Biologicals xxx (2015) 1e10
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Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation Mohammad Anwaruzzaman, Wensheng Wang, Eunice Wang, Lolita Erfe, Janice Lee, Shengjiang Liu* Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2014 Received in revised form 17 January 2015 Accepted 5 April 2015 Available online xxx
Infectivity and reverse transcriptase quantitative real-time polymerase chain reaction (qRT-PCR) assays have been optimized and validated for xenotropic murine leukemia virus (X-MuLV) detection. We have evaluated the assays systematically with regard to specificity, linearity, lower limit of detection (LLOD), lower limit of quantification (LLOQ), and precision. Both assays are specific for X-MuLV detection, with a linear detection range of 0.6e5.6 log10 TCID50/mL for the infectivity assay, and 1.4e6.5 log10 particles/mL for the qRT-PCR assay. The LLOD and LLOQ of the infectivity and the qRT-PCR assays are determined as 0.5 and 1.0 log10/mL, and 1.4 and 2.2 log10/mL. The inter-assay repeatability of qRT-PCR assay (4.2% coefficient of variation [CV]) is higher than the infectivity assay (7.9% CV). We have shown that both assays are closely correlated (r ¼ 0.85, P < 0.05, n ¼ 22). The particle/infectivity ratio is determined as 66. Both assays were applied to evaluate virus removal using virus clearance samples of chromatographic and filtration processes. Here, we have demonstrated that the qRT-PCR assay is much faster in testing and is approximately 8-fold more sensitive than the infectivity assay. Therefore, the qRT-PCR assay can replace the infectivity assay in many cases, but both assays are complementary in elucidating the mechanism of virus inactivation and removal in virus clearance validation. © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Reverse transcriptase real-time PCR assay Infectivity assay Xenotropic murine leukemia virus Virus clearance Chromatography Virus filtration
1. Introduction Genetically engineered rodent cells, such as Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells, are commonly used to produce recombinant proteins for human therapeutic use in the biopharmaceutical industry. These cells have been demonstrated to contain an intrinsic proviral genome that produces endogenous retrovirus-like particles (RVLPs) [1e6]. Two types of particles are consistently observed in CHO cells: intracytoplasmic type A particles, frequently associated with centrioles, and budding type C particles [4]. Type R particles are observed in BHK cells [5,6]. Based on the available information, none of these endogenous RVLPs is infectious [1e3,7]. However, because of the morphologic and biochemical resemblance to tumorigenic retroviruses, RVLPs * Corresponding author. Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, 800 Dwight Way, Berkeley, CA 94710, USA. Tel.: þ1 510 705 5506; fax: þ1 510 705 4557. E-mail address: [email protected] (S. Liu).
may pose a theoretical safety risk to biologics produced in CHO or BHK cells. Therefore, the detection of endogenous RVLPs in cell culture production and the removal and inactivation of endogenous RVLPs in the subsequent downstream purification process are required by regulatory agencies [8,9]. Because RVLPs are noninfectious, an infectivity assay cannot be used directly to detect these particles. Real-time polymerase chain reaction (RT-PCR) assay has been used for the quantification of the RVLP from CHO cells [10]. In the biopharmaceutical industry, xenotropic murine leukemia virus (X-MuLV) is commonly used as a specific model virus in evaluating the removal of RVLP by purification processes. The X-MuLV is an enveloped virus belonging to the Retroviridae family [9]. The viral genome is a single-stranded, positive-sense RNA molecule. Each viral genome contains 2 copies of identical RNA molecules [11]. From 50 to 30 , the genome contains the gag, pol, and env regions: coding for structural proteins; enzymes, including the RNA-dependent DNA polymerase (reverse transcriptase); and coat proteins, respectively [11]. The virus is sensitive to physicochemical treatment, such as heating, drying, low pH, and detergent [9].
http://dx.doi.org/10.1016/j.biologicals.2015.04.001 1045-1056/© 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
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Infectivity (or the 50% tissue culture infectious dose [TCID50]) assay is typically used to quantify infectious virus in the process sample. However, the TCID50 assay is labor intensive and time consuming. The TCID50 assay cannot be used alone to distinguish clearance by virus removal versus inactivation, limiting its ability to meet the increasing demands of the biopharmaceutical industry in virus clearance validation. In virus titration assays, virus is detected and quantified only when the virus level in the sample is at or above the lower limit of detection (LLOD) and lower limit of quantification (LLOQ), respectively. Virus titers are normally expressed with 95% confidence limits, which should not exceed 0.5 log10 of the expected titer [9]. The metrics for quantifying virus in biologics process samples is to report virus quantity in log10/mL when the virus levels in samples are greater than the LLOD. When virus levels in samples are less than the LLOD, then less than LLOD is taken as the assay result. Here we present optimization and validation of cell-based TCID50 and quantitative RT-PCR (qRT-PCR) assays for detection and quantification of X-MuLV to evaluate virus clearance validation. In the TCID50 assay, PG-4 cells were used as indicator cells, and the quantification of virus was performed by measuring the cytopathic €rber equation [12,13]. The qRTeffect (CPE) using the SpearmaneKa PCR assay was based on the 50 to 30 exonuclease activity of Taq DNA polymerase using env geneespecific primers and a dual-labeled probe [14e18]. The amount of X-MuLV particle was quantified from the virus RNA copy number. The qRT-PCR assay offers great advantages over the TCID50 assay, such as higher sensitivity and throughput, greater reliability, and reduced cost. Both the TCID50 and qRT-PCR assays were applied to evaluate X-MuLV removal by protein A column chromatography, membrane adsorption anion exchange (MA-AEX), and virus filtration processes of recombinant human therapeutic antibodies.
2. Materials and methods 2.1. Reagents All chemicals and reagents were of the highest molecular biology grade. Stock solutions were made in RNase/DNase-free sterile water. Elim Biopharmaceuticals, Inc. (Hayward, CA) synthesized oligonucleotides of desaltation grade. The dual-labeled qPCR probe contained 50 6-carboxy-fluorescein (FAM) and 30 Black Hole Quencher (BHQ) and was synthesized from Eurofins MWG/Operon (Huntsville, AL) and Biosearch Technologies (Novato, CA). The probe was high-performance liquid chromatography (HPLC) purification grade. Fisher Scientific (Pittsburgh, PA) supplied cell culture media used in the infectivity assay and bovine serum albumin (BSA) used in the qRT-PCR assay. Viral RNA purification spin columns and reagents including carrier RNA (poly A) were purchased from Qiagen (QIAamp Viral RNA Kit; Valencia, CA). Brilliant® QRT-PCR 2 Master Mix 1-Step Kit including reverse transcriptase/RT-block mix was purchased from Agilent Technologies (formerly Stratagene; Santa Clara, CA). Amplified PCR product from the env gene was purified using QIAquick Gel Extraction Kit (Qiagen) and cloned using the Strataclone™ PCR cloning kit (Agilent Technologies). The Easy-A® PCR High-Fidelity Master Mix Kit (Agilent Technologies) was used to screen orientation of the cloned insert. Immedia™Amp liquid medium used for bacterial growth was purchased from Life Technologies (formerly Invitrogen; Grand Island, NY). Plasmid was purified from the bacterial culture using the QIAFilter™ Plasmid Midi Kit (Qiagen). Promega RiboMax™ Large Scale RNA Production Systems (Madison, WI) was used to synthesize standard RNA. RNA was purified using RNeasy Mini Kit (Qiagen).
2.2. Cells The PG-4 indicator cell line (Felis catus, SþL, ATCC CRL-2032) was used in the X-MuLV infectivity assay. Additionally, the specificity Vero cell line (kidney, African Green Monkey, ATCC CCL-81) and the PK13 cell line (pig, Sus scrofa, ATCC CRL 6489) were used in the infectivity assay. 2.3. Virus Virus stocks (strain: PNFS Th-1) used were purchased from BioReliance (Rockville, MD) and produced internally at Bayer (Berkeley, CA). 2.4. Infectivity assay X-MuLV was quantified by the TCID50 assay in the PG-4 indicator cell line over 6e7 days; cells inoculated with medium (no virus) remained healthy and viable. PG-4 cells were seeded in 96-well microplates at a cell density of 3000 cells/well in McCoy's 5A medium containing 10% fetal bovine serum (FBS), 100 mg/mL penicillin/streptomycin, and 2 mM Lglutamine. Cells were incubated overnight in a humidified incubator at 37 C with 5% CO2. The following day, each virus-containing sample was serially diluted (1:3.2) in 11 wells in a dilution block with McCoy's 5A medium containing 3 mg/mL polybrene (the undiluted sample [1:3.20] through the eleventh dilution [1:3.210]). Then, 100 mL of each set of 11 serially diluted samples were inoculated in each well of the first 11 columns in a 96-well assay plate. The last (twelfth) column of the same assay plate was inoculated with the diluent medium, which served as the in-plate control. The control and diluted samples were inoculated in 8 replicate wells (row 1 through row 8) on each plate. TCID50 assay-positive control plates were also prepared from prediluted stock virus at the beginning and end of the assay. The prediluted virus was serially diluted in dilution blocks using McCoy's 5A medium containing polybrene as described above. The inoculated plate was incubated in a 37 C humidified CO2 incubator for 2.0 ± 0.5 h. Then, 100 mL of 2 assay medium (McCoy's 5A medium, 4% FBS, 200 mg/mL penicillin/streptomycin, and 4 mM L-glutamine) was added to each well on the plate. The plate was returned to the incubator for 6e7 days. Each well was scored for the cytopathic effect caused by virus infection under a light microscope, and virus was quantified as log10 TCID50/mL using the Microsoft Office Excel® worksheet according to SpearmaneK€ arber equation [12,13]. Hereafter, the unit for virus titer of the infectivity assay is simplified as log10/mL. For each sample the final virus titer (TCID50/mL) was calculated from the log10 titer, the sample volume per well (eg, 0.1 mL), and the sample predilution factor. The 95% confidence limit was calculated as ±1.96 SD. 2.5. Quantitative RT-PCR system 2.5.1. Standard X-MuLV RNA preparation The standard X-MuLV RNA was a 1.85-kb in vitro transcript generated from a plasmid harboring the X-MuLV env gene cDNA sequences using the in vitro transcription kit. X-MuLV virus stock (BioReliance) was used to extract and purify viral RNA using the QIAamp Viral RNA mini kit. RNA was quantified by UV spectrophotometry at A260. cDNA was made from the purified RNA (50 ng) using the Brilliant® QRT-PCR Master Mix 2-Step Kit (Agilent Technologies). Primers used in cDNA preparation were X-MuLV-AF (19-mer, 84e102 nucleotides [nt] [50 d(GGC AGG AGC CTC GGT ACA A)-30 ]) and X-MuLV-B-R (19-mer, 1810e1828 nt [50 d(CCA AGC GGT TGA GAA TGC A)-30 ]). These primers are located 1745
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
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nucleotides apart on the env gene sequences. The PCR product band detected around 1.7 kb on a 1% agarose gel was purified using the QIAexII kit and cloned using the Strataclone™ PCR cloning kit. After transformation into bacteria, colonies containing the X-MuLV insert in the correct orientation were screened by PCR using the kit Easy-A® PCR High-Fidelity Master Mix and primers, M13For (50 d(GTA AAA CGA CGG CCA GT)-30 ) and X-MuLV-B-R. The thermocycling conditions used were: 35 cycles at 95 C for 30 s, 55 C for 30 s, and 72 C for 2 min. The resulting plasmid, pXMULV-A7, was purified from the selected bacterial clone using the QIAFilter™ Plasmid Midi Kit. The plasmid sequence was then confirmed by sequencing for the env gene insert at Elim Biopharmaceuticals using M13For primer (see above). The sequence was analyzed using the software Lasergene 7 (DNASTAR, Inc., Madison, WI). To ensure the proper termination of the RNA transcript, the plasmid was linearized with Hind III. The digestion mixture was then run on a 1% agarose gel and the linearized plasmid (5.2 kb) was purified using QIAquick Gel Extraction Kit. In vitro transcription was then performed on the purified linear plasmid DNA using the RiboMax™ Large Scale RNA Production Systems. The resulting transcribed RNA was 1850 bases long comprising 1745 bp of the insert plus 105 bp of adjacent plasmid sequences between the T7 transcription start and the Hind III site. RNA was then purified using RNeasy Mini Kit and quantified by UV spectrophotometry at A260 nm. RNA copies/mL was calculated using the following formula: N (copies/mL) ¼ [C (RNA concentration in ng/mL) 109 Avogadro's constant (6.023 1023)]/[K (fragment size in bases, 1850 bases) average molecular weight of RNA base (339.5 Da)]. Serial dilutions of the standard RNA were performed to prepare a set of different copy numbers of standard RNA and were stored at e65 C. A standard curve was prepared using 5 mL of standard RNA (ranging from ~1.6 100 to ~1.6 108 copies). 2.6. RNA extraction and purification method Xenotropic murine leukemia virus genomic RNA was extracted and purified by a modified spin column procedure [19] using the QIAamp viral RNA kit. To 140 mL X-MuLV containing sample, 560 mL of AVL virus lysis buffer containing 1 mg/mL of BSA and 10 mg/mL carrier RNA was added, mixed by inversion, and kept at room temperature. Then 560 mL of 96e100% ethanol (RNase/DNase-free) was added and mixed well. Viral RNA was purified by loading the mixture onto a spin column. Viral RNA along with carrier RNA was bound to the column by centrifugation at 8000 rpm for 1 min in a microcentrifuge. The spin column was washed with 500 mL washing buffer (AW1) by centrifugation at 8000 rpm for 1 min. A second wash of the spin column with 500 mL washing buffer (AW2) was performed at 14,000 rpm for 3 min. The spin column was again spun at 14,000 rpm for 1 min to completely remove any residual wash buffer. Viral RNA and carrier RNA were then co-eluted into a sterile microcentrifuge tube using 100 mL of AVE buffer, equilibrated to room temperature and centrifuged at 8000 rpm for 1 min.
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2.8. qRT-PCR assay The qRT-PCR assay was performed in a 96-well plate format (Agilent Technologies or Life Technologies). Each well (20 mL/reaction) consisted of 5 mL unknown sample or standard RNA solution (ranging from approximately 100e108 copies/reaction), 10 mL Brilliant® II/III QRT-PCR 2 Master Mix 1-Step Kit containing 500 nM forward primer, 500 nM reverse primer, 100 nM probe and reverse transcriptase/RNase block mixture. The assays were initiated first with reverse transcription at 50 C for 30 min then enzyme activation at 95 C for 10 min. This was followed by 40 cycles of denaturation at 95 C for 20 s, annealing at 48 C for 20 s, and extension at 60 C for 20 s. All reactions were performed on Mx3005P quantitative qPCR instrument (Agilent Technologies). On each qRT-PCR 96-well plate, triplicate controls of no template and standard RNA were used along with unknown samples in triplicates. The qPCR product (103 bp) was verified on a 4% agarose gel for each assay (data not shown). The copy number of RNA in unknown samples was determined from the standard curve using the MxPro software version 4.10 (Agilent Technologies) and viral particles were calculated by dividing the copy number by 2. Virus titer of the qRT-PCR assay is determined as log10 particles/mL. Hereafter, the unit for virus titer of the qRT-PCR assay is simplified as log10/ mL. 3. Results 3.1. Optimization of X-MuLV TCID50 and qRT-PCR assays The TCID50 assay was optimized for culture medium, cell passage number, seeding cell density, and post virus-infection incubation time (data not shown). The assay sensitivity was improved by the addition of polybrene to the McCoy's 5A virus dilution medium. Fig. 1 shows that virus titer was 6.7 ± 0.3 log10/mL when no polybrene was added to the diluent medium. When the diluent medium had 3 mg/mL polybrene, the sensitivity of the assay increased by 25-fold. The measured titer with the polybrene was 8.1 ± 0.8 log10/mL. These results indicate that polybrene concentration at 3 mg/mL was important for higher sensitivity of the XMuLV TCID50 assay.
2.7. qRT-PCR primers and probe Two primers (forward and reverse) and the probe were designed from the env gene sequences of X-MuLV. The forward primer, XMuLV-A-Fwd6, was an 18-mer, 85e102 nt [50 -d(GCA GGA GCC TCG GTA CAA)-30 ]; the reverse primer, XMuLV-A-Rev3, was a 21-mer, 167e187 nt [50 -d(GGA GGG AGG TAG CGT TAG CTG)-30 ]. The probe was a 26-mer, 105e130 nt [50 -d((FAM)TGA CAG CCC TCA CCA GGT CTT CAA TG(BHQ))-30 ]. A combination of the forward and reverse primers in qRT-PCR produced a 103-bp fragment.
Fig. 1. Effect of polybrene on sensitivity of the X-MuLV TCID50 assay. Stock X-MuLV was prediluted in McCoy's 5A medium with or without 3 mg/mL polybrene. The prediluted virus was then serially diluted (1:3.2) in 11 wells in a dilution block with McCoy's 5A medium with or without 3 mg/mL polybrene. The TCID50 assay was then performed with these diluted virus samples using preseeded PG-4 cells in 96-well plates (see Materials and methods). TCID50 ¼ 50% tissue culture infectious dose; XMuLV ¼ xenotropic murine leukemia virus.
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The qRT-PCR assay system was optimized for spin column RNA purification and qRT-PCR steps (data not shown). Stability of standard RNA for quantification of viral RNA copy numbers in unknown samples was also optimized. Stability of the standard RNA was evaluated by amplification and efficiency of qRT-PCR assay. The range of standard RNA copy numbers used was from ~2.7 10 0 to ~2.7 108 copies/reaction. Fig. 2 shows that the lowest RNA levels for which qRT-PCR amplification (cycle threshold [Ct] value) was observed were 2.7 103, 2.7 102, and 2.7 101 copies/reaction for standard RNA in water, water with 1 mg carrier RNA/reaction and AVE buffer with 1 mg carrier RNA/reaction, respectively. These results indicate that stability of standard RNA was higher in AVE buffer plus 1 mg carrier RNA/reaction than in water or water with carrier RNA. A linear regression curve was generated for each standard RNA set by plotting Ct values against the standard RNA copy number (Fig. 2). The regression coefficient R2 value of the standard curve for RNA in water was slightly lower (0.976) than that of standard RNA set with carrier RNA in water (0.999) or AVE buffer (0.998). Efficiency of the qRT-PCR reaction for each set of standard RNA was calculated from the slope of the standard curve as [10(1/slope) 1]. The qRT-PCR efficiency was significantly lower for RNA in water (78%) than the RNA with carrier RNA in water (107%) or AVE buffer (108%; Fig. 2). These results further indicate that the carrier RNA was important for stability of standard RNA and efficiency of qRT-PCR. Therefore, buffer AVE plus 1 mg carrier RNA/reaction was used as diluent of standard RNA preparation. An aliquot of qRT-PCR assay mixtures from the standard RNA and purified viral genomic RNA was analyzed on a 4% agarose gel electrophoresis. Both viral standard RNA and genomic RNA yielded a 103-bp DNA product (data not shown). Recovery of virus RNA by the spin column purification method was evaluated by comparing viral RNA of a sample in the spin column eluate to the viral RNA in a sample directly disrupted with AVL solution. AVL solution had no inhibitory effect on the RT and PCR at 100-fold dilution with water (RNase/DNase-free) containing 700 units SUPERaseIn™ per mL (Life Technologies) and 10 mg/mL carrier RNA. Virus titers in the spin column eluate and AVLdisrupted samples were 7.70 ± 0.17 and 7.50 ± 0.06 log10/mL, respectively (data not shown), suggesting that there was no difference in RNA between the spin column eluate and the
Fig. 2. Effect of carrier RNA on stability of standard RNA. Stock standard RNA was diluted to make standards 2.7 100, 2.7 101, 2.7 102, 2.7 103, 2.7 104, 2.7 105, 2.7 106, 2.7 107, and 2.7 108 copies/reaction in RNase/DNase-free water, water with 1 mg carrier RNA/reaction, and buffer AVE with 1 mg carrier RNA/reaction. Subsequently, qRT-PCR was performed on these standards. Linear regression curves were made for these standards, and qPCR efficiency was calculated from the slope as described in Materials and methods. Ct ¼ cycle threshold; CR ¼ carrier RNA; qRT-PCR ¼ reverse transcriptase quantitative real-time polymerase reaction; R2 ¼ regression coefficient.
AVL-disrupted samples. The AVL solution contained 10 mg/mL carrier RNA or 5.6 mg carrier RNA in 560 mL extraction mixture. Considering full recovery of RNA by the spin column procedure, the estimated carrier RNA in 100 mL viral RNA would be approximately 5.6 mg. To evaluate if the carrier RNA had any influence on stability of the purified X-MuLV RNA, viral RNA was purified from 4 virus samples containing different virus amounts in triplicate for each sample. Purified viral RNA was stored at e65 C. The following day (day 1), qRT-PCR assay was performed in duplicate for each RNA sample. Subsequent qRT-PCR assays were performed on days 7, 14, and 21. Average virus titer was calculated for these qRT-PCR samples and plotted against the days stored at e65 C (Fig. 3). The quantity of viral RNA of all 4 samples measured during the storage period (21 days at e65 C) did not change, indicating that the purified viral RNA was stable under those conditions. The maximum 95% confidence limit (CL) was 0.67 log10 and the percentage coefficient variation (CV) for each virus sample for each day of qRT-PCR was only 4.6%. 3.2. X-MuLV TCID50 and qRT-PCR assay specificity Specificity of the X-MuLV TCID50 assay was evaluated using PK13, Vero, and PG-4 indicator cells and corresponding growth media for these cells. No growth of X-MuLV (4 log10-fold diluted stock virus) was observed on plates seeded with PK13 or Vero indicator cells growing in the corresponding growth media. Growth of X-MuLV was observed on plates seeded with PG-4 cells. The measured average infectivity titer in 4 log10 diluted virus sample was 4.09 ± 0.32 log10/mL. These results indicate the high specificity of X-MuLV growth and replication in PG-4 cells (Table 1). Specificity of the X-MuLV qRT-PCR assay was also evaluated using stock pseudorabies virus (PRV), porcine parvovirus (PPV), Reo 3 virus, and 5.53 log10-diluted stock X-MuLV. These virus samples were diluted 1:20 in phosphate-buffered saline during extraction. Nucleic acids of these lysed viruses were then purified according to the spin column method, and the qRT-PCR assay was performed as described above. Table 1 shows that no amplification (no Ct) of nucleic acids from PPV, PRV or Reo 3 virus was observed by the qRTPCR assay. Amplification was observed only with X-MuLV RNA. The
Fig. 3. Stability of purified X-MuLV RNA at e65 C. X-MuLV RNA was extracted and purified from triplicates of each of 4 virus samples containing different virus amounts according to the procedure described above. Purified RNA was stored at e65 C. The following day (day 1), the qRT-PCR assay was performed in duplicate for each RNA sample. Subsequent qRT-PCR assays were performed with RNA purified in days 7, 14 and 21. RNA copies of these samples were determined from the standard curve and log10 particles/mL virus titers were calculated. Average virus titer was calculated for these samples and plotted against the days stored at e65 C. X-MuLV ¼ xenotropic murine leukemia virus; qRT-PCR ¼ reverse transcriptase quantitative real-time polymerase reaction.
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
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Table 1 Specificity evaluation for X-MuLV TCID50 and qRT-PCR assays. TCID50 assay
qRT-PCR assay
Indicator cells
Number of replicate assay plates
Average TCID50 (Log10/mL ± 95% CL)
Virus
Number of replicate assays
Average particle (Log10/mL ± Max 95% CL)
PG-4 PK13 Vero
3 3 3
4.09 ± 0.32a 0.75a,b 0.75a,b
X-MuLV PPV PRV Reo 3
3 3 3 3
3.89 ± 0.20c 1.4b 1.4b 1.4b
CL ¼ confidence limit; CPE ¼ cytopathic effect; Ct ¼ cycle threshold; PPV ¼ porcine parvovirus; PRV ¼ pseudorabies virus; qRT-PCR ¼ reverse transcriptase quantitative realtime polymerase reaction; TCID50 ¼ 50% tissue culture infectious dose; X-MuLV ¼ xenotropic murine leukemia virus. a Predilution factor of stock viruses in TCID50 was 4 log10. b No CPE in TCID50 assay and no Ct in qRT-PCR assay was observed. c Predilution factor of stock viruses in qRT-PCR assays was 5.53 log10.
average measured titer in the 5.53 log10-diluted X-MuLV sample was 3.89 ± 0.20 log10/mL. These results indicate that the qRT-PCR assay was highly specific for X-MuLV. 3.3. X-MuLV TCID50 and qRT-PCR assay linearity The X-MuLV TCID50 and qRT-PCR assay linearity was evaluated using virus samples prepared from serial dilutions of X-MuLV stocks. Fig. 4 shows that in both the TCID50 (Fig. 4A) and qRT-PCR
(Fig. 4B) assays, there was a linear relationship between virus titers of diluted virus samples and corresponding dilution factors used to prepare the virus samples. For the TCID50 assay, linearity was observed with an R2 value of 0.996 for virus titers ranging from 0.6 to 5.6 log10/mL; for the qRTPCR assay, linearity was observed with an R2 value of 0.999 for virus titers ranging from 1.4 to 6.5 log10/mL. The CV of intra-assay was in the range of 2.9e29% in the TCID50 assay and 0.9e19.2% in the qRTPCR assay. In both assays the percentage CV was higher for low titer virus samples than for mid-level virus titers. The maximum 95% CL observed was 0.73 in the TCID50 assay and 0.47 in the qRT-PCR assay. 3.4. X-MuLV assay limit of detection and quantification
Fig. 4. Linearity of (A) TCID50 and (B) qRT-PCR assays. X-MuLV stock was diluted serially in virus suspension buffer, TNEB (50 mM TriseHCl, pH 7.2, 100 mM NaCl, 5 mM EDTA, and 1 mg/mL BSA). For the TCID50 assay, virus stock (average titer, 8.68 log10 TCID50/mL) was diluted 3, 4, 5, 6, 6.5, 7, 7.5, and 8 log10 fold. The TCID50 assay was performed on these samples using PG-4 cells and virus titers in log10 TCID50/mL were calculated from the cytopathic scores. For the qRT-PCR assay, 2 different virus stocks (average titer, 9.68 log10 particles/mL) were diluted 1.6, 2.6, 3.6, 4.6, 5.6 and 6.6 log10 fold. Viral RNA of these samples was purified and qRT-PCR assay was performed on each RNA sample. RNA copy number and viral titer in log10 particles/mL were calculated. Average virus titers were calculated for each dilution level of the 2 virus stocks. Subsequently, linear regression curves were plotted for both assay virus titers against the log10 dilution factor. BSA ¼ bovine serum albumin; qRT-PCR ¼ reverse transcriptase quantitative real-time polymerase reaction; R2 ¼ regression coefficient; TCID50 ¼ 50% tissue culture infectious dose; X-MuLV ¼ xenotropic murine leukemia virus.
The LLOD and LLOQ of the X-MuLV TCID50 and qRT-PCR assays were evaluated using diluted virus samples prepared by serial dilutions (1:3.2 for the TCID50 assay and 1:10 for the qRT-PCR assay) of high-titer X-MuLV. The LLOD of the TCID50 assay was determined based on the criterion that all replicate assay measurements have positive scores. The criterion for LLOD of the qRT-PCR assay was the lowest point (highest dilution) that gives a qRT-PCR signal (Ct value) but is no longer colinear with the other dilutions or the lowest point that each replicate RNA has at least one qRT-PCR signal. The LLOQ for both assays was determined based on the criteria that all replicate assay measurements have positive scores (TCID50 assay) or Ct values (qRT-PCR assay) and that the measured average virus titer not be more than 0.5 log10 from the expected titer. The expected titer was chosen based on the virus titers obtained from dilutions within the linearity range and was calculated from the average measured log10 titer of the mid-range dilution sample in triplicate and the corresponding dilution factor. Results of LLOD and LLOQ for TCID50 and qRT-PCR assays are summarized in Table 2. In the TCID50 assay, the virus samples at dilution levels of 7.5, 8.0, and 8.5 log10 had positive cytopathic scores for all replicates. However, the maximum 95% confidence limit and percentage CV values for these samples were higher than expected (Table 2). Therefore a conservative approach was taken considering the limited replicates of each dilution level, the high 95% CL and percentage CV for the measured titers of the highest dilution levels. The LLOD and LLOQ for the TCID50 assay were determined to be 0.5 log10 and 1.0 log10/mL, respectively. Therefore, the TCID50 assay can detect 2.53 TCID50 or 0.4 log10 TCID50. The observed CV for 7.5 and 8.0 log10 dilution levels were 25.9% and 29%, respectively. The TCID50 titration assay is not a quantitative assay, it is an estimated assay. Usually high-percentage CV is observed for low and very high dilutions of virus samples with this assay (19). The expected virus titer in undiluted virus stock was 8.68 log10/mL. The expected virus
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
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Table 2 LLOD and LLOQ of X-MuLV TCID50 and qRT-PCR assays. TCID50 assay
qRT-PCR assay
Dilution factor (Log10)
Measured TCID50 (Log10/mL ± Max 95% CL)
6.5 7.0 7.5 8.0 8.5 9.0
2.14 1.42 0.87 0.64 0.03 N/A
± ± ± ± ±
0.26 0.36 0.51 0.73 1.22
Expected TCID50 (Log10/mL)
Max % CV
LLOD, LLOQ (Log10/mL)
Dilution factor (Log10)
Measured particles (Log10/mL ± Max 95% CL)
2.18 1.67 1.17 0.66 0.16 0.35
3.0 10.5 25.9 29.0 0.0 N/A
0.5, 1.0
2.6 3.6 4.6 5.6 6.6 7.6
5.40 4.19 3.20 2.23 1.36 N/A
± ± ± ± ±
0.09 0.08 0.11 0.37 0.47
Expected particles (Log10/mL)
Max % CV
LLOD, LLOQ (Log10/mL)
5.45 4.45 3.45 2.45 1.45 0.45
0.9 1.0 1.8 8.2 19.2 N/A
1.4, 2.2
CL ¼ confidence limit; CV ¼ coefficient of variation; LLOD ¼ lower limit of detection; LLOQ ¼ lower limit of quantification; qRT-PCR ¼ reverse transcriptase quantitative realtime polymerase reaction; TCID50 ¼ 50% tissue culture infectious dose; X-MuLV ¼ xenotropic murine leukemia virus; N/A ¼ not applicable.
titer in 8 log10 dilution virus sample was 0.68 log10/mL. The measured titer for the 8 log10 dilution virus sample was observed as 0.64 log10/mL, which is only 0.04 log10/mL lower than the expected titer value. This observation indicates that the accuracy of the assay for the 8 log10 dilution virus sample was not compromised even though 29% CV was observed. Therefore, CV at the LLOQ level was determined not to be greater than 30% for the TCID50 assay. Two different X-MuLV stocks with 5 different titer levels for each stock were used to evaluate the LLOD and LLOQ of the qRT-PCR assay. The expected virus titers for these diluted virus samples were calculated as explained above (Table 2). Viral RNA from these samples was purified in triplicate and the qRT-PCR assay was performed in duplicate on each replicate RNA. Average measured virus titers for each dilution level of 2 different stock viruses were calculated. Table 2 shows that the lowest point or the highest diluted virus sample that had Ct value or amplification in qRT-PCR assay was the 6.6 log10 diluted sample (titer 1.36 log10/mL). Each of 3 RNA replicates of this sample had only one Ct value which satisfied the criterion of LLOD for the qRT-PCR assay. Virus titer of this sample was found to be colinear with the titers of other lower diluted samples (Fig. 4B). Because no sample after the 6.6 log10 dilution had a qRT-PCR signal, 1.4 log10/mL was determined to be the LLOD of the qRT-PCR assay. At 5.6 log10 dilution level (titer 2.23 log10/mL), all 3 RNA replicates had qRT-PCR amplification or Ct values, and the difference between the measured and expected virus titers of this sample was less than 0.5 log10 (Table 2). Also, the maximum 95% confidence limit was only 0.37 and the maximum inter-assay precision was only 8.2% CV. Because of the high
precision of qRT-PCR measurements for replicates of this sample, the measured titer of this sample, 2.2 log10/mL, was determined as the LLOQ for the qRT-PCR assay. Based on these LLOD and LLOQ values, the qRT-PCR assay can detect and quantify 25 and 158 virus particles/mL, respectively. 3.5. Reproducibility precision of X-MuLV assays To evaluate the precision of the X-MuLV TCID50 and qRT-PCR assays, 6 samples (X1eX6) were prepared and assayed independently by 3 operators for each assay. For the TCID50 assay, each operator performed triplicate assay plates for each sample in each of the 3 independent runs on 3 different days. For the qRT-PCR assay, each operator performed triplicate RNA purification on each sample and duplicate qRT-PCR on each RNA in each of the 3 independent runs on 3 different days using 2 Mx3005P real-time qPCR instruments. The results are shown in Table 3. The maximum observed repeatability precision (intra-assay precision) within each run for each sample was 5.5% CV among triplicate assay plates (TCID50 assay) and 8.1% CV among 6 qRT-PCR assays. The maximum intermediate precision (inter-assay precision) for each sample was 7.9% CV among 9 replicate assay plates (TCID50 assay) and 4.2% CV among 18 replicate qRT-PCR assays by 3 operators and 2 instruments in 3 independent runs. The maximum 95% confidence limit observed for each set of triplicates for each operator was ±0.37 in the TCID50 assay and ±0.32 in the qRT-PCR assay. Statistical one-way analysis of variance (ANOVA) was performed to further evaluate the precision of the TCID50 and qRT-PCR assays
Table 3 Reproducibility for X-MuLV TCID50 and qRT-PCR assays. Assay
Sample
Estimated titer (Log10/mL)
Measured titer (Log10/mL ± Max 95% CL)
X1 X2 X3 X4 X5 X6
5.17 3.17 2.17 5.51 4.51 3.51
5.42 3.60 4.38 5.52 4.39 3.31
± ± ± ± ± ±
X1 X2 X3 X4 X5 X6
8.16 6.16 4.16 8.13 6.13 4.13
7.81 5.81 3.81 7.66 5.67 3.76
± ± ± ± ± ±
Measured/estimated titer (Log10/mL)
Max % CV Intra-assay
Inter-assay
0.34 0.33 0.33 0.30 0.37 0.34
0.25 0.43 2.21 0.01 0.12 0.20
2.5 5.2 5.5 3.7 3.1 3.4
3.9 5.1 3.5 3.3 3.6 7.9
0.25 0.31 0.31 0.27 0.32 0.26
0.35 0.35 0.35 0.47 0.46 0.37
0.8 1.7 6.9 2.6 1.1 8.1
1.6 2.7 4.2 1.8 2.9 3.5
TCID50
qRT-PCR
CV ¼ coefficient of variation; CL ¼ confidence limit; qRT-PCR ¼ reverse transcriptase quantitative real-time polymerase reaction; TCID50 ¼ 50% tissue culture infectious dose; X-MuLV ¼ xenotropic murine leukemia virus. Note: samples X1 through X6 were prepared independently in each assay.
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Table 4 One-way ANOVA P (probability > F) values for TCID50 and qRT-PCR assays. Assay method
Operator
Independent assay number
X1
X2
X3
X4
X5
X6
1 3 2 3 3 3 Group by operator Group by experiment
0.1288 0.0191 0.0009 0.0022
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Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation Mohammad Anwaruzzaman, Wensheng Wang, Eunice Wang, Lolita Erfe, Janice Lee, Shengjiang Liu* Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2014 Received in revised form 17 January 2015 Accepted 5 April 2015 Available online xxx
Infectivity and reverse transcriptase quantitative real-time polymerase chain reaction (qRT-PCR) assays have been optimized and validated for xenotropic murine leukemia virus (X-MuLV) detection. We have evaluated the assays systematically with regard to specificity, linearity, lower limit of detection (LLOD), lower limit of quantification (LLOQ), and precision. Both assays are specific for X-MuLV detection, with a linear detection range of 0.6e5.6 log10 TCID50/mL for the infectivity assay, and 1.4e6.5 log10 particles/mL for the qRT-PCR assay. The LLOD and LLOQ of the infectivity and the qRT-PCR assays are determined as 0.5 and 1.0 log10/mL, and 1.4 and 2.2 log10/mL. The inter-assay repeatability of qRT-PCR assay (4.2% coefficient of variation [CV]) is higher than the infectivity assay (7.9% CV). We have shown that both assays are closely correlated (r ¼ 0.85, P < 0.05, n ¼ 22). The particle/infectivity ratio is determined as 66. Both assays were applied to evaluate virus removal using virus clearance samples of chromatographic and filtration processes. Here, we have demonstrated that the qRT-PCR assay is much faster in testing and is approximately 8-fold more sensitive than the infectivity assay. Therefore, the qRT-PCR assay can replace the infectivity assay in many cases, but both assays are complementary in elucidating the mechanism of virus inactivation and removal in virus clearance validation. © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Reverse transcriptase real-time PCR assay Infectivity assay Xenotropic murine leukemia virus Virus clearance Chromatography Virus filtration
1. Introduction Genetically engineered rodent cells, such as Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells, are commonly used to produce recombinant proteins for human therapeutic use in the biopharmaceutical industry. These cells have been demonstrated to contain an intrinsic proviral genome that produces endogenous retrovirus-like particles (RVLPs) [1e6]. Two types of particles are consistently observed in CHO cells: intracytoplasmic type A particles, frequently associated with centrioles, and budding type C particles [4]. Type R particles are observed in BHK cells [5,6]. Based on the available information, none of these endogenous RVLPs is infectious [1e3,7]. However, because of the morphologic and biochemical resemblance to tumorigenic retroviruses, RVLPs * Corresponding author. Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, 800 Dwight Way, Berkeley, CA 94710, USA. Tel.: þ1 510 705 5506; fax: þ1 510 705 4557. E-mail address: [email protected] (S. Liu).
may pose a theoretical safety risk to biologics produced in CHO or BHK cells. Therefore, the detection of endogenous RVLPs in cell culture production and the removal and inactivation of endogenous RVLPs in the subsequent downstream purification process are required by regulatory agencies [8,9]. Because RVLPs are noninfectious, an infectivity assay cannot be used directly to detect these particles. Real-time polymerase chain reaction (RT-PCR) assay has been used for the quantification of the RVLP from CHO cells [10]. In the biopharmaceutical industry, xenotropic murine leukemia virus (X-MuLV) is commonly used as a specific model virus in evaluating the removal of RVLP by purification processes. The X-MuLV is an enveloped virus belonging to the Retroviridae family [9]. The viral genome is a single-stranded, positive-sense RNA molecule. Each viral genome contains 2 copies of identical RNA molecules [11]. From 50 to 30 , the genome contains the gag, pol, and env regions: coding for structural proteins; enzymes, including the RNA-dependent DNA polymerase (reverse transcriptase); and coat proteins, respectively [11]. The virus is sensitive to physicochemical treatment, such as heating, drying, low pH, and detergent [9].
http://dx.doi.org/10.1016/j.biologicals.2015.04.001 1045-1056/© 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
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Infectivity (or the 50% tissue culture infectious dose [TCID50]) assay is typically used to quantify infectious virus in the process sample. However, the TCID50 assay is labor intensive and time consuming. The TCID50 assay cannot be used alone to distinguish clearance by virus removal versus inactivation, limiting its ability to meet the increasing demands of the biopharmaceutical industry in virus clearance validation. In virus titration assays, virus is detected and quantified only when the virus level in the sample is at or above the lower limit of detection (LLOD) and lower limit of quantification (LLOQ), respectively. Virus titers are normally expressed with 95% confidence limits, which should not exceed 0.5 log10 of the expected titer [9]. The metrics for quantifying virus in biologics process samples is to report virus quantity in log10/mL when the virus levels in samples are greater than the LLOD. When virus levels in samples are less than the LLOD, then less than LLOD is taken as the assay result. Here we present optimization and validation of cell-based TCID50 and quantitative RT-PCR (qRT-PCR) assays for detection and quantification of X-MuLV to evaluate virus clearance validation. In the TCID50 assay, PG-4 cells were used as indicator cells, and the quantification of virus was performed by measuring the cytopathic €rber equation [12,13]. The qRTeffect (CPE) using the SpearmaneKa PCR assay was based on the 50 to 30 exonuclease activity of Taq DNA polymerase using env geneespecific primers and a dual-labeled probe [14e18]. The amount of X-MuLV particle was quantified from the virus RNA copy number. The qRT-PCR assay offers great advantages over the TCID50 assay, such as higher sensitivity and throughput, greater reliability, and reduced cost. Both the TCID50 and qRT-PCR assays were applied to evaluate X-MuLV removal by protein A column chromatography, membrane adsorption anion exchange (MA-AEX), and virus filtration processes of recombinant human therapeutic antibodies.
2. Materials and methods 2.1. Reagents All chemicals and reagents were of the highest molecular biology grade. Stock solutions were made in RNase/DNase-free sterile water. Elim Biopharmaceuticals, Inc. (Hayward, CA) synthesized oligonucleotides of desaltation grade. The dual-labeled qPCR probe contained 50 6-carboxy-fluorescein (FAM) and 30 Black Hole Quencher (BHQ) and was synthesized from Eurofins MWG/Operon (Huntsville, AL) and Biosearch Technologies (Novato, CA). The probe was high-performance liquid chromatography (HPLC) purification grade. Fisher Scientific (Pittsburgh, PA) supplied cell culture media used in the infectivity assay and bovine serum albumin (BSA) used in the qRT-PCR assay. Viral RNA purification spin columns and reagents including carrier RNA (poly A) were purchased from Qiagen (QIAamp Viral RNA Kit; Valencia, CA). Brilliant® QRT-PCR 2 Master Mix 1-Step Kit including reverse transcriptase/RT-block mix was purchased from Agilent Technologies (formerly Stratagene; Santa Clara, CA). Amplified PCR product from the env gene was purified using QIAquick Gel Extraction Kit (Qiagen) and cloned using the Strataclone™ PCR cloning kit (Agilent Technologies). The Easy-A® PCR High-Fidelity Master Mix Kit (Agilent Technologies) was used to screen orientation of the cloned insert. Immedia™Amp liquid medium used for bacterial growth was purchased from Life Technologies (formerly Invitrogen; Grand Island, NY). Plasmid was purified from the bacterial culture using the QIAFilter™ Plasmid Midi Kit (Qiagen). Promega RiboMax™ Large Scale RNA Production Systems (Madison, WI) was used to synthesize standard RNA. RNA was purified using RNeasy Mini Kit (Qiagen).
2.2. Cells The PG-4 indicator cell line (Felis catus, SþL, ATCC CRL-2032) was used in the X-MuLV infectivity assay. Additionally, the specificity Vero cell line (kidney, African Green Monkey, ATCC CCL-81) and the PK13 cell line (pig, Sus scrofa, ATCC CRL 6489) were used in the infectivity assay. 2.3. Virus Virus stocks (strain: PNFS Th-1) used were purchased from BioReliance (Rockville, MD) and produced internally at Bayer (Berkeley, CA). 2.4. Infectivity assay X-MuLV was quantified by the TCID50 assay in the PG-4 indicator cell line over 6e7 days; cells inoculated with medium (no virus) remained healthy and viable. PG-4 cells were seeded in 96-well microplates at a cell density of 3000 cells/well in McCoy's 5A medium containing 10% fetal bovine serum (FBS), 100 mg/mL penicillin/streptomycin, and 2 mM Lglutamine. Cells were incubated overnight in a humidified incubator at 37 C with 5% CO2. The following day, each virus-containing sample was serially diluted (1:3.2) in 11 wells in a dilution block with McCoy's 5A medium containing 3 mg/mL polybrene (the undiluted sample [1:3.20] through the eleventh dilution [1:3.210]). Then, 100 mL of each set of 11 serially diluted samples were inoculated in each well of the first 11 columns in a 96-well assay plate. The last (twelfth) column of the same assay plate was inoculated with the diluent medium, which served as the in-plate control. The control and diluted samples were inoculated in 8 replicate wells (row 1 through row 8) on each plate. TCID50 assay-positive control plates were also prepared from prediluted stock virus at the beginning and end of the assay. The prediluted virus was serially diluted in dilution blocks using McCoy's 5A medium containing polybrene as described above. The inoculated plate was incubated in a 37 C humidified CO2 incubator for 2.0 ± 0.5 h. Then, 100 mL of 2 assay medium (McCoy's 5A medium, 4% FBS, 200 mg/mL penicillin/streptomycin, and 4 mM L-glutamine) was added to each well on the plate. The plate was returned to the incubator for 6e7 days. Each well was scored for the cytopathic effect caused by virus infection under a light microscope, and virus was quantified as log10 TCID50/mL using the Microsoft Office Excel® worksheet according to SpearmaneK€ arber equation [12,13]. Hereafter, the unit for virus titer of the infectivity assay is simplified as log10/mL. For each sample the final virus titer (TCID50/mL) was calculated from the log10 titer, the sample volume per well (eg, 0.1 mL), and the sample predilution factor. The 95% confidence limit was calculated as ±1.96 SD. 2.5. Quantitative RT-PCR system 2.5.1. Standard X-MuLV RNA preparation The standard X-MuLV RNA was a 1.85-kb in vitro transcript generated from a plasmid harboring the X-MuLV env gene cDNA sequences using the in vitro transcription kit. X-MuLV virus stock (BioReliance) was used to extract and purify viral RNA using the QIAamp Viral RNA mini kit. RNA was quantified by UV spectrophotometry at A260. cDNA was made from the purified RNA (50 ng) using the Brilliant® QRT-PCR Master Mix 2-Step Kit (Agilent Technologies). Primers used in cDNA preparation were X-MuLV-AF (19-mer, 84e102 nucleotides [nt] [50 d(GGC AGG AGC CTC GGT ACA A)-30 ]) and X-MuLV-B-R (19-mer, 1810e1828 nt [50 d(CCA AGC GGT TGA GAA TGC A)-30 ]). These primers are located 1745
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
M. Anwaruzzaman et al. / Biologicals xxx (2015) 1e10
nucleotides apart on the env gene sequences. The PCR product band detected around 1.7 kb on a 1% agarose gel was purified using the QIAexII kit and cloned using the Strataclone™ PCR cloning kit. After transformation into bacteria, colonies containing the X-MuLV insert in the correct orientation were screened by PCR using the kit Easy-A® PCR High-Fidelity Master Mix and primers, M13For (50 d(GTA AAA CGA CGG CCA GT)-30 ) and X-MuLV-B-R. The thermocycling conditions used were: 35 cycles at 95 C for 30 s, 55 C for 30 s, and 72 C for 2 min. The resulting plasmid, pXMULV-A7, was purified from the selected bacterial clone using the QIAFilter™ Plasmid Midi Kit. The plasmid sequence was then confirmed by sequencing for the env gene insert at Elim Biopharmaceuticals using M13For primer (see above). The sequence was analyzed using the software Lasergene 7 (DNASTAR, Inc., Madison, WI). To ensure the proper termination of the RNA transcript, the plasmid was linearized with Hind III. The digestion mixture was then run on a 1% agarose gel and the linearized plasmid (5.2 kb) was purified using QIAquick Gel Extraction Kit. In vitro transcription was then performed on the purified linear plasmid DNA using the RiboMax™ Large Scale RNA Production Systems. The resulting transcribed RNA was 1850 bases long comprising 1745 bp of the insert plus 105 bp of adjacent plasmid sequences between the T7 transcription start and the Hind III site. RNA was then purified using RNeasy Mini Kit and quantified by UV spectrophotometry at A260 nm. RNA copies/mL was calculated using the following formula: N (copies/mL) ¼ [C (RNA concentration in ng/mL) 109 Avogadro's constant (6.023 1023)]/[K (fragment size in bases, 1850 bases) average molecular weight of RNA base (339.5 Da)]. Serial dilutions of the standard RNA were performed to prepare a set of different copy numbers of standard RNA and were stored at e65 C. A standard curve was prepared using 5 mL of standard RNA (ranging from ~1.6 100 to ~1.6 108 copies). 2.6. RNA extraction and purification method Xenotropic murine leukemia virus genomic RNA was extracted and purified by a modified spin column procedure [19] using the QIAamp viral RNA kit. To 140 mL X-MuLV containing sample, 560 mL of AVL virus lysis buffer containing 1 mg/mL of BSA and 10 mg/mL carrier RNA was added, mixed by inversion, and kept at room temperature. Then 560 mL of 96e100% ethanol (RNase/DNase-free) was added and mixed well. Viral RNA was purified by loading the mixture onto a spin column. Viral RNA along with carrier RNA was bound to the column by centrifugation at 8000 rpm for 1 min in a microcentrifuge. The spin column was washed with 500 mL washing buffer (AW1) by centrifugation at 8000 rpm for 1 min. A second wash of the spin column with 500 mL washing buffer (AW2) was performed at 14,000 rpm for 3 min. The spin column was again spun at 14,000 rpm for 1 min to completely remove any residual wash buffer. Viral RNA and carrier RNA were then co-eluted into a sterile microcentrifuge tube using 100 mL of AVE buffer, equilibrated to room temperature and centrifuged at 8000 rpm for 1 min.
3
2.8. qRT-PCR assay The qRT-PCR assay was performed in a 96-well plate format (Agilent Technologies or Life Technologies). Each well (20 mL/reaction) consisted of 5 mL unknown sample or standard RNA solution (ranging from approximately 100e108 copies/reaction), 10 mL Brilliant® II/III QRT-PCR 2 Master Mix 1-Step Kit containing 500 nM forward primer, 500 nM reverse primer, 100 nM probe and reverse transcriptase/RNase block mixture. The assays were initiated first with reverse transcription at 50 C for 30 min then enzyme activation at 95 C for 10 min. This was followed by 40 cycles of denaturation at 95 C for 20 s, annealing at 48 C for 20 s, and extension at 60 C for 20 s. All reactions were performed on Mx3005P quantitative qPCR instrument (Agilent Technologies). On each qRT-PCR 96-well plate, triplicate controls of no template and standard RNA were used along with unknown samples in triplicates. The qPCR product (103 bp) was verified on a 4% agarose gel for each assay (data not shown). The copy number of RNA in unknown samples was determined from the standard curve using the MxPro software version 4.10 (Agilent Technologies) and viral particles were calculated by dividing the copy number by 2. Virus titer of the qRT-PCR assay is determined as log10 particles/mL. Hereafter, the unit for virus titer of the qRT-PCR assay is simplified as log10/ mL. 3. Results 3.1. Optimization of X-MuLV TCID50 and qRT-PCR assays The TCID50 assay was optimized for culture medium, cell passage number, seeding cell density, and post virus-infection incubation time (data not shown). The assay sensitivity was improved by the addition of polybrene to the McCoy's 5A virus dilution medium. Fig. 1 shows that virus titer was 6.7 ± 0.3 log10/mL when no polybrene was added to the diluent medium. When the diluent medium had 3 mg/mL polybrene, the sensitivity of the assay increased by 25-fold. The measured titer with the polybrene was 8.1 ± 0.8 log10/mL. These results indicate that polybrene concentration at 3 mg/mL was important for higher sensitivity of the XMuLV TCID50 assay.
2.7. qRT-PCR primers and probe Two primers (forward and reverse) and the probe were designed from the env gene sequences of X-MuLV. The forward primer, XMuLV-A-Fwd6, was an 18-mer, 85e102 nt [50 -d(GCA GGA GCC TCG GTA CAA)-30 ]; the reverse primer, XMuLV-A-Rev3, was a 21-mer, 167e187 nt [50 -d(GGA GGG AGG TAG CGT TAG CTG)-30 ]. The probe was a 26-mer, 105e130 nt [50 -d((FAM)TGA CAG CCC TCA CCA GGT CTT CAA TG(BHQ))-30 ]. A combination of the forward and reverse primers in qRT-PCR produced a 103-bp fragment.
Fig. 1. Effect of polybrene on sensitivity of the X-MuLV TCID50 assay. Stock X-MuLV was prediluted in McCoy's 5A medium with or without 3 mg/mL polybrene. The prediluted virus was then serially diluted (1:3.2) in 11 wells in a dilution block with McCoy's 5A medium with or without 3 mg/mL polybrene. The TCID50 assay was then performed with these diluted virus samples using preseeded PG-4 cells in 96-well plates (see Materials and methods). TCID50 ¼ 50% tissue culture infectious dose; XMuLV ¼ xenotropic murine leukemia virus.
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
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M. Anwaruzzaman et al. / Biologicals xxx (2015) 1e10
The qRT-PCR assay system was optimized for spin column RNA purification and qRT-PCR steps (data not shown). Stability of standard RNA for quantification of viral RNA copy numbers in unknown samples was also optimized. Stability of the standard RNA was evaluated by amplification and efficiency of qRT-PCR assay. The range of standard RNA copy numbers used was from ~2.7 10 0 to ~2.7 108 copies/reaction. Fig. 2 shows that the lowest RNA levels for which qRT-PCR amplification (cycle threshold [Ct] value) was observed were 2.7 103, 2.7 102, and 2.7 101 copies/reaction for standard RNA in water, water with 1 mg carrier RNA/reaction and AVE buffer with 1 mg carrier RNA/reaction, respectively. These results indicate that stability of standard RNA was higher in AVE buffer plus 1 mg carrier RNA/reaction than in water or water with carrier RNA. A linear regression curve was generated for each standard RNA set by plotting Ct values against the standard RNA copy number (Fig. 2). The regression coefficient R2 value of the standard curve for RNA in water was slightly lower (0.976) than that of standard RNA set with carrier RNA in water (0.999) or AVE buffer (0.998). Efficiency of the qRT-PCR reaction for each set of standard RNA was calculated from the slope of the standard curve as [10(1/slope) 1]. The qRT-PCR efficiency was significantly lower for RNA in water (78%) than the RNA with carrier RNA in water (107%) or AVE buffer (108%; Fig. 2). These results further indicate that the carrier RNA was important for stability of standard RNA and efficiency of qRT-PCR. Therefore, buffer AVE plus 1 mg carrier RNA/reaction was used as diluent of standard RNA preparation. An aliquot of qRT-PCR assay mixtures from the standard RNA and purified viral genomic RNA was analyzed on a 4% agarose gel electrophoresis. Both viral standard RNA and genomic RNA yielded a 103-bp DNA product (data not shown). Recovery of virus RNA by the spin column purification method was evaluated by comparing viral RNA of a sample in the spin column eluate to the viral RNA in a sample directly disrupted with AVL solution. AVL solution had no inhibitory effect on the RT and PCR at 100-fold dilution with water (RNase/DNase-free) containing 700 units SUPERaseIn™ per mL (Life Technologies) and 10 mg/mL carrier RNA. Virus titers in the spin column eluate and AVLdisrupted samples were 7.70 ± 0.17 and 7.50 ± 0.06 log10/mL, respectively (data not shown), suggesting that there was no difference in RNA between the spin column eluate and the
Fig. 2. Effect of carrier RNA on stability of standard RNA. Stock standard RNA was diluted to make standards 2.7 100, 2.7 101, 2.7 102, 2.7 103, 2.7 104, 2.7 105, 2.7 106, 2.7 107, and 2.7 108 copies/reaction in RNase/DNase-free water, water with 1 mg carrier RNA/reaction, and buffer AVE with 1 mg carrier RNA/reaction. Subsequently, qRT-PCR was performed on these standards. Linear regression curves were made for these standards, and qPCR efficiency was calculated from the slope as described in Materials and methods. Ct ¼ cycle threshold; CR ¼ carrier RNA; qRT-PCR ¼ reverse transcriptase quantitative real-time polymerase reaction; R2 ¼ regression coefficient.
AVL-disrupted samples. The AVL solution contained 10 mg/mL carrier RNA or 5.6 mg carrier RNA in 560 mL extraction mixture. Considering full recovery of RNA by the spin column procedure, the estimated carrier RNA in 100 mL viral RNA would be approximately 5.6 mg. To evaluate if the carrier RNA had any influence on stability of the purified X-MuLV RNA, viral RNA was purified from 4 virus samples containing different virus amounts in triplicate for each sample. Purified viral RNA was stored at e65 C. The following day (day 1), qRT-PCR assay was performed in duplicate for each RNA sample. Subsequent qRT-PCR assays were performed on days 7, 14, and 21. Average virus titer was calculated for these qRT-PCR samples and plotted against the days stored at e65 C (Fig. 3). The quantity of viral RNA of all 4 samples measured during the storage period (21 days at e65 C) did not change, indicating that the purified viral RNA was stable under those conditions. The maximum 95% confidence limit (CL) was 0.67 log10 and the percentage coefficient variation (CV) for each virus sample for each day of qRT-PCR was only 4.6%. 3.2. X-MuLV TCID50 and qRT-PCR assay specificity Specificity of the X-MuLV TCID50 assay was evaluated using PK13, Vero, and PG-4 indicator cells and corresponding growth media for these cells. No growth of X-MuLV (4 log10-fold diluted stock virus) was observed on plates seeded with PK13 or Vero indicator cells growing in the corresponding growth media. Growth of X-MuLV was observed on plates seeded with PG-4 cells. The measured average infectivity titer in 4 log10 diluted virus sample was 4.09 ± 0.32 log10/mL. These results indicate the high specificity of X-MuLV growth and replication in PG-4 cells (Table 1). Specificity of the X-MuLV qRT-PCR assay was also evaluated using stock pseudorabies virus (PRV), porcine parvovirus (PPV), Reo 3 virus, and 5.53 log10-diluted stock X-MuLV. These virus samples were diluted 1:20 in phosphate-buffered saline during extraction. Nucleic acids of these lysed viruses were then purified according to the spin column method, and the qRT-PCR assay was performed as described above. Table 1 shows that no amplification (no Ct) of nucleic acids from PPV, PRV or Reo 3 virus was observed by the qRTPCR assay. Amplification was observed only with X-MuLV RNA. The
Fig. 3. Stability of purified X-MuLV RNA at e65 C. X-MuLV RNA was extracted and purified from triplicates of each of 4 virus samples containing different virus amounts according to the procedure described above. Purified RNA was stored at e65 C. The following day (day 1), the qRT-PCR assay was performed in duplicate for each RNA sample. Subsequent qRT-PCR assays were performed with RNA purified in days 7, 14 and 21. RNA copies of these samples were determined from the standard curve and log10 particles/mL virus titers were calculated. Average virus titer was calculated for these samples and plotted against the days stored at e65 C. X-MuLV ¼ xenotropic murine leukemia virus; qRT-PCR ¼ reverse transcriptase quantitative real-time polymerase reaction.
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
M. Anwaruzzaman et al. / Biologicals xxx (2015) 1e10
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Table 1 Specificity evaluation for X-MuLV TCID50 and qRT-PCR assays. TCID50 assay
qRT-PCR assay
Indicator cells
Number of replicate assay plates
Average TCID50 (Log10/mL ± 95% CL)
Virus
Number of replicate assays
Average particle (Log10/mL ± Max 95% CL)
PG-4 PK13 Vero
3 3 3
4.09 ± 0.32a 0.75a,b 0.75a,b
X-MuLV PPV PRV Reo 3
3 3 3 3
3.89 ± 0.20c 1.4b 1.4b 1.4b
CL ¼ confidence limit; CPE ¼ cytopathic effect; Ct ¼ cycle threshold; PPV ¼ porcine parvovirus; PRV ¼ pseudorabies virus; qRT-PCR ¼ reverse transcriptase quantitative realtime polymerase reaction; TCID50 ¼ 50% tissue culture infectious dose; X-MuLV ¼ xenotropic murine leukemia virus. a Predilution factor of stock viruses in TCID50 was 4 log10. b No CPE in TCID50 assay and no Ct in qRT-PCR assay was observed. c Predilution factor of stock viruses in qRT-PCR assays was 5.53 log10.
average measured titer in the 5.53 log10-diluted X-MuLV sample was 3.89 ± 0.20 log10/mL. These results indicate that the qRT-PCR assay was highly specific for X-MuLV. 3.3. X-MuLV TCID50 and qRT-PCR assay linearity The X-MuLV TCID50 and qRT-PCR assay linearity was evaluated using virus samples prepared from serial dilutions of X-MuLV stocks. Fig. 4 shows that in both the TCID50 (Fig. 4A) and qRT-PCR
(Fig. 4B) assays, there was a linear relationship between virus titers of diluted virus samples and corresponding dilution factors used to prepare the virus samples. For the TCID50 assay, linearity was observed with an R2 value of 0.996 for virus titers ranging from 0.6 to 5.6 log10/mL; for the qRTPCR assay, linearity was observed with an R2 value of 0.999 for virus titers ranging from 1.4 to 6.5 log10/mL. The CV of intra-assay was in the range of 2.9e29% in the TCID50 assay and 0.9e19.2% in the qRTPCR assay. In both assays the percentage CV was higher for low titer virus samples than for mid-level virus titers. The maximum 95% CL observed was 0.73 in the TCID50 assay and 0.47 in the qRT-PCR assay. 3.4. X-MuLV assay limit of detection and quantification
Fig. 4. Linearity of (A) TCID50 and (B) qRT-PCR assays. X-MuLV stock was diluted serially in virus suspension buffer, TNEB (50 mM TriseHCl, pH 7.2, 100 mM NaCl, 5 mM EDTA, and 1 mg/mL BSA). For the TCID50 assay, virus stock (average titer, 8.68 log10 TCID50/mL) was diluted 3, 4, 5, 6, 6.5, 7, 7.5, and 8 log10 fold. The TCID50 assay was performed on these samples using PG-4 cells and virus titers in log10 TCID50/mL were calculated from the cytopathic scores. For the qRT-PCR assay, 2 different virus stocks (average titer, 9.68 log10 particles/mL) were diluted 1.6, 2.6, 3.6, 4.6, 5.6 and 6.6 log10 fold. Viral RNA of these samples was purified and qRT-PCR assay was performed on each RNA sample. RNA copy number and viral titer in log10 particles/mL were calculated. Average virus titers were calculated for each dilution level of the 2 virus stocks. Subsequently, linear regression curves were plotted for both assay virus titers against the log10 dilution factor. BSA ¼ bovine serum albumin; qRT-PCR ¼ reverse transcriptase quantitative real-time polymerase reaction; R2 ¼ regression coefficient; TCID50 ¼ 50% tissue culture infectious dose; X-MuLV ¼ xenotropic murine leukemia virus.
The LLOD and LLOQ of the X-MuLV TCID50 and qRT-PCR assays were evaluated using diluted virus samples prepared by serial dilutions (1:3.2 for the TCID50 assay and 1:10 for the qRT-PCR assay) of high-titer X-MuLV. The LLOD of the TCID50 assay was determined based on the criterion that all replicate assay measurements have positive scores. The criterion for LLOD of the qRT-PCR assay was the lowest point (highest dilution) that gives a qRT-PCR signal (Ct value) but is no longer colinear with the other dilutions or the lowest point that each replicate RNA has at least one qRT-PCR signal. The LLOQ for both assays was determined based on the criteria that all replicate assay measurements have positive scores (TCID50 assay) or Ct values (qRT-PCR assay) and that the measured average virus titer not be more than 0.5 log10 from the expected titer. The expected titer was chosen based on the virus titers obtained from dilutions within the linearity range and was calculated from the average measured log10 titer of the mid-range dilution sample in triplicate and the corresponding dilution factor. Results of LLOD and LLOQ for TCID50 and qRT-PCR assays are summarized in Table 2. In the TCID50 assay, the virus samples at dilution levels of 7.5, 8.0, and 8.5 log10 had positive cytopathic scores for all replicates. However, the maximum 95% confidence limit and percentage CV values for these samples were higher than expected (Table 2). Therefore a conservative approach was taken considering the limited replicates of each dilution level, the high 95% CL and percentage CV for the measured titers of the highest dilution levels. The LLOD and LLOQ for the TCID50 assay were determined to be 0.5 log10 and 1.0 log10/mL, respectively. Therefore, the TCID50 assay can detect 2.53 TCID50 or 0.4 log10 TCID50. The observed CV for 7.5 and 8.0 log10 dilution levels were 25.9% and 29%, respectively. The TCID50 titration assay is not a quantitative assay, it is an estimated assay. Usually high-percentage CV is observed for low and very high dilutions of virus samples with this assay (19). The expected virus titer in undiluted virus stock was 8.68 log10/mL. The expected virus
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
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M. Anwaruzzaman et al. / Biologicals xxx (2015) 1e10
Table 2 LLOD and LLOQ of X-MuLV TCID50 and qRT-PCR assays. TCID50 assay
qRT-PCR assay
Dilution factor (Log10)
Measured TCID50 (Log10/mL ± Max 95% CL)
6.5 7.0 7.5 8.0 8.5 9.0
2.14 1.42 0.87 0.64 0.03 N/A
± ± ± ± ±
0.26 0.36 0.51 0.73 1.22
Expected TCID50 (Log10/mL)
Max % CV
LLOD, LLOQ (Log10/mL)
Dilution factor (Log10)
Measured particles (Log10/mL ± Max 95% CL)
2.18 1.67 1.17 0.66 0.16 0.35
3.0 10.5 25.9 29.0 0.0 N/A
0.5, 1.0
2.6 3.6 4.6 5.6 6.6 7.6
5.40 4.19 3.20 2.23 1.36 N/A
± ± ± ± ±
0.09 0.08 0.11 0.37 0.47
Expected particles (Log10/mL)
Max % CV
LLOD, LLOQ (Log10/mL)
5.45 4.45 3.45 2.45 1.45 0.45
0.9 1.0 1.8 8.2 19.2 N/A
1.4, 2.2
CL ¼ confidence limit; CV ¼ coefficient of variation; LLOD ¼ lower limit of detection; LLOQ ¼ lower limit of quantification; qRT-PCR ¼ reverse transcriptase quantitative realtime polymerase reaction; TCID50 ¼ 50% tissue culture infectious dose; X-MuLV ¼ xenotropic murine leukemia virus; N/A ¼ not applicable.
titer in 8 log10 dilution virus sample was 0.68 log10/mL. The measured titer for the 8 log10 dilution virus sample was observed as 0.64 log10/mL, which is only 0.04 log10/mL lower than the expected titer value. This observation indicates that the accuracy of the assay for the 8 log10 dilution virus sample was not compromised even though 29% CV was observed. Therefore, CV at the LLOQ level was determined not to be greater than 30% for the TCID50 assay. Two different X-MuLV stocks with 5 different titer levels for each stock were used to evaluate the LLOD and LLOQ of the qRT-PCR assay. The expected virus titers for these diluted virus samples were calculated as explained above (Table 2). Viral RNA from these samples was purified in triplicate and the qRT-PCR assay was performed in duplicate on each replicate RNA. Average measured virus titers for each dilution level of 2 different stock viruses were calculated. Table 2 shows that the lowest point or the highest diluted virus sample that had Ct value or amplification in qRT-PCR assay was the 6.6 log10 diluted sample (titer 1.36 log10/mL). Each of 3 RNA replicates of this sample had only one Ct value which satisfied the criterion of LLOD for the qRT-PCR assay. Virus titer of this sample was found to be colinear with the titers of other lower diluted samples (Fig. 4B). Because no sample after the 6.6 log10 dilution had a qRT-PCR signal, 1.4 log10/mL was determined to be the LLOD of the qRT-PCR assay. At 5.6 log10 dilution level (titer 2.23 log10/mL), all 3 RNA replicates had qRT-PCR amplification or Ct values, and the difference between the measured and expected virus titers of this sample was less than 0.5 log10 (Table 2). Also, the maximum 95% confidence limit was only 0.37 and the maximum inter-assay precision was only 8.2% CV. Because of the high
precision of qRT-PCR measurements for replicates of this sample, the measured titer of this sample, 2.2 log10/mL, was determined as the LLOQ for the qRT-PCR assay. Based on these LLOD and LLOQ values, the qRT-PCR assay can detect and quantify 25 and 158 virus particles/mL, respectively. 3.5. Reproducibility precision of X-MuLV assays To evaluate the precision of the X-MuLV TCID50 and qRT-PCR assays, 6 samples (X1eX6) were prepared and assayed independently by 3 operators for each assay. For the TCID50 assay, each operator performed triplicate assay plates for each sample in each of the 3 independent runs on 3 different days. For the qRT-PCR assay, each operator performed triplicate RNA purification on each sample and duplicate qRT-PCR on each RNA in each of the 3 independent runs on 3 different days using 2 Mx3005P real-time qPCR instruments. The results are shown in Table 3. The maximum observed repeatability precision (intra-assay precision) within each run for each sample was 5.5% CV among triplicate assay plates (TCID50 assay) and 8.1% CV among 6 qRT-PCR assays. The maximum intermediate precision (inter-assay precision) for each sample was 7.9% CV among 9 replicate assay plates (TCID50 assay) and 4.2% CV among 18 replicate qRT-PCR assays by 3 operators and 2 instruments in 3 independent runs. The maximum 95% confidence limit observed for each set of triplicates for each operator was ±0.37 in the TCID50 assay and ±0.32 in the qRT-PCR assay. Statistical one-way analysis of variance (ANOVA) was performed to further evaluate the precision of the TCID50 and qRT-PCR assays
Table 3 Reproducibility for X-MuLV TCID50 and qRT-PCR assays. Assay
Sample
Estimated titer (Log10/mL)
Measured titer (Log10/mL ± Max 95% CL)
X1 X2 X3 X4 X5 X6
5.17 3.17 2.17 5.51 4.51 3.51
5.42 3.60 4.38 5.52 4.39 3.31
± ± ± ± ± ±
X1 X2 X3 X4 X5 X6
8.16 6.16 4.16 8.13 6.13 4.13
7.81 5.81 3.81 7.66 5.67 3.76
± ± ± ± ± ±
Measured/estimated titer (Log10/mL)
Max % CV Intra-assay
Inter-assay
0.34 0.33 0.33 0.30 0.37 0.34
0.25 0.43 2.21 0.01 0.12 0.20
2.5 5.2 5.5 3.7 3.1 3.4
3.9 5.1 3.5 3.3 3.6 7.9
0.25 0.31 0.31 0.27 0.32 0.26
0.35 0.35 0.35 0.47 0.46 0.37
0.8 1.7 6.9 2.6 1.1 8.1
1.6 2.7 4.2 1.8 2.9 3.5
TCID50
qRT-PCR
CV ¼ coefficient of variation; CL ¼ confidence limit; qRT-PCR ¼ reverse transcriptase quantitative real-time polymerase reaction; TCID50 ¼ 50% tissue culture infectious dose; X-MuLV ¼ xenotropic murine leukemia virus. Note: samples X1 through X6 were prepared independently in each assay.
Please cite this article in press as: Anwaruzzaman M, et al., Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation, Biologicals (2015), http://dx.doi.org/ 10.1016/j.biologicals.2015.04.001
M. Anwaruzzaman et al. / Biologicals xxx (2015) 1e10
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Table 4 One-way ANOVA P (probability > F) values for TCID50 and qRT-PCR assays. Assay method
Operator
Independent assay number
X1
X2
X3
X4
X5
X6
1 3 2 3 3 3 Group by operator Group by experiment
0.1288 0.0191 0.0009 0.0022
