US 12,735,460 B2 · Method for preparing and certifying novel coronavirus nucleocapsid protein

Abstract

Disclosed in the present invention is a method for preparing and certifying a novel coronavirus nucleocapsid protein (nCOV N protein) reference material, the method including: purification and dilution of a raw material of the nCOV N protein; preliminary uniformity test and packaging of solution of a candidate nCOV N protein reference material; characterization of physicochemical properties of the solution of the candidate nCOV N protein reference material in the packaging units; uniformity and stability test of the nCOV N protein reference material in the packaging unit; measurement of standard N protein content of the nCOV N protein reference material, determination and statistical test of standard value of the nCOV N protein reference material; and uncertainty assessment of certified result of the standard value of the nCOV N protein reference material.

A method for preparing and certifying a novel coronavirus nucleocapsid protein (nCOV N protein), comprising the following steps:

I. purification and dilution of a raw material of the nCOV N protein
(a) purification of the raw material of the nCOV N protein
the nCOV N protein is an N protein sequence of SEQ ID NO:
1 as set forth below, or a protein sequence with 3 to 10 histidine or lysine attached to one or both ends of the N protein sequence of SEQ ID NO:
1:
1
MSQNGPQNQR NAPRITFGGP SDSTGSNQNG ERSGARSKQR
RPQGLPNNTA SWFTALTQHG KEDLKFPRGQ GVPINTNSSP
81
DDQIGYYRRA TRRIRGGDGK MKDLSPRWYF YYLGTGPEAG
LPYGANKDGI IWVATEGALN TPKDHIGTRN PANNARIVLQ
161
LPQGTTLPKG FYAEGSRGGS QASSRSSSRS RNSSRNSTPG
SSRGTSPARM AGNGGDAALA LLLLDRLNQL ESKMSGKGQQ
241
QQGQTVTKKS AAEASKKPRQ KRTATKAYNV TQAFGRRGPE
QTQGNFGQCE LIRQGTDYKH WPQIAQFAPS ASAFFGMSRI
321
GMEVTPSGTW LTYIGAIKLD DKDPNFKDQV ILLNKHIDAY
KTFPPTEPKK QRKKKADETQ ALPCRQKKQQ TVFLLPAADL
481
DQFSKQLQCS MSSADSTQA
the raw material is from a commercially available nCOV N protein biochemical reagent with a purity of 90% to 95%, for obtaining a candidate nCOV N protein reference material with a high purity, the biochemical reagent is purified by an isoelectric focusing method and/or a molecular sieve method to give the candidate nCOV N protein reference material with a purity of 98.5% or above;
(b) preliminary measurement of concentration of the candidate nCOV N protein reference material
13C isotope-labeled valine and phenylalanine with a concentration of 0.001 to 1 mg/mL are added into to 1 to 100 μL of the candidate nCOV N protein reference material, the mixture is subject to centrifugal concentration or nitrogen blowing, and then 100 to 1000 μL of concentrated hydrochloric acid with a concentration of 6 to 8 mol/L is added thereto, and sealed while passing nitrogen for protection, the mixture is hydrolyzed in an oven at 110° C. to 150° C. for 24 h to 72 h to give a hydrolysate, and after hydrolysis, an amino acid content in the hydrolysate is measured by high performance liquid chromatography-isotopic dilution mass spectrometry (HPLC-IDMS) using national reference materials of valine and phenylalanine as standard, and an initial concentration of the candidate nCOV N protein reference material is calculated according to the N protein sequence of SEQ ID NO:
1;
(c) dilution of the candidate nCOV N protein reference material
the candidate nCOV N protein reference material, of which the concentration has been preliminarily measured, is diluted with PBS as solvent to a concentration ranging from 0.05 to 0.2 mg/g to give a diluted solution of the candidate nCOV N protein reference material;
(II). preliminary uniformity test and packaging of the solution of the candidate nCOV N protein reference material
(d) preliminary uniformity test of the solution of the candidate nCOV N protein reference material
3 sub-samples are taken from each of upper, middle, and lower parts of the diluted solution of the candidate nCOV N protein reference material in step (c), respectively, to obtain a total of 9 test samples, the 9 test samples are numbered and arranged in order, and the content of the nCOV N protein in each test sample is subject to a first test by isotopic dilution mass spectrometry (IDMS), amino acid analysis (AAA), or immunoassay, then the order is disrupted, and the content of the nCOV N protein in each test sample is subject to a second test, the above operations are repeated until each test sample is tested three times, obtaining 27 test results, and the uniformity of the content x of the nCOV N protein in the taken test sample is calculated according to the formulas below;
when m=9 samples are taken, 9 sets of precisely measured data are obtained according to the above test method under a condition of repeatability n=3, as shown below:
No. 1:
x11, x12 . . . x1n, with a mean value of x1
No. 2:
x21, x22 . . . x2n, with a mean value of x2
. . .
No. m:
xm1, xm2 . . . xmn, with a mean value as below:
mean
⁢
value
⁢
x
=
=
∑
i
=
1
m
x
_
i
m
Formula
⁢
1
Statistical
⁢
value
⁢
N
⁢
N
=
m
·
n
Formula
⁢
2
sum of squares of between-group differences:
Q
1
=
∑
i
=
1
m
n
⁡
(
x
¯
i
-
x
=
)
2
Formula
⁢
3
sum of squares of intra-group differences:
Q
2
=
∑
i
=
1
m
∑
j
=
1
n
n
⁡
(
x
ij
-
x
_
i
)
2
Formula
⁢
4
degree of freedom between groups:
v1=m−1;
degree of freedom within groups:
v
2
=
N
-
m
S
1
2
=
Q
1
v
1
Formula
⁢
5
S
2
2
=
Q
2
v
2
Formula
⁢
6
statistical value F:
F
=
S
1
2
S
2
2
Formula
⁢
7
wherein, S12 is variance between groups, S22 is variance within groups, according to degree of freedom v1, v2, and a given significance level of α=0.05, a critical value of Fα,(v1,v2) is obtained by table look-up, and an F value calculated from Formula 7 is compared with Fα, if F<Fα, it is considered that there is no significant difference between within-groups and between-groups, and the nCOV N protein in the taken test sample is uniform;
otherwise, the content of the nCOV N protein in the taken test sample is not uniform, and if the content of the nCOV N protein in the taken test sample is not uniform, the steps (a) to (c) are repeated until the solution of the candidate nCOV N protein reference material in the taken test sample passes the preliminary uniformity test;
(e) packaging of the solution of the candidate nCOV N protein reference material
the solution of the candidate nCOV N protein reference material passing the preliminary uniformity test of step (d) is packaged into silanized cryotubes with volume of 500 μL to 2 mL with 10 μL to 500 μL of the candidate solution in each silanized cryotube, the packaged solution of the candidate nCOV N protein reference material is labeled and consecutively numbered according to the packaging order to obtain a total of P packaging units, which are then stored in a refrigerator at −80° C.;
(III). characterization of physicochemical properties of the solution of the candidate nCOV N protein reference material in the packaging unit
(f) purity test of the nCOV N protein reference material in the packaging unit
the purity of the nCOV N protein reference material in the packaging unit is characterized by a universal protein purity analysis method;
(g) characterization of molecular weight of the nCOV N protein reference material in the packaging unit
a molecular weight of the nCOV N protein reference material in the packaging unit is determined by gel electrophoresis, and/or gel exclusion HPLC, and/or mass spectrometry, and mean value of the determination results should be consistent with theoretical molecular weight of the N protein sequence of SEQ ID NO:
1;
(h) protein identification of the nCOV N protein reference material in the packaging unit
the nCOV N protein is digested with trypsin and/or V8 protease, the peptide fragments generated from the protease digestion are identified by peptide mass fingerprinting or tandem mass spectrometry, and then a target protein is obtained by searching the SWISSPROT database through SQEUEST or MASCOT, and the result should be consistent with the protein corresponding to the N protein sequence of SEQ ID NO:1;
(i) de novo sequencing of the nCOV N protein reference material in the packaging unit
the nCOV N protein is digested with trypsin and/or V8 protease, and the digested peptide fragments are subject to de novo sequencing by HPLC-tandem mass spectrometry, and amino acid sequence of the peptide fragment is determined by b, y ion series generated from each peptide fragment;
amino acid sequences of all the obtained peptide fragments are spliced manually or by a random matching software or a third-party software to give a full-length sequence of the nCOV N protein which should be consistent with the protein sequence corresponding to the N protein of SEQ ID NO:
1;
or the nCOV N protein is digested with trypsin and/or V8 protease, the digested peptide fragments are isolated by reverse phase HPLC, all the peptide fragments with good isolation degree and correspondingly high peak signals are collected one by one, lyophilized, reconstituted in water containing 0.1% formic acid, and then spotted on a PVDF membrane, each collected peptide fragment is subject to de novo sequencing with a protein sequencer based on the Edman principle, sequences of all the peptide fragments are spliced to obtain a full-length sequence of the nCOV N protein, and the result should be consistent with the protein sequence corresponding to the N protein sequence of SEQ ID NO:
1;
(j) identification and certification of impurity proteins in the nCOV N protein reference material in the packaging unit
in the purity test of the nCOV N protein reference material in step (f), if an individual impurity has a content exceeding 1%, a fraction containing the impurity is collected out and identified according to steps (f) to (h) for impurity protein;
after identification, the impurity is purchased or expressed by recombination to made a standard of the impurity, and then a standard curve is plotted, with horizontal axis representing a concentration of the standard and vertical axis representing a corresponding signal;
otherwise, it is not required to identify and certify the impurity;
(IV). uniformity and stability test of the nCOV N protein reference material in the packaging unit
(k) uniformity test of the nCOV N protein reference material in the packaging unit the consecutively numbered packaging units are randomly sampled with a randbetween function in Excel, when P≤200, the number m of the sampled packaging units is not less than 11;
when 200<P≤500, the number m of the sampled packaging units is not less than 15;
when 500<P≤1000, the number m of the sampled packaging units is not less than 25;
when the packaging units P>1000, the number m of the sampled packaging units is not less than 30;
according to the sampling order, the m sampled packaging units are re-numbered and arranged in order, the N protein content of the nCOV N protein reference material in the sampled packaging unit is subject to a uniformity test by IDMS, or amino acid analysis, or immunoassay;
during the test, the N protein in the nCOV N protein reference material in each sampled packaging unit is subject to a first test according to the above arrangement order, and then the order is disrupted, and the N protein content in the nCOV N protein reference material in each sampled packaging unit is subject to a second test, the above operations are repeated until the N protein in the nCOV N protein reference material in each sampled packaging unit is subject to a nth test, wherein n is an integer greater than or equal to 3, to give n test results, and an uniformity of the N protein in the nCOV N protein reference material in the packaging units are calculated and counted out according to the formulas below;
when m samples are taken, m sets of precisely measured data are obtained according to the test method under a condition of repeatability n, as shown below:
No
.
1
:
x
11
,
x
12
⁢
…
⁢
x
1
⁢
n
,
with
⁢
a
⁢
mean
⁢
value
⁢
of
⁢
x
_
1
No
.
2
:
x
2
⁢
1
,
x
2
⁢
2
⁢
…
⁢
x
2
⁢
n
,
with
⁢
a
⁢
mean
⁢
value
⁢
of
⁢
x
_
2
No
.
m
:
x
m
⁢
1
,
x
m
⁢
2
⁢
…
⁢
x
¯
mn
,
with
⁢
a
⁢
mean
⁢
value
⁢
x
_
m
mean
⁢
value
⁢
x
=
=
∑
i
=
1
m
x
_
i
m
Formula
⁢
8
Statistical
⁢
value
⁢
N
⁢
N
=
m
·
n
Formula
⁢
9
sum of squares of between-group differences:
Q
1
=
∑
i
=
1
m
n
⁡
(
x
¯
i
-
x
=
)
2
Formula
⁢
10
sum of squares of intra-group differences:
Q
2
=
∑
i
=
1
m
∑
j
=
1
n
(
x
ij
-
x
¯
i
)
2
Formula
⁢
11
degree of freedom between groups:
v1=m−1;
degree of freedom within groups:
v
2
=
N
-
m
S
1
2
=
Q
1
v
1
Formula
⁢
12
S
2
2
=
Q
2
v
2
Formula
⁢
13
statistical value F:
F
=
S
1
2
S
2
2
Formula
⁢
14
wherein, S12 is variance between groups, S22 is variance within groups, according to degree of freedom, v1, v2, and a given significance level of α=0.05, a critical value of Fα,(v1,v2) is obtained by table look-up, and an F value calculated from Formula 14 is compared with Fα, if F<Fα, it is considered that there is no significant difference between within-groups and between-groups, and the N protein in the nCOV N protein reference material in the packaging unit is uniform;
otherwise, the N protein in the nCOV N protein reference material in the packaging unit is not uniform,
(l) long-term stability test of the nCOV N protein reference material in the packaging unit
a long-term stability test is performed for a period of not less than 6 months, in which 5 time points k are selected following a rule of “first more and then less”, and at each time point, the nCOV N protein reference material is sampled from at least 2 packaging units in step (e) to subject to test in triplicate by IDMS, and/or amino acid analysis, and/or immunoassay for the N protein content of the nCOV N protein reference material in each packaging unit, and then an arithmetic mean value Yi of the test results at each time point is calculated, and fitted with the corresponding measuring time Xi according to the linear model below:
Y
i
=
b
0
+
bX
i
Formula
⁢
15
in Formula 15:
b0—intercept, b—regression coefficient/slope;
Xi—test time, in month;
Yi—arithmetic mean value of test results of the N protein content of the nCOV N protein reference material at each time point;
the regression coefficient/slope and the intercept are calculated according to Formula 16 and Formula 17:
b
=
∑
i
=
1
k
(
X
i
-
X
¯
)
⁢
(
Y
i
-
Y
_
)
∑
i
=
1
k
(
X
i
-
X
¯
)
2
Formula
⁢
16
b
0
=
Y
_
-
b
·
X
¯
Formula
⁢
17
in Formula 16 and Formula 17:
Xi—test time at the ith time point, in month;
Yi—arithmetic mean value of test results of the N protein in the nCOV N protein reference material in the packaging units at the ith time point;
X—mean value of test time at all the time points, in month;
Y—arithmetic mean value of test results of the N protein in the nCOV N protein reference material in the packaging units at all the time points;
k—number of time points;
s is calculated according to Formula 18:
s
=
∑
i
=
1
k
(
Y
i
-
b
0
-
bX
i
)
2
k
-
2
Formula
⁢
18
various symbols in Formula 18 have the same meanings as those in Formula 15, Formula 16, and Formula 17,
s(b) is calculated according to Formula 19:
s
⁡
(
b
)
=
s
∑
i
=
1
k
(
X
i
-
X
¯
)
2
Formula
⁢
19
the value of t0.95,k−2 is obtained by look-up in the t distribution table;
if |b|<t0.95,k−2·s(b), the slope is not significant, indicating that the nCOV N protein reference material is stable for long term;
otherwise, it indicates that the nCOV N protein reference material is not stable;
(m) short-term stability test of the N protein content of the nCOV N protein reference material in the packaging unit
a short-term stability test is performed for a continuous period of not less than 7 days, in which 5 time points k are selected following a rule of “first more and then less”, and at each time point, the nCOV N protein reference material is sampled from at least 2 packaging units in step (e) and placed at a temperature ranging from −20° C. to 40° C. for inspection, the N protein content of the nCOV N protein reference material in each packaging unit is tested in triplicate by IDMS, and/or amino acid analysis, and/or immunoassay, and then an arithmetic mean value Yi of the test results at each time point is calculated, and fitted with the corresponding measuring time Xi according to the linear model below:
Y
i
=
b
0
+
bX
i
Formula
⁢
20
in Formula 20:
b0—intercept, b—regression coefficient/slope;
Xi—test time at the ith time point, in day;
Yi—arithmetic mean value of test results of the N protein content of the nCOV N protein reference material at the ith time point;
the regression coefficient/slope and the intercept are calculated according to Formula 21 and Formula 22:
b
=
∑
i
=
1
k
(
X
i
-
X
¯
)
⁢
(
Y
i
-
Y
_
)
∑
i
=
1
k
(
X
i
-
X
¯
)
2
Formula
⁢
21
b
0
=
Y
_
-
b
·
X
¯
Formula
⁢
22
in Formula 21 and Formula 22:
Xi—test time at the ith time point, in day;
Yi—arithmetic mean value of test results of the N protein content in the nCOV N protein reference material in the packaging units at the ith time point;
X—mean value of test time at all the time points, in day;
Y—arithmetic mean value of test results of the N protein content in the nCOV N protein reference material in the packaging units at all the time points;
k—number of time points;
s is calculated according to Formula 23:
s
=
∑
i
=
1
k
(
Y
i
-
b
0
-
bX
i
)
2
k
-
2
Formula
⁢
23
various symbols in Formula 23 have the same meanings as those in Formula 20, Formula 21, and Formula 22,
s(b) is calculated according to Formula 23:
s
⁢
(
b
)
=
s
∑
i
=
1
k
(
X
i
-
X
¯
)
2
Formula
⁢
23
the value of t0.95,k−2 is obtained by look-up in the t distribution table,
if |b|<t0.95,k−2·s(b), the slope is not significant, indicating that the nCOV N protein reference material is stable for short term;
otherwise, it indicates that the nCOV N protein reference material is not stable for short term;
(V). determination of standard value of the N protein content in the nCOV N protein reference material
(n) determination of N protein content in the nCOV N protein reference material by amino acid analysis-based HPLC-isotopic dilution mass spectrometer
first, nCOV N protein reference materials are randomly taken as samples from at least 5 packaging units in step (e), and the N protein content in each of the above packaging units is tested in triplicate according to the method of step (b) to give a total of at least 15 test results, which are tested according to the K-S single-sample normality test and for which Grabs method and/or Dixon method are used for outlier test;
for samples that pass the statistical test, the arithmetic mean value of all the results is taken as the standard value of the test results of HPLC-IDMS;
while for samples that fail to pass the statistical K-S single-sample normality test, Grabs method and/or Dixon method for outlier test, the number of samples is increased and the step (n) is repeated until passed;
(o) the N protein content of the nCOV N protein reference material is determined by peptide fragment analysis-based HPLC-IDMS, one or more of the following standard peptide fragments and corresponding isotope-labeled peptide fragments of the standard peptide fragments are synthesized, and the candidate reference material also comprises the following peptide fragments:
MSDNGPQNQR,
ITFGGPSDSTGSNQNGER,
PQGLPNNTASWFTALTQHGK,
GQGVPINTNSSPDDQIGYYR,
WYFYYLGTGPEAGLPYGANK,
DGIIWVATEGALNTPK,
NPANNAAIVLQLPQGTTLPK,
GFYAEGSR,
GGSQASSR,
NSTPGSSR,
MAGNGGDAALALLLLDR,
LNQLESK,
GQQQQGQTVTK,
SAAEASK,
AYNVTQAFGR,
GPEQTQGNFGDQELIR,
QGTDYK,
HWPQIAQFAPSASAFFGMSR,
IGMEVTPSGTWLTYTGAIK,
DQVILLNK,
HIDAYK,
TFPPTEPK,
ADETQALPQR,
QQTVTLLPAADLDDESK,
QLQQSMSSADSTQA
the purity of the standard peptide fragments was tested according to the method of step (f) to screen out standard peptide fragments with a purity not less than 99%, and at the same time the corresponding isotope-labeled peptide fragments of the standard peptide fragments are synthesized with 13C, 15N or D as label;
when the isotope-labeled standard peptide fragment has a molecular weight of 1000 or below, the number of labeled atoms is not less than 3;
and when the isotope-labeled standard peptide fragment has a molecular weight of 1000 or above, the number of labeled atoms is not less than 6;
the standard peptide fragments are dissolved in water containing 0.1% trifluoroacetic acid to formulate a solution containing 0.1 mg/g of the standard peptide fragments;
0.001 to 1 mg/mL of 13C or/and 15N or/and D isotope-labeled amino acid is added to each 100 μL of the solution of standard peptide fragments so that the content of the isotope-labeled amino acids is the same as the theoretical content of amino acids in the solution of standard peptide fragments after complete hydrolysis, and the number of the isotope-labeled atoms is not less than 3;
after centrifugal concentration or nitrogen blowing, 100 to 1000 μL of concentrated hydrochloric acid with a concentration of 6 to 8 mol/L is added thereto, and sealed while passing nitrogen for protection;
the mixture is hydrolyzed in an oven at 110° C. to 150° C. for 24 to 72 hours, and then the content of amino acids in the hydrolysate is determined by HPLC-IDMS using a national reference material of mixed amino acid solution as standard, and the purity of the standard peptide fragments is calculated according to the sequence of the standard peptide fragments;
nCoV N protein reference materials are taken as samples from at least 5 packaging units in step (c), from each of which 100 μL is taken to determine N protein concentration according to step (b), then an isotope-labeled peptide fragment equimolar to the N protein is added, followed by 5 μg to 100 μg of trypsin which is dissolved in 200 μL of ammonium bicarbonate with a concentration of 0.1 to 0.2 mol/L at pH 8.0 and containing 10% to 20% of acetonitrile;
after the addition of trypsin, the mixture is digested at a temperature of 37° C. for 24 to 72 hours to give a digested sample;
a mixture of standard peptide fragments and isotope-labeled peptide fragments with the same concentration of digested peptide fragments as that in the digested sample is formulated as a standard solution, and the concentration of the digested peptide fragments in the digested sample is tested using the following method;
the digested sample and the standard solution are analyzed by HPLC-tandem mass spectrometry using an aqueous solution containing 0.1% formic acid as mobile phase A and an acetonitrile solution containing 0.1% formic acid as mobile phase B;
the injection volume is 3 to 20 μL, and a 2.1 mm×150 mm C18 reversed-phase chromatography column is used for isolation at a flow rate of 0.2 mL/min, the gradient of mobile phase changes linearly from 100% mobile phase A to 100% mobile phase B to achieve the isolation of the digested peptide fragments;
a triple tandem quadrupole mass spectrometer is used for detection to harvest mass spectrometry signals from various standard peptide fragments and corresponding isotope-labeled peptide fragments, and each of the digested samples and the standard solutions are analyzed in triplicate, the concentrations of various standard peptide fragments in the digested samples are calculated according to the concentrations of the standard peptide fragments and the isotope-labeled peptide fragments in the standard solution and their mass respectively added, as well as the peak areas of the standard peptide fragments and isotope-labeled peptide fragments in the extraction chromatogram of the digested samples, the peak areas of the standard peptide fragments and isotope-labeled peptide fragments in the extraction chromatogram of the standard solution, the sample mass, and the concentration and mass of the isotope-labeled peptide fragments added in the sample, and the concentration of N protein in the sample is calculated according to the molecular weight of the standard peptide fragments and N protein according to the formulas below:
R
STD
=
c
pep
⁢
M
pep
c
pep
′
⁢
M
pep
′
c
s
=
R
STD
⁢
A
s
⁢
c
s
′
⁢
M
s
′
A
STD
⁢
M
s
c
N
=
c
s
MW
s
×
MW
N
in the formula, RSTD is a ratio of the mass of the standard peptide fragment to the mass of the isotope-labeled peptide fragment in the standard solution, cpep is a concentration of the standard peptide fragment, Mpep is a mass of the standard peptide fragment added, c′pep is a concentration of the isotope-labeled peptide fragment, M′pep is a mass of the isotope-labeled peptide fragment added, cs is a concentration of the standard peptide fragment in the digested sample, As is a ratio of peak area of the standard peptide fragment to the isotope-labeled peptide fragment in the sample, c′s is a concentration of the isotope-labeled peptide fragment added in the sample, M′s is a mass of the isotope-labeled peptide fragment added in the sample, ASTD is a ratio of peak area of the standard peptide fragment to the isotope-labeled peptide fragment in the standard solution, Ms is a sample mass, cN is a concentration of N protein in the sample, MWs is a molecular weight of the standard peptide fragment, and MWN is a molecular weight of the N protein;
the obtained measurement results are tested according to the K-S single-sample normality test and the Grabs method and/or Dixon method are used for outlier test;
for samples that pass the statistical test, the arithmetic mean value of all the results is taken as the standard value of test results of peptide fragment analysis-based HPLC-IDMS;
while for samples that fail to pass the statistical K-S single-sample normality test, Grabs method and/or Dixon method for outlier test, the number of samples is increased and the step (o) is repeated until passed;
(VI). determination and statistical test of standard value of nCOV N protein reference material
the at least 15 data of N protein content obtained by the amino acid analysis-based HPLC-IDMS in step (n) and the at least 15 data of N protein content obtained by the peptide fragment analysis-based HPLC-IDMS in step (o) are subject to independent-samples t-test;
if the |t| value calculated from Formula 24 is less than tα,(n1+n2−2), wherein tα,(n1+n2−2) is obtained by look-up in GB/T 4086.3-1983, the Tables for statistical distributions—t-distribution, then it is considered that the t-test result is passed, indicating that there is no systematic bias between the certified results of the two methods;
the arithmetic mean value of the two methods is taken as a certified value x, and when the t-test result fails, the step (V) is repeated;
t
=
X
1
¯
-
X
2
¯
s
1
2
n
1
+
s
2
2
n
2
Formula
⁢
24
in Formula 24:
—mean number of the first sample, that is, a standard value of the test results by the amino acid analysis-based HPLC-IDMS;
—mean number of the second sample, that is, a standard value of the test results by the peptide fragment analysis-based HPLC-IDMS;
s12—variance of the first sample,
s
1
2
=
∑
i
=
1
n
1
(
X
i
-
X
1
_
)
2
n
1
-
1
;
s22—variance of the second sample,
s
2
2
=
∑
j
=
1
n
2
(
X
j
-
X
2
_
)
2
n
2
-
1
;
n1—volume of the first sample, that is, a number determined by the amino acid analysis-based HPLC-IDMS;
n2—volume of the second sample, that is, a number determined by the peptide fragment analysis-based HPLC-IDMS;
(VII). assessment of uncertainty of the certified results of the standard value of the nCOV N protein reference material
the uncertainty U of the certified results of the nCOV N protein reference material is derived from an uncertainty uchar introduced during the certification, an uncertainty ubb introduced by the uniformity of reference material, an uncertainty usts introduced by short-term stability and an uncertainty ults introduced by long-term stability, which are combined according to Formula 25
U
=
k
′
⁢
u
c
⁢
h
⁢
a
⁢
r
2
+
u
b
⁢
b
2
+
u
lts
2
+
u
s
⁢
t
⁢
s
2
Formula
⁢
25
in the formula, k′is a coverage factor, typically k′=2;
wherein, uchar comprises an uncertainty uchar,HPLC-AAA-IDMS from the amino acid analysis-based HPLC-IDMS and an uncertainty uchar,HPLC-PEP-IDMS from the peptide fragment analysis-based HPLC-IDMS;
and
uchar,HPLC-AAA-IDMS comprises an uncertainty component uchar,HPLC-AAA-IDMS,A Formula 26 introduced by the repeatability of the test results of the amino acid analysis-based HPLC-IDMS, as well as an uncertainty component uchar,HPLC-AAA-IDMS,wi Formula 27 introduced by multiple balance weighing, and an uncertainty component uchar,HPLC-AAA-IDMS,P Formula 28 introduced by the amino acid reference material, which are combined according to Formula 29,
u
char
,
HPLC
-
AAA
-
IDMS
,
A
=
s
n
Formula
⁢
26
in Formula 26, s is a standard deviation, i.e.,
s
=
∑
i
=
1
n
(
x
i
-
x
¯
)
2
n
-
1
,
xi−x is a deviation between a certain data and the sample mean, and n is number of measurements;
u
char
,
HPLC
-
AAA
-
IDMS
,
wi
=
❘
"\[LeftBracketingBar]"
MPE
❘
"\[RightBracketingBar]"
3
Formula
⁢
27
in Formula 27, MPE is a maximum permissible error of balance verification certificate
u
char
,
HPLC
-
AAA
-
IDMS
,
P
=
U
k
′
Formula
⁢
28
in Formula 28, U is an uncertainty in the certificate of national amino acid reference material, k′ is a coverage factor, typically k′=2;
u
char
,
HPLC
-
AAA
-
IDMS
=
u
char
,
HPLC
-
AAA
-
IDMS
,
A
2
+
∑
i
=
1
q
(
c
i
⁢
u
char
,
HPLC
-
AAA
-
IDMS
,
wi
)
2
+
c
p
2
⁢
u
char
,
HPLC
-
AAA
-
IDMS
,
p
2
Formula
⁢
29
in Formula 29, q is a number of balance weighing, ci is a sensitivity coefficient at the ith weighing, and cp is a sensitivity coefficient of the purity of the reference material which is calculated according to JJF1059.1-2012, the Evaluation and Expression of Uncertainty in Measurement;
uchar,HPLC-PEP-IDMS comprises an uncertainty component uchar,HPLC-PEP-IDMS,A Formula 30 introduced by the repeatability of the test results of the amino acid analysis-based HPLC-IDMS, as well as an uncertainty component uchar,HPLC-PEP-IDMS,wi Formula 31 introduced by multiple balance weighing, and an uncertainty component uchar,HPLC-PEP-IDMS,P Formula 32 introduced by the peptide fragment reference material, which are combined according to Formula 33,
u
char
,
HPLC
-
pep
-
IDMS
,
A
=
s
n
Formula
⁢
30
in Formula 30, s is a standard deviation, i.e.,
s
=
∑
i
=
1
n
(
x
i
-
x
¯
)
2
n
-
1
,
xi−x is a deviation between a certain data and the sample mean, and n is number of measurements;
u
char
,
HPLC
-
pep
-
IDMS
,
wi
=
❘
"\[LeftBracketingBar]"
MPE
❘
"\[RightBracketingBar]"
3
Formula
⁢
31
in Formula 31, MPE is the maximum permissible error of balance verification certificate
u
char
,
HPLC
-
pep
-
IDMS
,
P
=
U
k
′
Formula
⁢
32
u
char
,
HPLC
-
PEP
-
IDMS
=
u
char
,
HPLC
-
PEP
-
IDMS
,
A
2
+
∑
i
=
1
q
(
c
i
⁢
u
char
,
HPLC
-
PEP
-
IDMS
,
wi
)
2
+
c
p
2
⁢
u
char
,
HPLC
-
PEP
-
IDMS
,
p
2
Formula
⁢
33
in Formula 33, q is a number of balance weighing, ci is a sensitivity coefficient at the ith weighing, and cp is a sensitivity coefficient of the purity of the reference material;
uchar is calculated according to Formula 34:
u
char
=
1
2
⁢
u
char
,
HPLC
-
AAA
-
IDMS
2
+
u
char
,
HPLC
-
PEP
-
IDMS
2
Formula
⁢
34
when mean square between groups is greater than or equal to mean square within group, the uncertainty introduced by uniformity is calculated according to Formula 35;
u
b
⁢
b
=
S
b
⁢
b
=
1
n
⁢
(
Q
1
v
1
-
Q
2
v
2
)
=
s
1
2
-
s
2
2
n
Formula
⁢
35
when the mean square between groups is less than the mean square within group, the uncertainty introduced by uniformity is calculated according to Formula 36;
u
b
⁢
b
=
S
b
⁢
b
=
s
2
2
n
⁢
2
v
2
4
Formula
⁢
36
the uncertainty introduced by short-term stability and the uncertainty introduced by long-term stability of the reference material are calculated according to Formula 37 and Formula 38:
u
sts
=
s
k
,
sts
·
t
sts
Formula
⁢
37
u
lts
=
s
k
,
lts
·
t
lts
Formula
⁢
38
in the formulas,
the sk,sts is s(b) in the short-term stability test obtained from Formula 19, and tts is the duration in the short-term stability test, in day;
the sk,lts is s(b) in the long-term stability test obtained from Formula 23, and tlts is the duration in the long-term stability test, in month;
the above results are combined according to Formula 25 to give an expanded uncertainty U of the certified value of the reference material, so that the certified result of the nCOV N protein reference material can be represented as:
x
±
U
in the formula, x is the certified value, and U is the expanded uncertainty of the certified value;
and finally, certified results of the uniformity, stability, and N protein content of the prepared nCOV N protein reference material are obtained.

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Method for preparing and certifying novel coronavirus nucleocapsid protein

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Method for preparing and certifying novel coronavirus nucleocapsid protein

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Abstract

Claims8