Quantitation of the residual DNA from rice-derived ... · Molecular pharming is the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals

Quantitation of the residual DNA from rice-derived recombinant humanserum albumin

Zhen Chen a, Huixia Dai a, Zhenwei Liu a, Liping Zhang a, Jianlei Pang a, Jiquan Ou b, Daichang Yang a,b,⇑a College of Life Sciences, State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan 430072, Chinab Hubei Engineering Research Center for Molecular Pharming, Biolake, Wuhan 430076, China

a r t i c l e i n f o

Article history:Received 30 September 2013Received in revised form 22 December 2013Accepted 23 December 2013Available online 3 January 2014

Keywords:qPCRHost cell residual DNAOsrHSAMolecular pharmingRice seed bioreactor

a b s t r a c t

Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinicalapplications; thus, maximum residual limit has been established by drug safety authorities. Assays forresidual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems havebeen established, but no rice residual DNA assay for rice expression systems has been designed. Todevelop an assay for the quantification of residual DNA that is produced from rice seed, we establisheda sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribo-somal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the templateconcentration was in the range of 2 ! 104 to 0.2 pg of DNA per reaction in TaqMan and SYBR Green Iassays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinanthuman serum albumin from Oryza sativa (OsrHSA) was less than 3.8 ng per dosage, which was lower thanthat recommended by the World Health Organization (WHO). Our results indicate that the current puri-fication protocol could efficiently remove residual DNA during manufacturing and processing. Further-more, this protocol could be viable in other cereal crop endosperm expression systems for developinga residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.

! 2013 Elsevier Inc. All rights reserved.

Molecular pharming is the use of genetic engineering to insertgenes that code for useful pharmaceuticals into host animals orplants, which offers the advantages to producers that it does notrequire expensive infrastructure and production capacity can bequickly scaled to meet demand at greatly reduced cost. The prod-ucts from molecular pharming for pharmaceutical applicationshave rapidly increased during recent years [1]. Plants provide apromising production platform for recombinant pharmaceuticalproteins in terms of scalability, safety, and cost effectiveness, andthey may be an alternative to conventional fermentation systemssuch as bacteria, yeast, and mammalian cell cultures. Cereal cropseeds, such as rice, barley, and maize, could be ideal hosts [2]. Re-cently, various recombinant proteins have been successfully ex-pressed in rice seeds, including human a-antitrypsin, T cellepitope peptides, cholera toxin B subunit, infectious bursal diseasevirus immunogen VP2, and large-scale-produced human serumalbumin [3–7], and many of these proteins have entered the clini-cal research stage [8,9].

For clinical use, biopharmaceutical manufacturing must bear inmind that final products produced from cell substrates should con-tain below a maximum threshold per dose of residual host cell

DNA. Current regulatory guidelines recommend that host residualDNA in a final product be less than 100 pg per dose or 10 ng perdose for some biopharmaceuticals requiring large doses (e.g.,monoclonal antibodies) [10,11]. Therefore, it is critical to strictlycontrol the amount of residual DNA in pharmaceutical-grade re-combinant protein pharmaceuticals because it is directly associ-ated with the safety of humans. The conventional methods toquantify residual DNA in the pharmaceutical industry have in-cluded the PicoGreen assay, hybridization assay, threshold tech-nology, and quantitative real-time polymerase chain reaction(qPCR)1 [12–14]. Of these detection methodologies, qPCR-based as-says are considered the most practical for residual DNA quantitationdue to their sensitivity, accuracy, precision, and time saving. qPCRhas been successfully developed to quantitatively detect the residualDNA of Escherichia coli, NS0, and Chinese hamster ovary (CHO) cells.However, no quantification assay for plant-derived host residualDNA has been reported. With the rapidly developing molecular

0003-2697/$ - see front matter ! 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ab.2013.12.033

⇑ Corresponding author. Fax: +86 27 87570660.E-mail address: [email protected] (D. Yang).

1 Abbreviations used: qPCR, quantitative real-time polymerase chain reaction; CHO,Chinese hamster ovary; rRNA, ribosomal RNA; LINE, long interspersed nucleotideelement; LTR, long terminal repeat; OsrHSA, recombinant human serum albuminfrom Oryza sativa; FAM, 6-carboxyfluorescein; BHQ1, black hole quencher 1; AGE,agarose gel electrophoresis; HPLC, high-performance liquid chromatography; UNG,uracil-N-glycosylase; SD, standard deviation; CV, coefficient of variation; Ct, cyclethreshold; WHO, World Health Organization.

Analytical Biochemistry 450 (2014) 4–10

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Analytical Biochemistry

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pharming industry using plant cells as hosts (e.g., rice grain) [3–9], itis necessary to develop a sensitive assay for quantifying residualDNA in rice-derived pharmaceuticals.

In qPCR analysis, the initial copy number of the target DNA se-quences must be proportional to achieve a threshold signal atshorter times. Therefore, it is advantageous to select target geneDNA sequences that are found in multiple copies in the genome.Multiple-copy elements such as long interspersed nucleotide ele-ment 1 (LINE-1), LINE-2, B1, B2, and long terminal repeat (LTR)have been typically used for residual DNA analysis [15]. Mean-while, ribosomal RNA (rRNA) genes are frequently evaluated forresidual DNA analysis because they are found in multiple copiesand are highly conserved [16]. Although other techniques are avail-able [17], most researchers who perform qPCR rely on TaqManprobes or SYBR Green dyes. The aim of this work was to developa qPCR protocol for the quantification of residual DNA for a molec-ular pharming product, recombinant human serum albumin fromOryza sativa (OsrHSA). In this study, the highly conserved, multi-ple-copy rice 5S rRNA gene was chosen as an internal referencefor residual DNA quantitation using qPCR. It has been estimatedthat the copy number of the 5S rRNA gene, based on the unitlengths of 5S rRNA and including the flanking spacer region(303 bp), is 919 copies for indica rice and 752 copies for japonicarice [18]. Here we evaluated the reliability, precision, and accuracyof TaqMan and SYBR Green I assays for 5S rRNA. Application of thisprotocol to the quantitation of residual DNA of OsrHSA indicatedthat it is reliable, precise, and accurate. The limit of detection ofthe residual DNA reached 3.8 ng per dosage, indicating that it couldeffectively be used for residual DNA detection in pharmaceuticalprotein products derived from rice grain.

Materials and methods

Standard and sample preparation

An ABI Prism 7900HT instrument was used as the sequencedetection system (Applied Biosystems, Foster City, CA, USA). Geno-mic DNA from a japonica rice variety, Taibei 309, was obtainedusing the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) accord-ing to the manufacturer’s instructions. Genomic DNA was quanti-fied by measuring its absorbance at 260 nm through a UV-2450visible spectrophotometer (Shimadzu, Tokyo, Japan). Rice genomicDNA was stored at "20 "C for use as a standard preparation forsubsequent experiments. To prepare the DNA standard template,rice genomic DNA was serially diluted 10-fold from 1 ! 104 to0.01 ng/ml in deionized water.

OsrHSA was prepared to test the residual DNA quantificationassay. A series of separation and purification procedures for OsrH-SA from crude seed extract to the final bulks are depicted in detailin the U.S. patent application [19,20]. The impurities from the mainsteps of the purification procedures were used to test the recovery,repeatability, and quantitation limit of the protocol. Each assaywas performed in triplicate with biological and technological re-peats. Protein solutions were from the three purification steps, de-noted as A, B, and C. Each protein solution was diluted to aconcentration of 3.0 mg/ml. The resulting protein solution was di-luted to 200 mg/ml and designated as D. The nucleic acids fromOsrHSA were extracted using the standard protocol following themanufacturer’s instructions.

Primer and probe preparation

Specific primers and TaqMan probe for the rice 5S rRNA gene(GenBank ID: D26370) were designed using Beacon Designersoftware and purchased from Invitrogen (Carlsbad, CA, USA). The

fluorescent probe was labeled at its 50 end with 6-carboxyfluores-cein (FAM) dye and at its 30 end with black hole quencher 1 (BHQ1)dye. The primers were purified by agarose gel electrophoresis(AGE), and the fluorescent probes were purified by high-perfor-mance liquid chromatography (HPLC). The sequences of the prim-ers and fluorescent probes are shown in Supplementary Table 1 ofthe online supplementary material.

Real-time PCR system

qPCRs were performed in duplicate using an ABI StepOne Real-Time PCR System and MicroAmp optical 48-well reaction platesthat were sealed with optical adhesive covers (Applied Biosys-tems). SYBR Green qPCR was carried out in a volume of 10 ll con-taining 5 ll of 2! SYBR Green Master Mix (Applied Biosystems),1 ll of forward and reverse primer, 2 ll of rice genomic DNA(1 lg/ml), and 1 ll of deionized water. The amplification efficiencywas optimized by adjusting the primer concentrations. Thecombinations of primers at different concentrations are listed inSupplementary Table 2. TaqMan PCR amplification was carriedout in a volume of 20 ll containing 10 ll of 2! TaqMan MasterMix (Applied Biosystems), 2 ll of the forward and the reverseprimers, 2 ll of probe (2.5 lM), 2 ll of rice genomic DNA templatein the range of 1 ! 106 to 100 pg/ml, and 2 ll of deionized water.qPCR using SYBR Green fluorescence was carried out as follows:one 10-min cycle at 95 "C (activation of enzyme), 40 cycles of15 s at 95 "C and 30 s at 60 "C, and one cycle from 60 to 95 "C withan increase of 0.7 "C/s for melting. TaqMan PCR amplification wasas follows: one 2-min cycle at 50 "C to activate uracil-N-glycosy-lase (UNG), followed by 40 cycles of 15 s at 95 "C and 30 s at60 "C. The amplification data were analyzed using ABI StepOneReal-Time PCR System software (version 2.0; Applied Biosystems).The detection limit was defined as the lowest detectable residualDNA concentration, corresponding to a signal with a mean of blankvalue plus 2 ! standard deviation (SD). The quantitation limit wasdetermined as the lowest concentration with a coefficient ofvariation (CV) under 20%.

Reproducibility and specificity of the qPCR assay

To determine the reproducibility of the real-time PCR, variousconcentrations of genomic DNA (1000, 100, 10, 1, and 0.1 ng/ml)were used for intra- and interassay comparisons. Intraassay varia-tions were assessed by testing three replicates of each concentra-tion within a single round of real-time PCR, and the interassayvariation was assessed by three rounds of real-time PCR.

To determine the specificity of the real-time PCR, human geno-mic DNA at a final concentration of 10 and 100 ng/ml was used astemplate, and the same PCR parameters as for the SYBR Green Ireaction were used for real-time PCR.

Fragmentation of rice genomic DNA

Genomic DNAs were sonicated by the Bioruptor UCD-200instrument and then resuspended in deionized water at a concen-tration of 36 lg/ml, after which 100 ll of each sample was vor-texed and centrifuged. The Bioruptor was used with thefollowing settings: middle power, 30 s on and 30 s off per cycle,and 2 min and 10 min of sonication. The temperature was main-tained at 4 "C during the entire sonication procedure.

Recovery of residual DNA extraction from OsrHSA

To ensure the efficient extraction of target residual DNA andqualify the samples from a large-scale production of OsrHSA, aspiked assay was used to determine the recovery of residual DNA

Quantitation of the residual DNA from rice-derived recombinant human serum albumin / Z. Chen et al. / Anal. Biochem. 450 (2014) 4–10 5

by the extraction procedure. Rice genomic DNA was spiked into theOsrHSA extraction A, the multimodal weak cation (Capto MMC)chromatography preparation B, the multimodal strong anion (Cap-to-Adhere) chromatography preparation C, and the hydrophobic(phenyl) chromatography preparation D at a final concentrationof 1000 pg/ml in each. Meanwhile, unspiked controls were ana-lyzed to determine the internal levels of rice genomic DNA in thefour OsrHSA preparations. Spiked and unspiked samples were pre-pared in duplicate, and residual DNA from the spiked and unspikedsamples was collected using the Wako DNA Extractor Kit (WakoChemicals, Richmond, VA, USA). For testing the recovery, rice geno-mic DNA spiked into preparation D solution at final concentrationsof 5, 20, 100, 500, and 2000 pg/ml was also studied. Briefly, 500 llof the spiked and unspiked samples with different concentrationswas used to extract DNA, and the DNA pellets were reconstitutedto 20 ll for qPCR analysis. Quantification recovery was calculatedby the formula

DNA recovery ð%Þ ¼ ða" bÞ=c ! 100

where a refers to the observed DNA concentration, b refers to theinternal DNA concentration; and c refers to the spiked DNAconcentration.

Repeatability and quantitation limit of ribosomal DNA recovery

The repeatability and quantitation limit of DNA recovery wasevaluated using a Wako DNA Extractor Kit. Briefly, rice genomicDNA was spiked into preparation D solution at a final concentra-tion of 100 pg/ml. Spiked control was then analyzed by qPCR.The quantitation limit was determined by preparing spiked controlat 4 pg/ml. An inherent DNA concentration of the preparation Dsolution was also analyzed as unspiked control. A difference of lessthan 0.2log10 between the spiked concentration and the calculatedconcentration is acceptable. All tests were done with three techno-logical replicates and three biological replicates.

Results and discussion

Primer determination and optimization

It is critical to obtain the best amplification efficiency, accuracy,and reliability by determining the best primer pair and optimiza-tion parameters for primer concentration. To achieve this goal,the amplification efficiencies of different primer pairs and concen-trations were investigated. Three pairs of primers for the rice 5SrRNA gene were designed using Beacon Designer software (Supple-mentary Table 1), and their performance was compared (data notshown). Primer pair 3 showed the highest efficiency of all primers,and the highest efficiency and lowest cycle threshold (Ct) valueswere obtained at a concentration of 10 lM forward primer and4 lM reverse primer. A single band of the expected size (99 bp),without primer dimers or off-target amplifications, was furtherconfirmed by gel electrophoresis (Fig. 1A). The melting curve forthe reaction showed a single symmetrical peak, indicating that asingle gene-specific product was produced (Fig. 1B).

Determination of the standard curve for residual nucleotidequantification

To accurately quantitate the residual nucleotides of OsrHSA, weassessed and compared the two qPCR methods, SYBR Green I andTaqMan, using rice genomic DNA. As shown in Figs. 1C to 1F,amplification plots and standard curves were generated in theSYBR Green I and TaqMan methods. The results indicated thatstrong linear relationships (R2 = 0.9991 for SYBR Green and

0.9993 for TaqMan) were obtained between the 10-fold dilutionseries and the cycle threshold. Linearity between the Ct valuesand the corresponding concentration was observed over a rangeof 2 ! 104 to 0.2 pg of DNA per reaction when using the TaqManand SYBR Green I methods, which is equivalent to approximately2 genomes [21]. In addition, the detection limits reached 0.02 pgfor both assays. The quantification limit of residual DNA for the as-says was usually within 0.5 to 5 pg per reaction or 50 to 100 pg/ml(10–20 genome equivalents) [22]. Our results show that the sensi-tivity of qPCR increased using the 5S rRNA gene as the target.

In general, to reach accurate and reliable results in qPCR, theamplification efficiency should be greater than 90%. The slope ofthe standard curve is mathematically correlated to the PCR effi-ciency based on the equation E = 10"1/slope, (where E is the PCRamplification efficiency. The results showed that the slopes of thecurves that were generated by the SYBR Green and TaqMan assayswere "3.12 and "3.26, respectively, indicating that both real-timePCR assays were highly efficient. Correspondingly, the E values forthe assays reached 103 and 104%, respectively, both close to 100%.Therefore, similar results were obtained from the TaqMan andSYBR Green I qPCR in terms of linearity, quantitative range, detec-tion limit, and amplification efficiency. We chose the SYBR Green Iassay for the following experiments due to cost savings.

Reproducibility and specificity of real-time PCR

To test the reproducibility and specificity of the SYBR Green Iassay, a series of dilutions of the template (1000, 100, 10, 1, and0.1 ng/ml) were tested. The results indicated that the intra- andinterassay CVs of the Ct values ranged from 0.51 to 2.2% and theCVs of the calculated concentrations ranged from 2.9 to 20%, whichwere within the acceptable level (Table 1).

To test the specificity of this protocol, human genomic DNA wasused as template. The standard curve was not altered when humangenomic DNA template was spiked into the sample, and no inter-ference was observed in the current protocol when contaminatinghuman genomic DNA was added (Fig. 2).

Amplification efficiency of intact genomic and fragmentized DNA

The amplification efficiency was obtained for intact genomicDNA; however, during the harsh purification process, the DNA mol-ecules in the OsrHSA preparations are sheared into small pieces, soit was essential to understand the potential difference in amplifica-tion efficiency between intact genomic and fragmented DNA. Tomimic harsh protein processing, the intact rice genomic DNA wasfragmented by sonication for 2 min and 10 min to obtain DNA frag-ment size ranges of 300 to 20,000 bp (Fig. 3A). We then comparedthe standard curves of the two samples. The curves showed no sig-nificant difference in amplification efficiency between the intactrice genomic and fragmented DNA (Fig. 3B), but the Ct values fromthe fragmented DNA were slightly lower than those of the intactgenomic DNA. This result indicated that the protocol for the quan-titation of residual DNA is reliable, accurate, and reproducible.

Characterization of residual DNA extraction from OsrHSA

Efficient DNA extraction from a protein solution is a key pointfor the precise quantification of residual DNA. In this study, weused the Wako DNA Extractor Kit to isolate residual DNA [23].Four OsrHSA preparations from different processing steps weretested using this protocol. Rice genomic DNA (1000 pg/ml) wasspiked into the OsrHSA preparations and was then extracted.The resulting DNA samples were then amplified. The recoveryrates ranged from 55.0 to 125.3% for the TaqMan qPCR systemand from 80.7 to 122.2% for the SYBR Green I qPCR system,

6 Quantitation of the residual DNA from rice-derived recombinant human serum albumin / Z. Chen et al. / Anal. Biochem. 450 (2014) 4–10

indicating that the recovery rate of the SYBR Green I qPCR wasbetter than that of TaqMan qPCR (Table 2). As shown in Table 3,at least 80% recovery from different levels of spiked DNA in prep-aration D was achieved. Our results showed the overestimatedquantification values of residual DNA in specific samples (Tables2 and 3); however, the mechanism is still obscure. A supplementof carrier RNA is capable of preventing nonspecific absorption andreducing the loss of DNA, and this is closely associated with anenhancement in DNA recovery [18,21]. In addition, the possibility

of other interfering factors cannot be excluded, for example, otherresiduals from the sample matrix and DNA extraction kits [21].Normally, the accepted criterion for the recovery assay is the abil-ity to distinguish a 2- to 4-fold difference in the target DNA con-tent [18]. Our results confirmed that the accuracy of the protocolfor quantitating residual DNA from OsrHSA met that criterion,suggesting that the residual DNA that was purified fromthe OsrHSA solution was acceptable for further quantificationanalysis.

Fig.1. Rice genomic DNA amplification using qPCR based on TaqMan and SYBR Green I. The template DNA concentration ranged from 2 ! 104 to 0.2 pg by serial 10-folddilution. (A) Amplification products of qPCR by 1% AGE. Lane a: products of qPCR based on the SYBR Green I reaction; lane b: products of qPCR based on the TaqMan reaction.(B) Melting curves of the qPCR products based on the SYBR Green I reaction. (C,D) Amplification plot for qPCR based on the SYBR Green I reaction and TaqMan reaction. (E,F)Standard curve for qPCR based on SYBR Green I and the TaqMan reaction.

Quantitation of the residual DNA from rice-derived recombinant human serum albumin / Z. Chen et al. / Anal. Biochem. 450 (2014) 4–10 7

We also evaluated the repeatability of the assay by preparingthree replicated spiked controls at 100 pg/ml in the preparationD solution. As shown in Table 4, the results indicated that it hasgood repeatability. The SDs of the Ct values and calculated concen-tration were 0.13 and 6.9, respectively. The CVs of the Ct values andcalculated concentration were 0.61 and 7.3%, respectively. Thequantitation limit was performed at 4 pg/ml. As shown in Table 5,the difference log10 of the spiked control at 4 pg/ml is below 0.2.Therefore, the extraction method has quantitation limits of at least4 pg/ml.

Table 1Intra- and interassay reproducibility of real-time PCR.

Sample (ng/ml) Intraassay Interassay

Ct value Calculated (ng/ml) Ct value Calculated (ng/ml)

Mean ± SD CV (%) Mean ± SD CV (%) Mean ± SD CV (%) Mean ± SD CV (%)

1000 13.08 ± 0.08 0.61 933.13 ± 54.59 5.85 12.73 ± 0.28 2.20 971.47 ± 28.0 2.90100 16.01 ± 0.09 0.56 109.69 ± 7.61 6.94 15.85 ± 0.26 1.64 108.90 ± 8.80 8.0810 19.41 ± 0.12 0.62 10.61 ± 0.94 8.86 19.11 ± 0.25 1.31 11.23 ± 0.92 8.191 22.85 ± 0.12 0.51 0.99 ± 0.13 13.13 22.62 ± 0.19 0.84 0.95 ± 0.05 5.260.1 26.00 ± 0.27 1.04 0.10 ± 0.015 15.00 25.87 ± 0.42 1.62 0.10 ± 0.02 20.00

Fig.2. Standard curves generated from qPCR with or without human genomic DNA.

Fig.3. Standard curves using different rice genomic DNAs as standards. (A) AGE of rice genomic DNA fragments after sonication. (B) Gray indicates rice genomic DNA withoutsonication, purple indicates rice genomic DNA after 2 min of sonication on middle power at 30-s intervals, and green indicates rice genomic DNA after 10 min of sonication onmiddle power with 30-s intervals. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 2Recovery assay for different solution.

Sample SYBR Green I TaqMan

Actual (pg/ml) Calculated (pg/ml ± SD) Recovery (%) Calculated (pg/ml ± SD) Recovery (%)

A 1000 1100 ± 84 110.0 1050 ± 104 105.0B 1000 1045 ± 65 104.5 780 ± 90 78.0C 1000 1222 ± 15 122.2 1253 ± 61 125.3D 1000 807 ± 100 80.7 550 ± 75 55.0

Note. Samples A, B, C, and D represent the products from different step purifications as mentioned above.

Table 3Recovery of different spike concentrations.

Actual (pg/ml) Calculated (pg/ml ± SD) Recovery (%)

10 8 ± 2 80.040 32 ± 6 80.0200 200 ± 58 100.02000 2410 ± 236 120.5

8 Quantitation of the residual DNA from rice-derived recombinant human serum albumin / Z. Chen et al. / Anal. Biochem. 450 (2014) 4–10

Application for residual DNA quantification in OsrHSA

To assess the removal efficiency of residual DNA from eachpurification step and whether residual DNA meets the minimumlimit that is recommended by regulatory authorities, we carriedout the quantitative assay using a standard operating protocol(see Supplementary Document 1 in supplementary material) ofSYBR Green I qPCR. As shown in Table 6, the removal efficiencyof residual DNA from each processing step ranged from 92 lg to152 ng. The final OsrHSA product contained 76 pg/ml residualDNA in a total volume of 2 L (200 mg/ml), which amounted to3.8 ng per dosage (10 g/dosage) of OsrHSA in clinical applications.The result was lower than the 10 ng per dosage of residual DNAcontamination in recombinant biopharmaceutical protein that isrecommended by the World Health Organization (WHO) [10].Our results also demonstrate the highly efficient removal of resid-ual DNA from OsrHSA during manufacturing and processing.

In addition, the sequence analysis showed that the rice 5S rRNAgenes have only two SNPs compared with the 5S rRNA genes ofwheat, rye, and maize [24]. Therefore, it is possible that this estab-lished protocol could be used to quantify the residual DNA fromother cereal crop endosperm expression systems.

Conclusion

A reliable and precise assay is required to assess residual DNA inbiopharmaceutical products that are produced in different expres-sion systems. In this study, we developed a qPCR protocol forresidual DNA quantitation in the rice endosperm expression sys-tem. The sensitivity, accuracy, and precision of the qPCR assaywere optimized using the highly repetitive and conserved 5S rRNA

gene. The sensitivity and accuracy of the SYBR Green I qPCR andTaqMan qPCR assays reached 2 ! 104 to 0.2 pg of DNA, and thedetection limit reached as low as 0.02 pg of residual DNA. Thereproducibility and high specificity when using SYBR Green I qPCRwere studied. The quantitation limit of extraction reached approx-imately 4 pg/ml, which satisfies the requirement of limit of resid-ual DNA detection in biologics. Furthermore, our results indicatethat the rice endosperm expression platform is ideal for thelarge-scale production of recombinant pharmaceutical proteinsand has the advantage of very less residual DNA in final pharma-ceuticals because the programmed cell death happened in riceendosperm cells during seed maturation. Using this protocol, theresidual DNA from primary purification solution to the final OsrH-SA product was efficiently removed in quantities of 92 lg to152 ng, and the final concentration was 3.8 ng per dosage, whichwas lower than that required by the WHO.

Acknowledgments

This work was supported by the Major Projects of NationalEssential New Drug Research and Development of China(2013ZX09102038) and the National High-Tech R&D Program863 of China (2011AA100604).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ab.2013.12.033.

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Table 4Repeatability of residual DNA extraction at 100 pg/ml.

Replicate Ct value Calculated (pg/ml)

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Table 5Quantitation limits of residual DNA purification.

Replicate Actual (pg/ml) Calculated (pg/ml) Log10 difference

1 4 2.66 0.182 4 2.93 0.143 4 3.44 0.07Mean 4 3.01 0.13

Table 6Residual DNA contents of the four samples quantified by SYBR Green qPCR.

Preparation Volume(L)

OsrHSAconcentration(mg/ml)

Calculatedresidual DNAconcentration(pg/ml)

TotalresidualDNA(lg)

Removalefficiency(%)

A 2300 3 40 ± 6.5 92.0 –B 400 3 118 ± 18.1 47.2 48.7C 160 3 117 ± 12.0 18.7 60.2D 2 200 76 ± 9.0 0.152 99.2

Note. Volume is total sample volume in the large-scale purification process. Totalresidual DNA is measured from calculated residual DNA concentration ! volume.

Quantitation of the residual DNA from rice-derived recombinant human serum albumin / Z. Chen et al. / Anal. Biochem. 450 (2014) 4–10 9

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10 Quantitation of the residual DNA from rice-derived recombinant human serum albumin / Z. Chen et al. / Anal. Biochem. 450 (2014) 4–10