CN114891861A - RAA kit for detecting CHO cell residual DNA in antibody and detection method thereof - Google Patents

Abstract

The invention relates to a kit, in particular to an RAA kit for detecting CHO cell residual DNA in an antibody and a detection method thereof. An RAA kit for detecting host cell residual DNA in an antibody comprises a primer pair and a probe; the invention completes the detection of the host cell residual DNA in the monoclonal antibody sample within 20min by applying the real-time fluorescence RAA method for the first time, has the detection sensitivity meeting the corresponding requirement, can be used for quickly detecting the residual DNA in the sample, and provides great convenience for intermediate quality control of production enterprises and quick supervision of supervision departments. Although the real-time fluorescent quantitative PCR technology is mature and can quantitatively detect residual DNA in a sample, the method needs longer time for detection, which can reach 1.5-3 h, and the price of a commercialized kit is relatively higher, compared with a qPCR method RAA, the method has the advantages of lower price and short detection time, and can be used as a beneficial supplement of qPCR.

Description

RAA kit for detecting CHO cell residual DNA in antibody and detection method thereof

Technical Field

The invention relates to a kit, in particular to an RAA kit for detecting CHO cell residual DNA in an antibody and a detection method thereof.

Background

Host cell residual DNA refers to DNA fragments or longer molecules that may be present in a biological preparation produced by the cells of the host tissue. Currently, commonly used hosts for biologics include: among mammalian cell lines, baby hamster kidney cell lines, Chinese hamster ovary Cells (CHO), Vero, mouse myeloma NS0 cell lines, human embryonic kidney cell lines (HEK-293), yeast, Escherichia coli, and the like. Biological products such as recombinant protein drugs, antibody drugs, vaccines and the like are produced by microorganisms or animal cells which are passed through successive passages, and although a complicated purification process is performed, host cell residual DNA may remain in the products. The remaining host cell DNA generally has the same basic structural unit, but exists in different lengths and forms, all of which can lead to risks of infectivity, carcinogenicity, immunogenicity, and mutability; in view of the above risks, domestic and foreign regulatory authorities have developed corresponding guidelines and provided methods for detecting residual DNA. Three methods are already loaded in the 'Chinese pharmacopoeia' 2020 edition for detecting exogenous residual DNA, wherein a qPCR technology is a more common method, the method can not only accurately quantify, but also has higher sensitivity reaching fg level, and the detection accuracy is greatly improved. However, it is difficult to implement in the basement level and remote areas because it requires a precise and expensive qPCR instrument and the price of the related detection kit is high, and it has complicated sample pretreatment and relatively long detection time, and the difficulty of applying to quick detection is large. For real-time monitoring of enterprise production, there is a particular need for a rapid, inexpensive kit that can detect whether residual DNA in an antibody meets standards without the need for expensive equipment.

Recombinase-mediated nucleic acid amplification (RAA) relies primarily on recombinases, single-stranded binding proteins (SSBs), and DNA polymerases. The whole reaction is then completed with the aid of some auxiliary components, such as recombinase loading factors, dNTPs, Adenosine Triphosphate (ATP), high molecular weight polyethylene glycol, salt molecules, etc. The recombinase in the RAA is a recombinase obtained in a bacterium or a fungus. When the recombinase isothermal amplification technology reaction is started, recombinase is combined with a primer under the assistance of a loading factor to form a recombinase primer complex, the formed complex searches for a homologous sequence in double-stranded DNA, the homologous sequence invades the double-stranded DNA after positioning to form a D-loop structure, the double-stranded DNA is untied, and a single-stranded binding protein (SSB) is combined with a replaced DNA strand to prevent primer dissociation. Subsequently, the recombinase in the recombinase primer complex is hydrolyzed, and the 3' -end of the primer is exposed and binds to a DNA polymerase (Bsu or Sau), thereby starting the synthesis of the DNA strand. Finally, the two parent strands are separated to obtain two new complementary double strands, and the hydrolyzed recombinase can be immediately combined with a new primer to start another strand displacement reaction. The repetition of the above process allows the DNA amplification to accumulate exponentially. It completed the detection within 30 minutes. However, the 'fast' is an advantage and a disadvantage, and has a small linear range, and accurate quantification is difficult to realize when the content of the sample is extremely low, but the provided limit range still has reference value for quality control of the production process of an enterprise and government regulation.

Disclosure of Invention

Object of the Invention

The invention provides an RAA kit for detecting CHO cell residual DNA in an antibody and application thereof, the kit can finish detection within 20min, the detection precision accords with the regulation of national pharmacopoeia, and expensive instruments are not needed.

Technical scheme

A RAA kit for detecting CHO cell residual DNA in antibody is characterized by comprising

Primer F2: 5'-CATCAGTCACCCACATTTGTCGAGATGTTAATTACG-3'

Primer R2: 5'-CCTATAAATGCTGTTGCTATTACTGCGAATAG-3'

Probe P2: 5 '-CTATTCCTTCATGTAGGACGAGGAGTTTATTACGG (FAM-dt) T (THF) A (BHQ1-dt) ACACTA TAGTAG (3' -phosphate).

The kit is characterized by also comprising a detection unit tube filled with reaction dry powder, reaction buffer solution, magnesium acetate and positive control; the reaction dry powder is freeze-dried powder obtained by adopting a low-temperature freeze-drying technology, and mainly comprises recombinase, single-chain binding protein, DNA polymerase, dNTPs and ATP; the reaction buffer comprises the following components in percentage by weight: Tris-HCl buffer (pH 8.0) at 300mM, 120mM potassium acetate and 20% PEG 20000.

The RAA kit for detecting CHO cell residual DNA in antibody is characterized in that the following components are added into a reaction tube filled with the freeze-dried powder of claim 2: 2.1. mu.l of upstream primer (10. mu.M), 2.1. mu.l of downstream primer (10. mu.M), 0.6. mu.l of probe (10. mu.M), 25. mu.l of reaction buffer, 15.7. mu.l of double distilled water, 2. mu.l of template and 2.5. mu.l of magnesium acetate (280 mmol/L); the reaction conditions are as follows: at 39.2 deg.C, 60 cycles of 30 seconds each.

Advantageous effects

The invention completes the detection of the host cell residual DNA in the monoclonal antibody sample within 20min by applying the real-time fluorescence RAA method for the first time, has the detection sensitivity meeting the corresponding requirement, can be used for quickly detecting the residual DNA in the sample, and provides great convenience for intermediate quality control of production enterprises and quick supervision of supervision departments. Although the real-time fluorescent quantitative PCR technology is mature and can quantitatively detect residual DNA in a sample, the method needs longer time for detection, which can reach 1.5-3 h, and the price of a commercialized kit is relatively higher, compared with a qPCR method RAA, the method has the advantages of lower price and short detection time, and can be used as a beneficial supplement of qPCR.

Drawings

FIG. 1: RAA amplification profiles of different primer combinations;

FIG. 2: gel imaging maps of different primer combinations are shown, wherein M holes are 2k markers, and 1-3 holes are CHO1-F1/R1, F3/R3 and F4/R4 respectively; 5-6 wells are CHO2-F1/R1, F2/R2,7 wells negative control;

FIG. 3: RAA amplification plots at different primer concentrations;

FIG. 4: RAA amplification plots at different magnesium acetate concentrations in CHO 1;

FIG. 5: RAA amplification plots at different magnesium acetate concentrations in CHO 2;

FIG. 6: RAA amplification plots under different temperature conditions;

FIG. 7: real-time fluorescent RAA amplification specificity analysis map;

FIG. 8: real-time fluorescence RAA amplification sensitivity analysis graph;

FIG. 9: real-time fluorescent RAA samples were examined for amplification.

Detailed Description

Example 1 host cell DNA isolation

Extracting according to the instruction of a host cell residual DNA sample pretreatment kit (magnetic bead method), and comprising the following steps:

first, sample digestion

1. 100. mu.l of the sample to be tested was taken into a 1.5ml clean centrifuge tube.

2. For each 100. mu.l sample, 10. mu.l of 5M NaCl was added.

3. Adding protease K digestive juice, shaking, mixing, and water bath at 55 deg.C for 1 h.

Two, DNA binding

1. Taking out the sample from the water bath, quickly centrifuging for 30s, adding the working binding solution, and uniformly mixing by oscillation.

2. After 10s of rapid centrifugation, 200. mu.l of isopropanol and 10. mu.l of magnetic beads were added to the sample mixture.

3. Placing the centrifuge tube containing all the mixture on a vortex oscillator, oscillating for 5min, rapidly centrifuging for 10s, and standing on a magnetic separation rack.

4. After the solution was clarified and the beads were completely separated, the supernatant was carefully removed with a pipette tip. The time for waiting for the complete separation of the magnetic beads is about 3-5 min.

Third, DNA washing

1. Taking down the centrifuge tube containing the magnetic beads from the magnetic separation frame, adding 700 mu l of washing solution A, and oscillating for 10s to uniformly mix the magnetic beads and the washing solution A; after 10s of rapid centrifugation, the centrifuge tubes were replaced on a magnetic separation rack. And after the solution is clarified and the magnetic beads are completely separated, removing the supernatant by using a gun head, and finishing the washing of the magnetic beads for the 1 st time.

2. Taking down the centrifuge tube containing the magnetic beads from the magnetic separation frame, adding 700 mu l of washing solution B, and oscillating for 40s to uniformly mix the magnetic beads and the washing solution B; after 10s of rapid centrifugation, the centrifuge tubes were replaced on a magnetic separation rack. And after the solution is clarified and the magnetic beads are completely separated, removing the supernatant by using a gun head, and finishing the washing of the magnetic beads for the 2 nd time.

3. In order to ensure that the liquid is sufficiently removed, the centrifugal tube can be quickly centrifuged again for 10s and placed on a magnetic separation rack, and after the magnetic beads are completely separated, residual liquid is carefully sucked and removed by using a 10-microliter gun head.

4. And (4) taking the centrifugal tube from the magnetic separation frame, opening a tube cover, drying at room temperature for 30 s-3 min, and removing residual ethanol.

Fourth, DNA elution

1. And adding 100 mu l of eluent preheated at 70 ℃ along the wall of the centrifugal tube, slightly oscillating for 5s by using a vortex oscillator to uniformly mix the magnetic beads and the eluent, carrying out water bath at 70 ℃ for 7min, and uniformly oscillating for 2-3 times in the water bath process.

2. After incubation, the centrifuge tubes were centrifuged at high speed for 1min, then placed statically on a magnetic separation rack, and after magnetic beads were separated, the solution was carefully transferred to a clean centrifuge tube with a pipette tip.

3. And (3) rapidly centrifuging the centrifuge tube obtained in the last step for 10s, standing the centrifuge tube on a magnetic separation frame, and transferring the solution to a clean centrifuge tube again by using a gun head after the magnetic beads are separated, so as to obtain the sample purification solution.

EXAMPLE 2 selection of amplified fragments of interest and design and screening of primer probes

1. Design of primers and probes

Referring to the Chinese Pharmacopoeia, United States Pharmacopeia, a conserved sequence in CHO cell DNA was selected as a target sequence, and the target sequence is shown in Table 1 and includes a target sequence 1(CHO cell Alu-like repeat Clone250) (GenBank accession No. J00052.1) and a target sequence 2(cytochrome b (cytb) gene) (GenBank accession No. AJ 973385.1); then searching in NCBI gene bank, designing corresponding primer and probe by combining with TwistDx company design manual, and synthesizing by committee biological engineering (Shanghai) corporation. The corresponding primer probes are shown in table 2.

Table 1: target sequence in CHO cell DNA

Table 2: CHO cell DNA primers and probes

2. Primer and probe screening process

2.1 composition of the kit

The reaction was performed using a RAA nucleic acid amplification kit (fluorescent type) (purchased from Hangzhou Mass Biotechnology Ltd.) comprising a detection unit tube containing reaction dry powder, a reaction buffer, magnesium acetate, a positive control, primers and a probe. The reaction dry powder is freeze-dried powder obtained by adopting a low-temperature freeze-drying technology, the main components of the reaction dry powder are recombinase, single-strand binding protein, DNA polymerase, dNTPs and ATP, and the dry powder is relatively stable in form components and convenient to store and transport. The reaction buffer comprises the following components in percentage by weight: Tris-HCl buffer (pH 8.0) at a concentration of 300mM, 120mM potassium acetate and 20% PEG 20000 by mass; the magnesium acetate concentration was 280mM, and the primer and probe concentrations were 10. mu.M.

2.2 real-time fluorescent RAA detection reaction System

The reaction system is as follows: 2.1 mul of upstream primer (10 mul), 2.1 mul of downstream primer (10 mul), 0.6 mul of probe (10 mul), 25 mul of reaction buffer solution and 15.7 mul of double distilled water are respectively added into a detection unit tube filled with reaction dry powder, 2 mul of extracted sample DNA is added into the detection unit tube (the negative control is changed into 2 mul of double distilled water), finally 2.5 mul of magnesium acetate (280mM) is added on the tube cover of the detection unit tube, the tube cover is covered, the upper part and the lower part are slightly thrown and evenly mixed for 5 to 6 times, the mixture is centrifuged at low speed for 10 seconds, the detection unit tube is put into a real-time fluorescence quantitative PCR instrument for constant temperature amplification at 39.2 ℃ for 60 cycles, and each cycle is 30 seconds. Different primer pairs are combined and screened by adopting the method to obtain a pair of primers with the highest amplification efficiency, namely the optimal amplification primer.

2.3 establishment and optimization of real-time fluorescent RAA detection System

In order to obtain the optimal amplification result, the reaction conditions of the real-time fluorescence RAA are optimized, and the real-time fluorescence RAA reaction system is as follows: 2.1. mu.l of forward primer (10. mu.M), 2.1. mu.l of reverse primer (10. mu.M), 0.6. mu.l of probe (10. mu.M), 25. mu.l of reaction buffer, 15.7. mu.l of double distilled water, 2. mu.l of template, and 2.5. mu.l of magnesium acetate (280 mmol/L). The reaction conditions are as follows: at 39.2 deg.C, 60 cycles of 30 seconds each. Wherein, what needs to be optimized is: the concentration of the primers, the concentration of magnesium acetate and the reaction temperature.

2.4 primer concentration optimization

Using CHO cell DNA as a template, changing the adding amount of the primer according to a reaction system of 1.3 to make the final concentration of the primer be 0.25 mu M, 0.68 mu M and 0.84 mu M respectively to carry out RAA amplification, and screening out the optimal amplification concentration of the primer according to an amplification curve.

2.5 magnesium acetate concentration optimization

Using CHO cell DNA as a template, adding 2.2.5.1 screened optimal primer concentration according to a reaction system of 2.3, changing the adding amount of magnesium acetate to make the final concentration respectively 16.8mmol/L, 22.4mmol/L, 28mmol/L and 33.6mmol/L for RAA amplification, and screening the optimal amplification concentration of magnesium acetate according to an amplification curve.

2.6 reaction temperature optimization

Using CHO cell DNA as template, adding the selected optimal concentration of primer and optimal concentration of magnesium acetate according to 2.3 reaction system, changing reaction temperature to amplify the reaction at 38 deg.C, 39.2 deg.C, 40 deg.C and 41 deg.C, and selecting optimal amplification temperature according to amplification curve.

2.7 real-time fluorescent RAA specificity verification

In order to verify the specificity of the screened primers and probes, RAA amplification is carried out on positive recombinant plasmid plates containing CHO cell target sequences according to the screened optimal reaction conditions, and the specificity is judged according to the amplification result.

2.8 real-time fluorescent RAA sensitivity validation

The reference sample was diluted to 9 ng/. mu.l with a mother solution of a recombinant plasmid (900 ng/. mu.l) in sterile water, and then sequentially diluted in duplicate to six concentrations of 0.9 ng/. mu.l, 0.09 ng/. mu.l, 0.009 ng/. mu.l, 0.0009 ng/. mu.l, 0.00009 ng/. mu.l, and 0.000009 ng/. mu.l as templates, and the selected real-time fluorescent RAA optimal reaction conditions were examined. And determining the lower detection limit of the method according to the takeoff time and the fluorescence intensity of the amplification curve, and performing corresponding sensitivity analysis.

2.9 application of real-time fluorescence RAA technology in monoclonal antibody

And extracting DNA of the three batches of single antibody samples according to the instruction of the host cell extraction kit, amplifying the extracted DNA by adopting the screened optimal reaction conditions, and judging the condition of residual DNA in the samples according to the amplification result.

3. As a result:

3.1 screening results of primer pairs

In a real-time fluorescence RAA amplification experiment, other conditions are unchanged, different primer pairs are added into a reaction octant tube to carry out amplification primer screening, the screened primer pairs comprise four pairs of primers F1/R1, F2/R2, F3/R3 and F4/R4 in a target sequence 1 and three pairs of primers F1/R1, F2/R2 and F3/R3 in a target sequence 2, the final amplification result is shown in figure 1, and in the four pairs of primers in the target sequence 1, the primers F1/R1 and F4/R4 are amplified, wherein F4/R4 has short amplification time, strong fluorescence intensity and good amplification effect; in the target sequence 2, the primers F1/R1, F2/R2 and F3/R3 are amplified, wherein the take-off time of the primers F1/R1 and the take-off time of the primers F2/R2 are short, the fluorescence intensity is strong, and the amplification fluorescence intensity of the primers F2/R2 is strongest, so that F4/R4 in the target sequence 1 and F2/R2 in the target sequence 2 are selected as CHO cell DNA amplification primers respectively, and the amplification effect of the primers F2/R2 in the target sequence 2 in the two pairs of amplification primers is better.

The corresponding primer sequences were:

target sequence 1, F4: 5'-CTACCAGAGGTCCTGAGTTCAATTCCCAGCAACTAC-3'

R4:5’-CTGTCTTCACAACATAGTTAGAAAAGACAGGTATC-3’

Target sequence 2, F2: 5'-CATCAGTCACCCACATTTGTCGAGATGTTAATTACG-3'

R2:5’-CCTATAAATGCTGTTGCTATTACTGCGAATAG-3’

3.2 electrophoretic detection results

The ultraviolet gel imaging result of the PCR amplification product is shown in FIG. 2, wherein the primer pair F1/R1 and F4/R4 in the target sequence 1 and the primer pair F1/R1 and F2/R2 in the target sequence 2 show specific amplification bands, the size of the amplified target fragment is between 100-250bp, wherein the amplification band of the primer in the target sequence 2 is clearer, and no specific amplification band appears in other primer pairs and negative control. Preliminary verification shows that the primers F4/R4 in the target sequence 1 and F2/R2 in the target sequence 2 can be amplified to obtain a target fragment in real-time fluorescence RAA amplification, and the amplification effect of the primers F2/R2 in the target sequence 2 is better.

The optimal primer pair of CHO cell DNA is selected as F4/R4 in a target sequence 1 and F2/R2 in a target sequence 2 by real-time fluorescent RAA amplification and PCR amplification, namely:

target sequence 1, F4: 5'-CTACCAGAGGTCCTGAGTTCAATTCCCAGCAACTAC-3'

R4:5’-CTGTCTTCACAACATAGTTAGAAAAGACAGGTATC-3’

Target sequence 2, F2: 5'-CATCAGTCACCCACATTTGTCGAGATGTTAATTACG-3'

R2:5’-CCTATAAATGCTGTTGCTATTACTGCGAATAG-3’

3.3 concentration optimization results of primers

At 39.2 ℃, the optimal primers of the target sequence are selected to amplify F4/R4 in the target sequence 1 and F2/R2 in the target sequence 2 by using CHO cell DNA as a template. The amplification results are shown in FIG. 3, when the amounts of primers added were 1.3. mu.l (0.26. mu.M), 1.7. mu.l (0.34. mu.M), and 2.1. mu.l (0.42. mu.M), respectively. For the primer pair F4/R4 of the target sequence 1, amplification is carried out when the adding amount of the primers is 1.3. mu.l, 1.7. mu.l and 2.1. mu.l, and the takeoff time of the amplification curve is slightly short and the fluorescence intensity is strong when the adding amount of the primers is 1.7. mu.l and 2.1. mu.l. For the primer pair F2/R2 of the target sequence 2, amplification is realized when the adding amount of the primers is 1.3. mu.l, 1.7. mu.l and 2.1. mu.l, the takeoff time of the amplification curve is similar, wherein the fluorescence intensity is strongest when the adding amount is 2.1. mu.l, and the amplification effect is best.

In conclusion, when the addition amount of the primers is 2.1. mu.l, both pairs of primers have better amplification, but compared with the primer pair F4/R4 in the target sequence 1, the curve obtained by amplifying the primer pair F2/R2 in the target sequence 2 has low take-off time, strong fluorescence intensity and better overall amplification effect, so the optimal primers are the primers F2/R2 in the target sequence 2, and the optimal dosage of the primers is 2.1. mu.l.

3.4 optimization of magnesium acetate

The amounts of magnesium acetate added were varied to 1.3. mu.l (7.28mmol/L), 1.5. mu.l (8.4mmol/L), 1.7. mu.l (9.25mmol/L), 2.0. mu.l (11.2mmol/L), 2.1. mu.l (11.76mmol/L), 2.5. mu.l (14mmol/L) and 3.0. mu.l (16.8mmol/L), respectively. At 39.2 ℃, the primers F4/R4 in the target sequence 1 and F2/R2 in the target sequence 2 are selected and amplified by taking CHO cell DNA as a template.

As shown in FIGS. 4 and 5, the amplification results were shown in the case of the primer F4/R4 of the target sequence 1, wherein the magnesium acetate was added in an amount of 1.3. mu.l, 1.7. mu.l, 2.1. mu.l, or 2.5. mu.l, and the takeoff time of the amplification curve was short and the fluorescence intensity was strong in the case of 1.7. mu.l, 2.1. mu.l, or 2.5. mu.l, and the takeoff time of the amplification curve was shortest in the case of 2.5. mu.l. For the primer pair F2/R2 in target sequence 2, amplification was achieved at magnesium acetate additions of 1.3. mu.l, 1.5. mu.l, 1.7. mu.l, 2.0. mu.l, 2.1. mu.l, 2.5. mu.l and 3.0. mu.l, with similar takeoff times for the amplification curves, with slightly faster amplification and less difference in fluorescence intensity at 2.0. mu.l, 2.5. mu.l and 3.0. mu.l.

Compared with the primer F4/R4 in the target sequence 1, the primer pair F2/R2 in the target sequence 2 has shorter takeoff time of an amplification curve, stronger fluorescence intensity and better amplification effect, so the optimal primer is the primer F2/R2 in the target sequence 2, the two pairs of primers in the two target sequences have better amplification effect when the addition of magnesium acetate is 2.5 mul, and the optimal dosage of the magnesium acetate is 2.5 mul.

3.5 reaction temperature optimization

The reaction temperature was varied to amplify at 38 ℃, 39.2 ℃, 40 ℃ and 41 ℃ respectively, and the amplification results are shown in FIG. 6. only at 39.2 ℃, the primer pair F4/R4 in the target sequence 1 and the primer pair F2/R2 in the target sequence 2 amplified, and the primers F2/R2 in the target sequence 2 had a shorter takeoff time and stronger fluorescence intensity than the primers F4/R4 in the target sequence 1. Therefore, the optimal amplification primer is the primer F2/R2 in the target sequence 2, and the optimal amplification temperature is 39.2 ℃.

And (4) conclusion: real-time fluorescent RAA optimal detection system

In order to obtain an optimal reaction system, two pairs of amplification primers obtained by preliminarily screening primer pairs with different combinations are F4/R4 in a target sequence 1 and F2/R2 in a target sequence 2, and then the concentrations of the two pairs of primers, the magnesium acetate concentration and the reaction temperature are respectively optimized. In the optimization of primer concentration, the amplification was carried out while varying the final concentrations of primers to 0.26. mu.M, 0.34. mu.M and 0.42. mu.M, respectively, and the results showed that the amplification effect was the best when the concentration was 0.42. mu.M. When the final concentration of magnesium acetate is changed to 7.28mmol/L, 8.4mmol/L, 9.25mmol/L, 11.2mmol/L, 11.76mmol/L, 14mmol/L and 16.8mmol/L respectively, the optimal amplification concentration of magnesium acetate is 14 mmol/L. When the reaction temperature was changed so that the amplification was carried out at four different temperatures of 38 deg.C, 39.2 deg.C, 40 deg.C and 41 deg.C, respectively, the optimum amplification temperature was 39.2 deg.C.

In the optimization of the above three conditions, the amplification effect of the primers F2/R2 in the target sequence 2 is better than that of the primers F4/R4 in the target sequence 1. Therefore, the optimal primer pair of the reaction is the primer pair F2/R2 in the target sequence 2, the size of the amplified target fragment is 199bp, the optimal primer concentration is 0.42 mu M, the optimal amplification temperature is 39.2 ℃, the optimal concentration of magnesium acetate is 14mmol/L, and the final reaction system is shown in Table 3.

Table 3: real-time fluorescence RAA optimal reaction system (50. mu.l)

3.6 real-time fluorescent RAA specificity assay

The recombinant plasmid for reference containing CHO cell target gene segment and the recombinant plasmid for reference containing Vero cell target segment are used as templates respectively, RAA amplification is carried out according to the screened reaction systems, the amplification result is shown in figure 7, and the primer pair F2/R2 in the target sequence 2 has better amplification effect on the recombinant plasmid for reference containing CHO cell target gene segment and has no amplification on the recombinant plasmid for reference containing Vero cell target segment, so that the screened primer probe has better specificity.

3.7 real-time fluorescent RAA sensitivity analysis

Diluting the reference substance with the recombinant plasmid (with the concentration of 900 mu g/ml) to 9 mu g/ml by using sterile water, and then sequentially diluting the reference substance by using six concentrations of 0.9 mu g/ml, 0.09 mu g/ml, 0.009 mu g/ml, 0.0009 mu g/ml, 0.00009 mu g/ml and 0.000009 mu g/ml as templates to carry out real-time fluorescence RAA detection, wherein the detection result is shown in figure 8, the fluorescence intensity is gradually increased along with the increase of the concentration, and the fluorescence intensity is not increased when the concentration is higher; the takeoff time is gradually prolonged along with the reduction of the concentration, the takeoff time is decreased in a certain proportion when the concentration is higher, the change is smaller when the concentration is lower, and when the concentration is lower than 0.00009 mu g/ml, amplification does not exist any more, namely the detection lower limit of the method is 0.18 ng/dose, and the amplification takeoff time is within 10 min.

3.8 detection results of real-time fluorescence RAA technique in monoclonal antibody

And (2) extracting DNA of three batches of single antigen liquid by using a host cell DNA extraction kit, respectively taking the extracted DNA and the recombinant plasmid (the concentration is 0.00009 mu g/ml) for the reference as templates, and performing RAA amplification according to the conditions, wherein the amplification result is shown in figure 9, two batches of three batches of sample extracted DNA are slightly amplified, the takeoff time of an amplification curve is slightly lower than that of the recombinant plasmid for the reference, the fluorescence intensity is weak, and the monoclonal antibody stock solution with DNA residues can be obtained. The protein content of the test solution is known to be 10mg/ml, and according to the formula: the exogenous DNA residual quantity (ng/dose) ═ the mean value of the DNA detection value of the sample solution/the final protein content of the sample solution x dose, the obtained DNA residual quantity is lower than 0.18 ng/dose, and the limit requirement that the residual DNA in the monoclonal antibody stock solution is less than 1 ng/dose specified in the enterprise quality standard is met. As can be seen from the sensitivity analysis, when the concentration of the recombinant plasmid for reference is higher, the amplification takeoff time and the template concentration have a certain proportional relationship. And (3) detecting the extracted DNA of the three samples by adopting a qPCR method, wherein the result shows that the residual quantity of the DNA of the three samples is lower than 0.04 ng/dose, and the limit requirement that the residual DNA in the monoclonal antibody stock solution is less than 1 ng/dose specified in the enterprise quality standard is met.

Sequence listing

<110> food and drug supervision and inspection research institute of Jiangsu province

<120> RAA kit for detecting CHO cell residual DNA in antibody and detection method thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 240

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<213> CHO cell DNA target sequence 1(2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 1

acgattatct gaaaagatgc cccgctacag ggctggagag atggctcaga ggttaagagc 60

actgactgct ctaccagagg tcctgagttc aattcccagc aactacatgg tggctcacaa 120

ccatccgtta tgagacctgg tgccctcttc tggtgtgcag atatacatgg aagcagaatg 180

ttgtatacat taataaataa ataaaatctt tttaaaaaaa agatgctcag atacctgtct 240

<210> 2

<211> 480

<212> DNA

<213> CHO cell DNA target sequence 2(2 Ambystoma laterale x Ambystoma jeffersonanum)

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tatacattac acatcagata ctactacagc attctcatca gtcacccaca tttgtcgaga 120

tgttaattac ggctgactaa tccgctacct acacgctaac ggagcttcaa tattctttat 180

ctgcctattc cttcatgtag gacgaggagt ttattacggt tcatacacta tagtagaaac 240

ttgaaacgta ggtattgtcc tactattcgc agtaatagca acagcattta taggctatgt 300

actaccatga ggtcaaatat cattctgagg agctacagta atcactaacc ttttatcagc 360

tatcccttat ataggtacca ctctagtaga atgaatctga gggggattct ctgtagacaa 420

agccacacta acacgcttct tcgcattcca tttcatccta ccattcatta tcgctgccct 480

<210> 3

<211> 35

<212> DNA

<213> F1(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 3

cagggctgga gagatggctc agaggttaag agcac 35

<210> 4

<211> 36

<212> DNA

<213> R1(2 Ambystoma laterale x Ambystoma jeffersonianum)

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ccatgttatc tgcacaccag aagagggcac caggtc 36

<210> 5

<211> 36

<212> DNA

<213> F2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 5

cagaggttaa gagcactgac tgctctacca gaggtc 36

<210> 6

<211> 36

<212> DNA

<213> R2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 6

cacaacatag ttagaaaaga caggtatctg agcatc 36

<210> 7

<211> 35

<212> DNA

<213> F3(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 7

ctgctctacc agaggtcctg agttcaattc ccagc 35

<210> 8

<211> 36

<212> DNA

<213> R3(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 8

tgtatataca acattctgct tccatgtata tctgca 36

<210> 9

<211> 36

<212> DNA

<213> F4(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 9

ctaccagagg tcctgagttc aattcccagc aactac 36

<210> 10

<211> 35

<212> DNA

<213> R4(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 10

ctgtcttcac aacatagtta gaaaagacag gtatc 35

<210> 11

<211> 37

<212> DNA

<213> F1-2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 11

ctactacagc attctcatca gtcacccaca tttgtcg 37

<210> 12

<211> 36

<212> DNA

<213> R1-2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 12

catcagtcac ccacatttgt cgagatgtta attacg 36

<210> 13

<211> 36

<212> DNA

<213> F2-2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 13

catcagtcac ccacatttgt cgagatgtta attacg 36

<210> 14

<211> 32

<212> DNA

<213> R2-2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 14

cctataaatg ctgttgctat tactgcgaat ag 32

<210> 15

<211> 35

<212> DNA

<213> F3-2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 15

catttgtcga gatgttaatt acggctgact aatcc 35

<210> 16

<211> 33

<212> DNA

<213> R3-2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 16

ctactagagt ggtacctata taagggatag ctg 33

<210> 17

<211> 48

<212> DNA

<213> Probe P1(2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 17

cccagcaact acatggtggc tcacaaccat ccgtgagacc tggtgccc 48

<210> 18

<211> 49

<212> DNA

<213> Probe P2(2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 18

ctattccttc atgtaggacg aggagtttat tacggtaaca ctatagtag 49

Claims (3)

1. A RAA kit for detecting CHO cell residual DNA in antibody is characterized by comprising

Primer F2: 5'-CATCAGTCACCCACATTTGTCGAGATGTTAATTACG-3'

Primer R2: 5'-CCTATAAATGCTGTTGCTATTACTGCGAATAG-3'

Probe P2: 5 '-CTATTCCTTCATGTAGGACGAGGAGTTTATTACGG (FAM-dt) T (THF) a (BHQ1-dt) ACACTA TAGTAG (3' -phosphate).

2. The RAA kit for detecting CHO cell residual DNA in antibody according to claim 1, characterized in that it further comprises a detection unit tube containing reaction dry powder, reaction buffer, magnesium acetate, positive control; the reaction dry powder is freeze-dried powder obtained by adopting a low-temperature freeze-drying technology, and mainly comprises recombinase, single-chain binding protein, DNA polymerase, dNTPs and ATP; the reaction buffer comprises the following components in percentage by weight: Tris-HCl buffer pH =8.0 at a concentration of 300mM, 120mM potassium acetate and PEG 20000 at a mass fraction of 20%.

3. The method for detecting the RAA kit for detecting the residual DNA of the CHO cells in the antibody according to claim 2, wherein the following components are added into a reaction tube filled with the lyophilized powder according to claim 2: 10 mu M2.1 mu L of upstream primer, 10 mu M2.1 mu L of downstream primer, 10 mu M0.6 mu L of probe, 25 mu L of reaction buffer, 15.7 mu L of double distilled water, 2 mu L of template and 280 mmol/L2.5 mu L of magnesium acetate; the reaction conditions are as follows: at 39.2 deg.C, 60 cycles of 30 seconds each.

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