture comprising a plurality of oligonucleotides and probes useful for detecting a biological contaminant in cell culture, feedstock, partially purified and purified biological molecules and the like. In one embodiment, the biological contaminant is a DNA virus, more particularly a parvovirus, and even more particularly a rodent parvovirus such as MVM. The parvovirus comprises an NS1 gene having a nucleic acid sequence that is at least 88% identical to any of the sequences set forth in Table 1. In another embodiment, the parvovirus comprises an NS1 gene having a nucleic acid sequence that is at least 97% identical to the sequence set forth in SEQ ID NO: 9 (i.e., the small mouse virus (MVM) NS1 gene). In another embodiment, the parvovirus comprises an NS1 gene comprising the consensus sequence of SEQ ID NO: 37.

- 13 048105

Table 1

Parvoviridae NS1 sequences

Gene name SEQ ID NO Identity to SEQ ID NO: 9 Gene name SE Q ID NO Identical to SEQ ID NO: 9 Lymph strophic variant MVM 12 97% MVM, strain M 25 95% Mouse parvovirus 4b 13 96% Parvovir with LuIII 26 90% Mouse parvovirus 4a 14 96% Parvovir from rat UT 27 89% Mouse parvovirus 1b 15 96% KilHEM virus rats 28 89% Immunosuppressive variant of MVM 16 96% Small rat virus 1c 29 89% Mouse parvovirus 1 17 96% Small rat virus 1b 30 89% Mouse parvovirus 5a 18 96% Small virus 31 89%

rats 1a Mouse parvovirus UT 19 96% Parvovir with H-1 32 89% Mouse parvovirus 1e 20 96% NTUI isolate of small rat virus 33 89% Mouse parvovirus 1c 21 95% Parvovir with h-1 34 89% Hamster parvovirus 22 95% Isolate NTU2 of small rat virus 35 88% Mouse parvovirus 3 23 95% Small rat virus 2 a 36 88% Small mouse virus 24 95% Consensus with NS1 37 100%

In a specific embodiment, the mixture comprises: (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 oligonucleotide reverse primer. Such a mixture can be used in both a test sample and a positive control sample. In a more specific embodiment, the oligonucleotide primers and the parvovirus specific oligonucleotide detection probe hybridize to an NS1 sequence, such as, for example, the NS1 sequence set forth in SEQ ID NO: 9. In a more specific embodiment, (1) the forward primer comprises the sequence of SEQ ID NO: 1; (2) the parvovirus specific oligonucleotide detection probe comprises the sequence of SEQ ID NO: 2; (3) the artificial probe is USP and comprises the sequence of SEQ ID NO: 3; and (4) the reverse primer comprises the sequence of SEQ ID NO: 4.

As described above, the USP contains a fluorophore and a quencher such that amplicons containing such a sequence, i.e., PAC DNA, can be detected in real time by QPCR. Similarly, the parvovirus specific detection probe contains an oligonucleotide with a covalently attached fluorophore and quencher. In practice, the emission wavelength of the parvovirus detection probe fluorophore should be different from the emission wavelength of the USP in order to differentiate between a true positive parvovirus signal and a false positive signal due to contamination of the test sample with a PAC plasmid or another PAC (e.g., an amplicon contaminated with PAC). Thus, in such embodiments in which the USP fluorophore is FAM, the fluorophore attached to the parvovirus detection oligonucleotide probe should have an emission wavelength other than about 520 nm. In a specific embodiment, the fluorophore attached to the rodent parvovirus specific oligonucleotide detection probe is VIC® Dye (Life Technologies, Inc., Carlsbad, CA), which has an absorption maximum of 538 nm and an emission maximum of about 554 nm. In this case, the quencher can be a FRET quencher or a contact quencher. In one embodiment, the quencher is a non-fluorescent quencher, minor groove binding protein (MGBNFQ) (see Sylvain et al., Rapid Screening for HLA-B27 by a TaqMan-PCR Assay Using SequenceSpecific Primers and a Minor Groove Binder Probe, a Novel Type of TaqMan™ Probe, 287(1-2) Journal of Immunological Methods 179-186 (2004)).

The primer and probe mixture described above is used to test and differentiate between a positive amplification control construct and a true parvovirus contaminant. In some embodiments, the primer mixture also includes a set of primers and probes for detecting a nucleic acid extraction control (NEC). In a particular embodiment, the NEC is bacteriophage M13, such as strain M13K07 (SEQ ID NO: 8). Thus, in some embodiments, in addition to the parvovirus primers and probe and the UPS probe, the primer and probe mixture comprises (5) an M13-specific forward oligonucleotide primer; (6) an M13-specific oligonucleotide detection probe; and (7) an M13-specific reverse oligonucleotide primer.

In some embodiments, the primers and probes for M13 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; and the M13 specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO: 7. As with the non-overlapping fluorophore emission spectrum of the parvovirus probe and USP, the NEC probe comprises a fluorophore that emits light in a non-overlapping spectrum. In a specific embodiment, the fluorophore attached to the specific oligonucleotide detection probe on M13 is Cy5, a cyanine dye having an absorption maximum at about 650 nm and an emission maximum at about 670 nm (see Southwick et al., Cyanine Dye Labeling Reagents: Carboxymethylindocyanine Succinimidyl Esters, 11 Cytometry, 418-430 (1990)). In this case, the quencher can act through FRET or contact quenching. In one embodiment, the quencher is Black Hole Quencher®-2 (BHQ®-2, Biosearch Technologies, Inc., Petaluma, CA), which absorbs at a maximum at about 579 nm and quenches in the range of about 550 nm to about 650 nm.

Another aspect of the present invention provides a method for detecting a biological contaminant during the production of a biological molecule. More particularly, the biological contaminant comprises a parvovirus, more particularly a rodent parvovirus, and most particularly such parvoviruses have at least 97% identity to the NS1 gene of MVM. In some embodiments, the NS1 gene comprises the sequence of SEQ ID NO: 9. In one embodiment, the process for producing a biological molecule is a process for culturing mammalian cells to produce an antibody, decoy molecule, or other therapeutic antibody. A test sample is taken from the cell culture (or bulk ingredients) and extracted for nucleic acids. In some cases, the test sample comprises a NEC, such as M13 (e.g., SEQ ID NO: 8), to serve as a control for the correct extraction of nucleic acids prior to Q-PCR. The method comprises the steps of: (1) combining (a) a nucleic acid sample extracted from a 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) performing a polymerase chain reaction (PCR) on the combination; and (3) monitoring the production of different amplicons by the amplitude of the emitted fluorescence.

The formation of specific amplicons correlates with the presence and amount of various template nucleic acids in the test sample. Specific amplicons include (1) target amplification polynucleotides (TAPs), such as, for example, rodent parvovirus sequences (e.g., biological contaminants that contain NS1 sequences), (2) nucleic acid extraction controls (NECs), such as, for example, M13 (e.g., M13K07) polynucleotides (NECPs), and (3) plasmid amplification control polynucleotides (PACPs), such as the original artificial plasmid-specific sequence (UAPS). If TAPs and NECPs are produced, but PACPs are not produced, then it can be concluded that the test sample contains

- 15 048105 is a biological contaminant and does not contain cross-contamination by amplification of the control plasmid. However, if both TAPs and PACPs (i.e., UAPs) are produced in the Q-PCR reaction of the test sample, it can be concluded that the test sample has been cross-contaminated with the PAC plasmid and the TAP result may be a false positive.

In a specific embodiment, wherein 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 comprising the sequence of SEQ ID NO: 2, a VIC fluorophore and an MGBNFQ quencher, (3) an artificial oligonucleotide detection probe such as USP comprising the sequence of SEQ ID NO: 3 and labeled with 6-FAM and BHQ®-1, (4) a rodent parvovirus-specific reverse oligonucleotide primer comprising the sequence of SEQ ID NO: 4, (5) an M13-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO: 5, (6) an M13-specific oligonucleotide detection probe, containing the sequence of SEQ ID NO: 6 and labeled with cy5 and BHQ®-2, and (7) a specific reverse oligonucleotide primer to M13 containing the sequence of SEQ ID NO: 7 - TAP production was monitored at [wavelength] about 533 nm - about 580 nm, PACP production was monitored at about 465 nm - about 510 nm, and NECP production was monitored at about 618 nm - about 660 nm.

In one embodiment, the test sample is taken from a CHO cell culture product, such as a EESYR® cell product transformed with nucleic acids encoding a protein of interest, 96-72 hours before the time of harvesting the culture material. If TAP and NECP are detected in the test sample and PACP is not detected in the test sample, then a confirmatory test can be performed on a second test sample obtained from the same cell culture product 48 hours before the time of harvesting. If TAP and NECP are again detected in the second test sample and PACP is not detected in the test sample, then the cell culture is considered contaminated and cannot be processed further. Alternatively, the second test can be omitted, the cell culture is considered contaminated and the culture cannot be processed further.

In one embodiment, nucleic acid is extracted from a test sample taken from a cell culture product. In this case, one milliliter of the test sample is subjected to cell lysis, proteolysis, and heat denaturation, followed by coupling of the sample with a nucleic acid extraction control (NEC) sample, and then extraction of nucleic acids from the sample. In one embodiment, nucleic acids are extracted from the test sample (or test sample supplemented with NEC) using an automated nucleic acid extraction system, such as the QIAsymphony® instrument (Qiagen, Inc., Valencia, CA) (see Lee et al., Comparative evaluation of the QIAGEN QIAsymphony® SP system and bioMerieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory, 52(4) J. Clin. Virol. 339-43 (2011)).

In one embodiment, the enzyme uracil-N-glycosidase (UNG) is added to the Q-PCR reaction mixture prior to 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, in some cases, for 2 minutes or 5 minutes.

In a specific embodiment, after further treatment with UNG, the reaction mixture was incubated at 95°C for 2 minutes, followed by eight cycles of (1) denaturation at 95°C for 10 seconds, followed by (2) annealing and elongation for 30 seconds, with the first annealing temperature being 70°C, then the annealing temperature was decreased by 1°C in each cycle so that at the eighth annealing it was 62°C. After the first eight cycles, 40 cycles of DNA amplification were performed comprising the steps of (1) denaturation at 95°C for 10 seconds, followed by (2) annealing and elongation at 62°C for 30 seconds. In a specific embodiment, the rate of change of temperature from the denaturation temperature to the annealing temperature was about 4.4°C per second, and from the annealing temperature to the denaturation temperature was about 2.2°C per second.

In some embodiments, a method for detecting a biological contaminant in a cell culture production environment or product thereof includes performing an external positive amplification control (PAC) assay performed separately from the test sample assay and performing an external negative control assay separate from the test sample assay and the PAC assay. If either the negative control or the positive control fails, the result obtained from the test sample assay is discarded.

In one embodiment, the external positive control comprises the steps of (1) combining, including without a test sample, (a) a positive amplification control (PAC) plasmid, which in a specific embodiment comprises the sequence of SEQ ID NO: 11, (b) a rodent parvovirus-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO: 1, (c) a pair-specific oligonucleotide detection probe.

- 16 048105 rodent parvovirus comprising the sequence of SEQ ID NO: 2 labeled with VIC and MGBNFQ, (d) an artificial oligonucleotide detection probe, such as USP, comprising the sequence of SEQ ID NO: 3 labeled with 6-FAM and BHQ®-1, (e) a rodent parvovirus-specific oligonucleotide reverse 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) performing a positive control polymerase chain reaction (PCR) with the positive control mixture; and (3) control of the production of (a) target amplification polynucleotides (TAPs), (b) nucleic acid extraction control polynucleotides (NECPs), and (c) plasmid amplification control polynucleotides (PACPs) during PCR. TAP production is controlled at about 533 nm to about 580 nm, PACPs production is controlled at about 465 nm to about 510 nm, and NECP production is controlled at about 618 nm to about 660 nm.

The positive control PCR amplification reaction is performed identically to the test sample PCR reaction (as described above). If TAPs and PACPs are produced in the positive control reaction, then it can be concluded that the PCR amplification procedure is functioning properly. If TAPs are not produced in the positive control amplification reaction, then any negative TAPs in the test sample can be discarded 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 exhibit a positive signal for NECP (see Table 1).

In one embodiment, the external negative control comprises the steps of (1) combining, including without the test sample and without the PAC plasmid, (a) a control, which may be a buffer that mimics the buffer system of the test sample, 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 USP, comprising the sequence of SEQ ID NO: 3 labeled with 6-FAM and BHQ®-1, (e) a rodent parvovirus-specific oligonucleotide reverse 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) performing a positive control polymerase chain reaction (PCR) with a positive control mixture; and (3) monitoring the production of (a) target amplification polynucleotides (TAPs), (b) nucleic acid extraction control amplification polynucleotides (NECPs), and (c) plasmid amplification control polynucleotides (PACPs) during PCR. TAP production is monitored at about 533 nm to about 580 nm, and PACP production is monitored at about 465 nm to about 510 nm.

The negative control PCR reaction is performed identically to the test sample PCR reaction (as described above). If TAPs and PACPs are produced in the negative control reaction, then it can be concluded that the PCR reagents are contaminated with the PAC plasmid. If TAPs but not PACPs are produced in the negative control reaction, then it can be concluded that the PCR reagents are contaminated with parvovirus. In both cases, the test sample results are rejected. However, if both TAP and PACP production are negative in the negative control reaction, TAP and PACP production are positive in the positive amplification control, and TAP (and NECP, if applicable) production is positive and PACP production is negative in the test sample reaction, then the examiner can conclude that the test sample is contaminated (see Table 2 and Table 3).

Another aspect of the present invention provides a positive amplification control plasmid (PAC plasmid) and a positive control reagent mixture including 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 specific to the plasmid (either UAPS or an original 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 nucleic acid sequence of M13K07 comprises the sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and the original 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 presented in SEQ ID NO: 1.

In some embodiments, the positive control reagent mixture comprises, among others, the PAC plasmid described above, 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 oligonucleotide reverse primer, an M13 specific forward oligonucleotide primer, an M13 specific oligonucleotide detection probe, and an M13 specific reverse oligonucleotide primer, and a buffer. The mixture optionally contains dNTPs and Taq polymerase.

Table 2

Analysis controls and test session status

Analysis controls Parvovirus signal (TAP) UAP signal (RASR) Signal M13K07 (NECP) NEC 0 0 + RAS + + + Negative control (buffer or water) 0 0 0 Test session status □ true □ INCORRECT

Table 3

Test session status

Description of the sample Parvovirus Signal UAP M13K07 Is the sample suitable? Negative test sample 0 0 + Yes False negative test sample 0 0 0 No Positive test sample + 0 + Yes False positive test sample + + + Yes

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 binding protein quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO: 2, USP comprises a VIC fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO: 3, the rodent parvovirus specific oligonucleotide reverse 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 fluorophore Cy5, BHQ-2 quencher and nucleic acid sequence SEQ ID NO: 6, and the specific reverse oligonucleotide primer to M13 contains the nucleic acid sequence SEQ ID NO: 7.

Example 1: Oligonucleotides and nucleic acid reagents

Rodent parvovirus M13K07 and synthetic original oligonucleotides (Oligos) were obtained from different suppliers in different sizes and forms, which are described in Table 4. All oligos had a three-year expiration date from the date of receipt from the supplier.

Oligonucleotides were diluted in water to a concentration of 100 μM to generate stock solutions. Stock solutions were then diluted to yield 10x the stock solution before running qPCR reactions. Table 5 illustrates 10x and 1x concentrations of qPCR oligonucleotides (primers and probes).

- 18 048105

Table 4

Parvovirus M13 and original artificial primer and probe sequence

Oligo type Name Sequential t (5 ' - 3 ' ) Supplier/Quantity/Quantity/Form Direct oligonucleotide primer FWD 5'/TGC ATA AAA GAG TAA OST CAC CAG/3' (SEQ ID NO: 1) IDT/IgM/High Performance Chromatography (HPLC)/lyophilized or ready-made Life Technologies/8OKpMol HPLC/dry or liquid Detection probe sequence MVM-MGBI 5'/VIC/ ACT GGA TGA TGA TGC AGC /MGBNFQ/З' (SEQ ID NO:2) Firm Life Technologies/ 50K/NRDS/liquid; 100 pmol/µl Oligo type Name Sequential t (5’ - 3’) Supplier/Quantity/Quantity/Form Detection probe sequence parvovir US antismys lovoy indicato R 5'/6-FAM/TGT CGA TGG CGA ATG GCT A /BHQ®-l/3' SEQ ID NO:3 IDT/1 mkM/HPLC/lyophilized or ready-to-use Reverse Oligonucleotype Primer REV 5'/CCA CCT GGT TGA GCC ATC/3' (SEQ ID NO:4) IDT/I 1 mkM/HPLC/lyophilized or ready-made Life Technologies/80K pmol/HPLC/dry or liquid Direct Oligonucleotype Primer Ml3FWD 5' /AAG CCT CAG CGA CCG AAT AT/3' (SEQ ID NO: 5) IDT/1 mkM/HPLC/lyophilized or ready-made Detection probe sequence Probe M13 /5'/CY5/ TAT GCG TGG GCG ATG GTT GTT GTC A/BHQ2/3' (SEQ ID NO:6) IDT/1 mkM/HPLC/lyophilized or ready-to-use Reverse oligonucleotide primer M13-REV 5'/TCA GCT TGC TTT CGA GGT GAA T/3' (SEQ ID NO:7) IDT/1 mkM/HPLC/lyophilized or ready-to-use

Table 5

Oligonucleotide concentrations

Oligonucleotide name Oligonucleotide concentration for PCR reaction 10x concentration Oligonucleotide volume a Stock solution for 1 ml of 10-fold concentration MVM-fwd 0.15 μM 1.5 μM 15 µl MVM-MGB 0.15 μM 1.5 μM 15 µl Antisense indicator of parvovirus 0.15 μM 1.5 μM 15 µl MVM-rev 0.15 μM 1.5 μM 15 µl M13-Fwd 0, 1 μM 1.0 µM 10 µl M13-probe 0, 1 μM 1.0 µM 10 µl M13-Rev 0.1 μM 1.0 µM 10 µl

Example 2: Positive control amplification plasmid

The positive control plasmid for amplification of parvo-M13 (PAC) was prepared in the plasmid pUC57-19 048105

Kan (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 x 10 ⁸ copies/ng, calculated by the following formula: [amount = (amount * number/mole) / (bp * ng/g * g/ mole of bp)]; where amount, number/mole = 6.022 x 10 ²³ , bp = 4372, ng/g = 1 x 10 ⁹ , ag/mole of bp = 650. Plasmid concentration was calculated and expressed as ng/µL and serially diluted to a 10-fold concentration of 10 ² copies/µL. From a dilution of ¹⁰² copies/µl, 50 µl aliquots were prepared and stored in 2 ml sterile screw-capped tubes. All aliquots were stored at <-60°C.

Example 3: Preparation of phage M13K07

Ten-fold serial dilutions of phage M14K07 were prepared using minimal essential medium (MEM) as a diluent to obtain M13K07 with a titer of 100 pfu (plaque forming units)/µl. Aliquots of 200 µl were prepared and stored at <-60°C for a three-year shelf life.

Example 4: Preparation of reagents for Q-PCR reaction

The rodent parvovirus real-time PCR detection assay is a fully automated TaqMan® PCR workflow consisting of automated DNA purification with the QIAsymphony® (Qiagen), followed by nucleic acid amplification and detection of the real-time PCR product with the LightCycler® 480 instrument (Roche Diagnostic). Each test sample automatically received phage M13K07 as an internal control (IC) to assess for the presence of PCR inhibitors. The rodent parvovirus primer oligonucleotides are designed to hybridize to a highly conserved region of the rodent parvovirus genome (NS-1 region) to provide broad-range detection. The assay was performed in doublet format (i.e., on two targets), and the primers for rodent parvovirus and M13K07 generated PCR products of approximately 110 and 97 base pairs (bp), respectively. The PCR product was 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. The positive amplification control (PAC) plasmid was used at a concentration of 100 copies/μl. This plasmid contains a unique (indicator) sequence (USP) that distinguishes it from wild-type parvovirus by a specific probe with the FAM fluorophore.

The master mix was prepared in a 2 ml tube according to Table 6 in a quantity sufficient for at least three times the number of samples tested, including controls. The tubes were stored at 2-8°C. A vial of negative amplification control (NAC) was prepared by adding 50 µl of water to a 2 ml tube.

Table 6 Reaction mixtures

Concentration of the reagent stock solution Reagent concentration for one reaction Maternal concentration of reagent for one reaction Double qPCR master mix (TaqMan or VeriQuest) 2X IX 12.5 µl Cvtcm Parvo-qPCR- Oligo YUKH IX 2.5 µl Volume of mixture for each nest 15 µl Sample volume per nest (extracted DNA) 10 µl

Example 5: Preparation of the test sample

Since all samples could potentially contain or be contaminated with a contaminant, aseptic techniques were used for all samples during the pre-processing steps. The subsequent steps were performed in a clean biological safety cabinet. Test items (samples) were prepared from EESYR® cell cultures producing the antibody or trap molecule and were frozen or used immediately.

A 1000 μl aliquot of phosphate-buffered saline (PBS) was added to appropriate 2 ml tubes to serve as a negative extraction control (NEC). When PCR was used as the endpoint of the cell culture step, the positive and negative cell culture controls were used as the positive extraction control and negative extraction control, respectively.

All test items were thawed at room temperature. These samples in 60 ml containers were transferred to 50 ml Falcon tubes and aliquoted. 1000 µl of each sample

- 20 048105 was pipetted into 2 ml tubes for pre-treatment. 400 µL of ChargeSwitch® Lysis Buffer L13 (Invitrogen, Cat# CS11202 or equivalent, Carlsbad, CA) was added to each tube and vortexed for at least 10 seconds. 20 µL of Proteinase K (>10 mg/mL, >800 units/mL in 40% glycerol (v/v) containing 10 mM Tris-HCl, pH 7.5, with 1 mM calcium acetate; followed by SAFC, Cat# P4850-5ML, Sigma Aldrich, St. Louis, MO) was added to each sample and vortexed for at least 10 seconds. Samples were then incubated at 65°C for 30 minutes. Following incubation, samples mixed by vortexing and centrifuged at 17,000 g for 10 minutes.

In parallel, the positive amplification control (PAC; minimum 50 µl of ¹⁰² copies/µl plasmid in a 2 ml tube) and the internal control (IC; minimum 150 µl/12 samples) M13K07 ( ¹⁰² PFU/ml) were thawed.

Example 6: Nucleic acid extraction

An internal control (IC) was required for the programmable extraction protocol on the QIASymphony® Instrument (Qiagen, Valencia, CA). The instrument automatically added 120 μL of resuspended IC to each sample. For every 12 samples, the instrument required 1.8 mL of IC (i.e., 1 vial). IC vials were prepared by adding 1650 μL of AVE buffer (RNase-free water containing 0.04% sodium azide) to 150 μL of M13K07 IC.

Each prepared sample was loaded into the instrument sample carrier in the biosafety cabinet (BSC). The IC vial(s) were loaded into a separate (special) sample carrier in the BSC at a ratio of one IC vial per 12 samples. All moving parts were closed and the instrument was run according to the manufacturer's recommended protocol (see QIAsymphony DNA Handbook, 09/2010, available at http://www.algimed.by/download/ENQIAsymphony-DNA-Handbool.pdf).

The reagent prep cassette was prepared using the QIAsymphony@ DSP Virus/Pathogen reagent kit, which contains all the reagents required to perform the extraction (see QIAsymphony@ DSP Virus/Pathogen Kit Instructions for Use (manual, April 2013, available at https://www.qiagen.com/us/resources/download.aspx?id=f8bc0b3c-0aff-46ee-8807-5ed145f9e969&lang=en). The reagent prep cassette contained proteinase K and had a shelf life of approximately two weeks.

Nucleic acid extraction was performed in a 96-well reaction plate format to facilitate coupling to Q-PCR. After the automated nucleic acid extraction procedure was complete and condition-checked, the reaction plate was cooled and sealed with LightCycler® 480 Sealing Foil (Roche, Branchburg, NJ). The 96-well plate was placed in a plate mixer, balanced with a suitable counterweight (e.g., another 96-well plate), and mixed for 30-60 seconds. Wells were checked for bubbles and mixing was repeated if necessary.

Example 7: Q-PCR

Q-PCR was performed on a LightCycler® 480 Instrument (Roche, Branchburg, NJ) using either TaqMan Triplex and/or Veriquest Triplex software. The TaqMan Triplex protocol used TaqMan Fast Advance Custom Master Mix without reference dye (ROX). Three fluorescence channels (FAM, VIC, and CY5) were selected and the UNG step was 2 minutes. The reaction parameters used are listed in Table 7.

When using VeriQuest@ Master Mix, also without ROX, a UNG step time of 5 minutes was used. The reaction parameters used are presented in Table 8.

Table 7

Stages of the TaqMan Triplex PCR Rodent Parvovirus program

Stage name Cycles Type of analysis Temperature Time Block tracking speed (°C/sec) Step size UNG 1 No 50 2 min 4,4 Preincubation 1 No 95 2 min 4,4 TD 8 No 95 70 62 10 sec 30 sec 4.4 2.2 1°С/step Amplification 40 Quantitative 95 62 10 sec 30 sec 4.4 2.2 Cooling 1 No 40 30 sec 2,2

The intersection point (IP) of the fluorescent signals for each of the M13 rodent parvovirus internal controls and the positive amplification control plasmid was determined using one or both of two algorithms. The first algorithm is an automated second derivative method. This method does not require user input and generally results in greater consistency, and is therefore considered the preferred method. The second algorithm is a smoothing method

- 21 048105 curve. This method allows the user to set a threshold level in cases of divergent baseline data. The point at which the linear-logarithmic curve intersects the threshold level becomes the intercept point. The following filter combinations were used to monitor fluorescent signals: 533-580 nm (VIC signal) for rodent parvovirus (Sample-Parvo parameter set); 618-660 nm (Cy5 signal) for the internal control M13K07 (Sample-M13 parameter set); and 465-510 nm (FAM signal) for the positive control amplification plasmid (Sample-FAM parameter set). The baseline fluorescence level was determined by curve smoothing in case of unclear fluorescent signal. An acceptable baseline fluorescent signal level was considered to be <1 amplification scale unit. Any fluorescent signal <1 unit was considered to be at an acceptable baseline level and thus negative.

Table 8

Stages of the Rodent Parvovirus PCR VeriQuest Triplex Program

Stage name Cycles Type of analysis Temperature Time Block tracking speed (°C/sec) Step size UNG 1 No 50 5 min 4,4 Preincubation 1 No 95 2 min 4,4 8 No 95 70 62 10 sec 30 sec 4.4 2.2 1°С/step Amplification -tion 40 Quantitative 95 62 10 sec 30 sec 4.4 2.2 Coolin 1 No 40 30 sec 2,2

Example 8: Test conditions with a significant result

In order for the test work to be considered significant, the following conditions must be met: The negative amplification control (NAC, i.e., water) must be negative for fluorescent signals in all three channels.

The negative extraction control (NEC, i.e., PBS) or cells in the negative control culture flasks should be negative for the fluorescent signal in the [533-58 0] channel (i.e., the signal of the VIC probe to rodent parvovirus), negative for the fluorescent signal in the [465-510] channel (i.e., the signal of the positive amplification control [PAC] antisense indicator-FAM probe), and positive for the fluorescent signal in the [618-660] channel (i.e., the signal of the CY5 probe to M13K07). PAC should be positive for fluorescent signals in all three channels. The Cp value of M13K07 in the NEC was used as a reference for assessing the presence of inhibitory substances in the sample.

If PCR was used as the endpoint of a cell culture stage test sample with a positive control for rodent parvovirus, the positive viral control should be positive for the fluorescent signal in the [533-580] channel (i.e., the signal of the VIC probe for rodent parvovirus), positive for the fluorescent signal in the [618-660] channel (i.e., the signal of the CY5 probe for M13K07), and negative for the fluorescent signal in the [4 65-510] channel (i.e., the signal of the PAC probe for antisense-FAM). The Cp C13K07 value in the cell culture positive control sample was expected to be within ±4 cycles of the Cp NEC-M13K value.

For all plasmid-containing RAS control reactions, fluorescent signals should be positive in all three channels.

Example 9: Test conditions with non-significant result

The assay was considered non-significant when any one or more of the following conditions were met: (1) very low amplification curve (< 1 fluorescence scale unit) for RAS, (2) confirmed significant error (hardware, software or human error), (3) NAC was positive in any of the three channels, (4) NEC was positive in amplification in the [533-58 0] VIC channel, positive in amplification in the [4 65-510] FAM channel or negative in amplification in the [618-660] Cy5 channel and (5) RAS was negative in amplification signal in any of the three channels. Examination and retesting of the test sample was performed whenever the assay was considered non-significant.

Example 10: Conditions for a negative sample result in a significant test

All of the following conditions must be met for a significant negative parvovirus test result. The specimen must be positive for M13K07 DNA amplification in the M13 [618-660] channel within the expected range of NEC-M13 Cp values + 4 cycles, assuming the absence of PCR inhibitors. The specimen must be negative for parvovirus DNA amplification in the [533-580] channel. The fluorescent signal must be less than 1 fluorescence scale unit, regardless of the magnitude of the

- 22 048105 ny Cp was considered as an acceptable baseline fluorescence level and was recorded as negative for parvovirus DNA amplification. The sample must be negative for the PAC amplification signal in the antisense indicator channel [465-510]. It should be noted that a fluorescent signal lower than 1 fluorescence scale unit, regardless of the Cp value (automatically generated by the instrument), was considered as an acceptable baseline fluorescence level and was recorded as negative for PAC plasmid DNA amplification.

Example 11: Conditions for a no sample result in a significant test

A sample missing result occurs when any one or more of the following conditions are met. Whenever a sample is negative for M13K07 DNA amplification signal in the Cy5 [618-660] channel, negative for parvovirus DNA amplification signal in the parvovirus [533-58 0] channel, and negative for PAC amplification in the antisense [465-510] channel, the sample or PCR reagent was tested according to the standard operating procedure. This condition suggests the presence of PCR inhibitors or failure of complete nucleic acid extraction. Samples may be diluted (1:2, 1:5, 1:10) to overcome inhibition. A Cp value for the fluorescent signal in the M13 [618-660] channel that is outside the range (NEC M13 Cp ± 4 cycles) indicates partial PCR inhibition or phage loading error and requires retesting. Sample dilution (1:2, 1:5, 1:10) can be considered to overcome any PCR inhibition. Any evidence of significant error or an unexpectedly very low fluorescent signal (< 1 fluorescence scale unit) observed in the M13 [618-660] channel that does not allow an unambiguous assessment of sample stability is considered to be a lack of sample and suggests an error in DNA amplification or extraction. This requires retesting.

Example 12: Condition for the result in a significant test on an initially out-of-specification sample (IOOS)

A sample was considered as IOOS when it was (i) positive for parvovirus DNA amplification signal in the parvovirus channel [533-580] (i.e., the VIC probe signal for rodent parvovirus) with a fluorescent signal greater than 1 fluorescence scale unit (in at least one of the two doublet wells), and (ii) negative for PAC amplification in the FAM channel of the antisense tracer [465-510], indicating that there was no cross-contamination from PAC. As a result, the specialist should (i) initiate a GLIF (global laboratory inspection), (ii) notify the unit that submitted the virological QC specimen for testing, (iii) initiate a NOE, (iv) save the amplification tube (freeze at -20°C) for further investigation (e.g. screening or indicator sequence sequencing), and (v) repeat the analysis and retest the specimen.

Example 13: Repetition and Retest Plan

A retest was initiated in all cases where the assay was insignificant or the sample result was considered as missing. A retest was performed to confirm a case of IOOS (i.e., the sample result was positive for the DNA amplification signal in the parvovirus channel [533-580] for the first time). Retesting should be performed using fresh aliquots of reagents (i.e., reagent master mix, extraction kit).

If NAC was fluorescence positive in any channel, the entire test panel should be repeated starting with the amplification (PCR) step using the same purified DNA samples and freshly prepared master mix. If NEC was fluorescence negative in the M13 [618660] channel, the entire test panel should be repeated starting with the DNA extraction step. If NEC was fluorescence positive in the parvovirus [533-580] channel or the antisense [465-510] channel, the entire test panel should be repeated starting with the DNA extraction step. If PAC was fluorescence negative in any channel, the test panel should be repeated starting with the amplification (PCR) step using the same purified DNA samples and freshly prepared master mix.

For samples that return a sample missing result, the test battery should be repeated for the affected samples, starting with the DNA extraction step. Samples may be diluted (1:2, 1:5 or 1:10) (in addition to the undiluted sample) prior to DNA extraction as part of the screening and problem solving process to demonstrate the presence of inhibitors.

A repeat test to confirm the initial positive result (iOOS) was performed using four separate aliquots as follows: two aliquots of the test item from the original sample source (e.g., one day after the final feed), and two aliquots of the test item from another sample collection (e.g., two days after the final feed, if possible) or from another set of samples. If any of the additional tests among the 4 aliquots yielded a positive signal in the absence of evidence of significant error (demonstrated by testing), the lot was considered non-compliant for the absence of rodent parvovirus viral genomic material. The infectivity assay served as the basis for the final decision on such a positive Q-PCR result. CHO-K1 cells were used as an indicator cell line to determine the infectivity status

- 23 048105 detected nucleic acid.

Whenever a repeat test result is negative, a confirmatory test under different sample collection conditions (e.g., three days after the final feed) is required to confirm the absence of rodent parvovirus genomic material.

Example 14: Retest plan for other sample types with IOOS

Other sample types, including, for example, bulk unprocessed material, cells at the end of the production run, cells at the limit of in vitro cell age for production, and soy material that was initially positive (iOOS), were retested as follows using four separate aliquots.

Two aliquots of the test item from the original sample container (i.e., e.g., bag) and two aliquots from another sample container were retested according to standard operating procedures. CHO-K1 indicator cells were inoculated with one aliquot of the test item sample from the original sample container. The inoculated CHOK1 indicator cells were collected after 1-3 days of culture according to standard operating procedures to determine the infectivity status of the detected nucleic acid. A standard curve for quantification of the re-extracted nucleic acid may be used.

Whenever additional PCR testing of four aliquots is positive without evidence of significant error (demonstrated by the assay), the infectivity assessment determines the final decision on the item. Once the initial OOS is confirmed, nucleic acid sequencing and transmission electron microscopy (TEM) can resolve the issue of identifying the organism and ruling out any laboratory error.

Evaluation of the infectivity of CHO-K1 as an indicator cell for suspected contaminated material is required to determine the infectivity of the nucleic acid detected. Whenever the assay failed to confirm the possibility of parvovirus contamination and all additional PCR tests of four aliquots of the test article and the CHOK1 culture were negative, the lot was considered to be free of infectious rodent parvovirus.

Example 15: Parvovirus testing during recombinant protein production

The Q-PCR procedure described above was performed as a critical in-process control on cell culture fluid testing, i.e., unprocessed bulk materials from bioreactor production containing CHO cell derivatives containing a heterologous antibody heavy chain and light chain construct. Each heterologous monoclonal antibody (mAb) binds to different targets or epitopes. Good Manufacturing Process (GMP) checks were performed by a virology quality control scientist at a large-scale bioprocess manufacturing facility. Sixteen (16) of these tests are listed in Table 9. In each case, test panel results were significant, assay stability criteria were met, and no false positive detections were observed. Test panel #13 and #14 experienced false negative detections, suggesting the presence of PCR inhibitors or a nucleic acid extraction defect.

Table 9

pcr tests for rodent parvovirus

Test Sample type Product Party Testing 1 UPB EA mAbl '51 Parvo PCR Rodents 2 UPB mAbl ' 52 Parvo PCR Rodents 3 UPB tAЬZ '07 Parvo PCR Rodents 4 UPB mAb I '51 Parvo PCR Rodents 5 UPB EA mAb2 '35 Parvo PCR Rodents 6 UPB EA mAb4 '05 Parvo PCR Rodents 7 UPB tAЬ2 '36 Parvo PCR Rodents

- 24 048105

8 UPB mAbll '53 Parvo PCR Rodents 9 UPB mAbl '53 Parvo PCR Rodents 10 UPB EA shaЬZ '08 Parvo PCR Rodents 11 UPB EA шАЬ5 '01 Parvo PCR Rodents 12 UPB EA mAbl '54 Parvo PCR Rodents 13 UPB shabb '01 Parvo PCR Rodents 14 UPB tAЬ4 ' 05 Quantitative Analysis of Parvo PCR in Rodents 15 UPB tAЬ2 '35 Quantitative Analysis of Parvo PCR in Rodents 16 UPB шАЬ2 '36 Quantitative Analysis of Parvo PCR in Rodents

rB=raw bulk material; EA=early warning; Rodent Parvo PCR=Q-PCR procedure described above.

Claims (8)

1. A composition for use as a positive control for quantitative polymerase chain reaction (Q-PCR) in the examination of a test sample for parvovirus contamination, comprising a) a positive amplification control (PAC) plasmid comprising: (i) a parvovirus nucleotide sequence comprising a parvovirus NS-1 sequence having at least 88% identity to any of the sequences of SEQ ID NOs: 12-36, 97% identity to the sequence of SEQ ID NO: 9, or comprising the sequence of SEQ ID NO: 37, (ii) a nucleotide sequence of bacteriophage M13 (M13) SEQ ID NO: 8, and (iii) an artificial nucleotide sequence, wherein the artificial nucleotide sequence comprises a sequence of 17-20 nucleotides, wherein no more than 7-10 internal consecutive nucleotides and no more than 6 consecutive 3'-nucleotides are identical to any of the parvovirus sequences represented by SEQ ID NOs: 9 and 12-37; b) direct oligonucleotide primer specific for rodent parvovirus; c) an oligonucleotide detection probe specific for rodent parvovirus; d) an artificial oligonucleotide detection probe capable of hybridizing with an artificial nucleotide sequence; e) reverse oligonucleotide primer specific for rodent parvovirus; f) forward oligonucleotide primer specific for M13; g) an oligonucleotide detection probe specific for M13; h) and a reverse oligonucleotide primer specific for M13; and i) buffer; wherein the rodent parvovirus-specific primers and the detection probe hybridize to the NS-1 sequence, and wherein the M13 primers and probe hybridize to the sequence of SEQ ID NO: 8. 2. The composition according to claim 1, wherein the NS-1 sequence of the parvovirus is selected from the group consisting of the prototypic strain of small mouse virus (MVMp), the immunosuppressive strain of small mouse virus (MVMi), the Cutter strain of small mouse virus (MVMc); mouse parvovirus 1b (MPV1b), mouse parvovirus 1a (MPV-1a), mouse parvovirus 1c (MPV-1c), hamster parvovirus (HaPV), Tulane parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a, small rat virus and the University of Massachusetts strain of rat virus L (RV-Umass). 3. The composition according to claim 1, wherein the artificial oligonucleotide detection probe comprises a nucleotide sequence that has no more than five consecutive nucleic acids at its 3' end that are identical to any sequence from SEQ ID NO: 9 and 12-37. 4. The composition according to claim 1, wherein the oligonucleotide sequences according to b, c and e hybridize with SEQ ID NO: 37. 5. The composition of claim 1, wherein each detection probe according to c, d and g comprises a fluorophore and a quencher such that the fluorophore of each detection probe emits light at different wavelengths. - 25 048105 6. The composition according to claim 1, wherein the nucleotide sequence of the parvovirus comprises the sequence of SEQ ID NO: 37, and the artificial nucleotide sequence comprises the sequence of SEQ ID NO: 10. 7. The composition according to claim 6, wherein the plasmid (PAC) contains the nucleotide sequence SEQ ID NO: 11. 8. The composition according to claim 7, wherein the direct oligonucleotide primer specific for rodent parvovirus comprises the nucleotide sequence SEQ ID NO: 1; an oligonucleotide detection probe specific for rodent parvovirus comprises a VIC fluorophore, a non-fluorescent quencher - minor groove binding protein (MGBNFQ), and the nucleotide sequence of SEQ ID NO: 2; the artificial oligonucleotide detection probe comprises the VIC fluorophore, the non-fluorescent quencher BHQ and the nucleotide sequence of SEQ ID NO: 3; the reverse oligonucleotide primer specific for rodent parvovirus contains the nucleotide sequence of SEQ ID NO: 4; a forward oligonucleotide primer specific for M13 comprising the nucleotide sequence of SEQ ID NO: 5; an oligonucleotide detection probe specific for M13 comprises a Cy5 fluorophore, a BHQ-2 quencher and the nucleotide sequence of SEQ ID NO: 6; and a reverse oligonucleotide primer specific for M13 comprises the nucleotide sequence of SEQ ID NO: 7.