CN107045019B - The measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG - Google Patents
The measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention belongs to biological medicine detection technique fields; it is related to the measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG; double-strand Fc segment, IVIG removal carbohydrate protective agent processing, saliva acid dissociation, fluorescent derivatization, high performance liquid chromatography detection, the drafting of standard curve and testing result are obtained through digestion and chromatography by IVIG to substitute into standard curve and calculate step, obtain the sialic acid content of IVIG sialic acid total content and Fc segment；Fab section sialic acid content is=(sialic acid total content × IVIG molecular weight-Fc segment sialic acid content × Fc fragments molecules amount in IVIG)/Fab fragments molecules amount, and IVIG molecular weight is 150,000 Da in formula, Fc fragments molecules amount is 55,000 Da, Fab fragments molecules amount are 95,000 Da.The present invention is simple, accurate, high sensitivity, and repeatability and accuracy are high.
Description
Technical field
The invention belongs to biological medicine detection technique fields, and in particular to IgG Fab segment and Fc segment in a kind of IVIG
The measuring method of sialic acid content.
Background technique
Intravenous human immunoglobulin(HIg) pH4 (intravenous immunoglobulins, IVIG) is a kind of important people
Plasma protein products, main component are Immunoglobulin IgGs.
Sialic acid (Sialic acid) is a kind of natural carbohydrate, usually with oligosaccharide, glycolipid or glycoprotein
Form exists, and is the derivative of 9- carbon monosaccharide.It is a kind of negatively charged ions that saliva can be made to generate smooth feeling.It not only has
Play the role of " inducing " invasion germ, cognition is the transmitting mediator of gangliosides at present, and is the component part of brain.Saliva
Liquid acid can prevent germ from invading, while be also the receptor of influenza virus, be the combination that influenza virus is incorporated in mucilage cell
Site.The concentration of sialic acid modification has functioned very important influence to large biological molecule, obtains and carries out to sialic acid
Quickly and accurately quantitative analysis method has important application value for the clinical use of drug, quality testing etc..
IgG is as a kind of glycoprotein, and wherein sialic acid is located at its Fab section and the Fc sections of end N- sugar chain terminals.Fab section, that is, antigen
Binding fragment (fragment antigen binding, Fab), is equivalent to two arms of antibody molecule, complete light by one
VH the and CH1 structural domain of chain and heavy chain composition；In conjunction with foreign protein.Fc sections i.e. crystallizable fragment (fragment
Crystallizable, Fc), it is equivalent to CH2 the and CH3 structural domain of IgG.In conjunction with phagocyte.IgG can under the action of enzyme
It is degraded to Fab segment and Fc segment.
IgG Fc segment Degree of sialylation will affect the combination of itself and Fc receptor system in IVIG, play IVIG anti-inflammatory
Curative effect is most important, and the number of IgG Fc segment sialic acid content is its important indicator for treating various inflammation diseases in IVIg.
And the Degree of sialylation of Fab section will affect the combination of antigen-antibody, influence effect of the IVIG when treating immune deficiency disorder.
At present to the detection of sialic acid content, it is limited to the detection of sample or biological products Total silicic acid content more, there is no specifically for Fab
Segment and Fc segment are separately detected, and the difference of the sialic acid content of the two has different meanings in clinical use.
According to current pharmacopeia relevant regulations and the spectrophotometry delivered, HPLC method is accurate, sensitivity is higher.But mesh
It is preceding that there is no literature reported on the detections that HPLC- fluorescence method is applied to sialic acid content in IVIG product.Therefore, it is necessary to establish one kind
Method for measuring IgG Fab segment and Fc segment sialic acid content in IVIG.
Summary of the invention
Contain in order to solve the above technical problems, the present invention provides IgG Fab segment and Fc segment sialic acid in a kind of IVIG
The measuring method of amount can measure IVIG product IgG Fab segment and Fc segment sialic acid content, and method is simple, accurate, sensitive
Degree is high, and repeatability and accuracy are high.
The measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG of the above technical problem is solved,
The measuring method is that purification step, the IVIG by double-strand Fc segment remove carbohydrate protective agent processing step, saliva acid dissociation
Step, fluorescent derivatization step, the plot step of high performance liquid chromatography detection and standard curve are to get sialic acid total content and Fc piece
The sialic acid content of section；Wherein, Fab section sialic acid content is=(sialic acid total content × IVIG molecular weight-Fc piece in IVIG
Sialic acid content × Fc fragments molecules amount of section)/Fab fragments molecules amount, IVIG molecular weight is 150,000Da, Fc segment in formula
Molecular weight is that 55,000Da, Fab fragments molecules amount is 95,000Da.
Carbohydrate protective agent refers to that monosaccharide such as glucose, disaccharide such as sucrose, trehalose, lactose, glycan have glucan etc., all has
There is a large amount of free hydroxyl.
In the present invention in a kind of IVIG IgG Fab segment and Fc segment sialic acid content measuring method, including following tool
Body step:
Step 1: sample to be tested pretreatment:
(1) purifying of double-strand Fc segment: IVIG is taken, obtains double-strand Fc piece after digestion, Protein G chromatography and sieve chromatography
Section；The double-strand Fc segment solution wherein chromatographed is after measured after protein concentration, and dilution or concentration, obtaining protein concentration is 0.5
The solution of~4mg/ml is to be measured；The effect of molecular sieve be by Fc from digestion and by Protein G chromatography after product in purify
Out.
(2) IVIG removes the processing of carbohydrate protective agent；IVIG is taken, sieve chromatography removes carbohydrate protective agent；
IVIG to be detected is splined on molecular sieve, removes carbohydrate components, the solution chromatographed protein concentration after measured
Afterwards, it dilutes or is concentrated, it is to be measured to obtain the solution that protein concentration is 0.5~4mg/ml；The effect of molecular sieve is by the IgG in IVIG
It is separated with carbohydrate protective agent.
The sample IVIG detected in the present invention contains the glucides such as a large amount of maltose, sucrose as IVIG in ingredient
The protective agent and stabilizer of storage, and sialic acid is also a kind of carbohydrate derivative.In present sialic acid content detection method not
By being chromatography or spectrophotometric method, it is based on reacting for chromonic material or fluorescent material and carbohydrate, contained in sample
The even seldom residual of glucide also affect measurement accuracy height.Detection method takes molecule first in the present invention
Sieve chromatography removes the glucide in sample, and removal interference improves detection accuracy.
The IVIG after removal carbohydrate protective agent and double-strand Fc segment after purification are subjected to step 2 to step 5 respectively again
Processing, obtains the sialic acid content of the sialic acid total content and Fc segment in IVIG；
Step 2: saliva acid dissociation: the sialic acid of sample to be tested is disintegrated down with acid flux material；
Step 3: fluorescent derivatization benzoic acid dimethylamino ethyl ester (DMB) derivative liquid is added in step 2,50 DEG C of gold
Belong to bath and is protected from light 2.5h；Individual sialic acid is to be detected well by high performance liquid chromatography (HPLC) instrument, is needed
After the fluorescent derivatization agent reaction reacted with sialic acid (glucide) is added, then upper HPLC detection.
Step 4: the drafting of high performance liquid chromatography detection and standard curve: being detected with high performance liquid chromatograph, then with
Standard curve sample gradient concentration is abscissa, and derivative peak area of the standard curve sample through high performance liquid chromatography detection is vertical sit
Mark draws standard curve using linear equation；
Step 5: the calculating of sialic acid content:
The derivative peak area through high performance liquid chromatography detection of sample to be tested is substituted into standard curve, calculates every milli in sample
Solution sialic acid content is risen, by the result of calculating divided by the protein concentration of sample solution to get the sialic acid into every mg sample
Content；
Wherein, Fab section sialic acid content is=(sialic acid total content × IVIG molecular weight-Fc segment saliva in IVIG
Liquid acid content × Fc fragments molecules amount)/Fab fragments molecules amount；In formula, IVIG molecular weight is 150,000Da, Fc fragments molecules amount
It is 95,000Da for 55,000Da, Fab fragments molecules amount.
Digestion condition is that EDTA-Na is added in IVIG in the step 12, L-cysteine hydrochloride and Papain
Enzyme, 37 DEG C, 4h, EDTA-Na2, L-cysteine hydrochloride and papain amount ratio are 1:5:20.
Sieve chromatography is the segment containing Fc that will be collected after Protein G chromatographic column chromatography in (1) step in the step 1
Component is splined on the molecular sieve that separating ranges include 5000Da-300000Da, each component in sample is separated according to molecular weight
It comes, collects the albumen that molecular weight is 50000Da, as complete double-strand Fc segment.
Testing sample solution protein concentration is 0.5~4mg/ml in the step 1, and optimization concentration is 0.5~2mg/ml,
More optimized concentration is 0.5mg/ml.
The sialic acid dissociation steps are to take the 1mg/ml sample to be tested of 300 μ L that 100 μ L 0.1M trifluoroacetic acids, gold is added
Belong to 80 DEG C, 1h of bath, product is through 0.22 μm of membrane filtration.
Chromatographic column in the high performance liquid chromatograph: 4.6 × 250 5 μm of Waters Symmetry C18 liquid-phase chromatographic column,
30 DEG C of column temperature, fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, mobile phase is methanol, the main ultrapure water of acetonitrile, wherein
Methanol: acetonitrile: ultrapure water=7:8:85, flow velocity 0.9mL/min.
1mg sialic acid standard adds 10mL water to be configured to the sialic acid standard of 0.1mg/mL in the drafting of the standard curve
Solution adds water to be diluted to 5,10,20,30,40ug/mL respectively, takes each concentration standards solution of 300 μ L, be separately added into 50 μ L
DMB derives liquid, and 50 DEG C of metal baths are protected from light 2.5h, 20 μ L sample detections.
The present invention measures IVIG product IgG Fab segment and Fc segment sialic acid content, and method is simple, accurate, sensitivity
Height, repeatability and accuracy are high.
Detailed description of the invention
Fig. 1 is the sialic acid content examination criteria curve of test one in the present invention
Fig. 2 is the sialic acid content examination criteria curve of test two in the present invention
Fig. 3-Fig. 9 is the chromatogram in the present invention in test one
Specific embodiment
The present invention is described in further detail With reference to embodiment.
Embodiment 1
The measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG, comprising the following specific steps
Step 1: sample to be tested pretreatment:
(1) purifying of double-strand Fc segment: IVIG is taken, obtains double-strand Fc piece after digestion, Protein G chromatography and sieve chromatography
Section；The double-strand Fc segment solution wherein chromatographed is after measured after protein concentration, and dilution or concentration, obtaining protein concentration is 0.5
The solution of~4mg/ml is to be measured；There are two effects for molecular sieve, at the beginning of experiment, to IgG desalination；Or by Fc from digestion and process
It is purified in product after ProteinG chromatography.
Sieve chromatography is that the component for the segment containing Fc collected after Protein G chromatographic column chromatography is splined on separating ranges packet
Molecular sieve containing 5000Da-300000Da separates each component in sample according to molecular weight, collects molecular weight and is
The albumen of 50000Da, as complete double-strand Fc segment.
Testing sample solution protein concentration is 0.5~4mg/ml, and optimization concentration is 0.5~2mg/ml, and more optimized concentration is
0.5mg/ml。
(2) IVIG removes the processing of carbohydrate protective agent；IVIG is taken, sieve chromatography removes carbohydrate protective agent；
IVIG to be detected is splined on molecular sieve, removes carbohydrate components, the solution chromatographed protein concentration after measured
Afterwards, it dilutes or is concentrated, it is to be measured to obtain the solution that protein concentration is 0.5~4mg/ml；
The sample IVIG detected in the present invention, containing glucides such as a large amount of maltose, sucrose in ingredient, and saliva
Acid is also a kind of carbohydrate derivative.Whether chromatography or spectrophotometric method in present sialic acid content detection method,
Reacting based on chromonic material or fluorescent material and carbohydrate, the even seldom residual of glucide contained in sample also shadow
Ring measurement accuracy height.Detection method takes the glucide in sieve chromatography removal sample first in the present invention, goes
Except interference, detection accuracy is improved.
The IVIG after removal carbohydrate protective agent and double-strand Fc segment after purification are subjected to step 2 to step 5 respectively again
Processing, obtains the sialic acid content of the sialic acid total content and Fc segment in IVIG；
Step 2: saliva acid dissociation: the sialic acid of sample to be tested is disintegrated down with acid flux material；Sialic acid dissociation steps
To take the 1mg/ml sample to be tested of 300 μ L that 100 μ L 0.1M trifluoroacetic acids are added, 80 DEG C of metal bath, 1h, product is filtered through 0.22 μm
Film filtering.
Step 3: the derivative liquid of DMB is added in step 2 fluorescent derivatization, and 50 DEG C of metal baths are protected from light 2.5h；Individually
Sialic acid is to be detected well by HPLC, needs that the fluorescent derivatization agent reacted with sialic acid (glucide) is added anti-
Ying Hou, then upper HPLC detection.
Step 4: the drafting of high performance liquid chromatography detection and standard curve: being detected with high performance liquid chromatograph, then with
Standard curve sample gradient concentration is abscissa, and derivative peak area of the standard curve sample through high performance liquid chromatography detection is vertical sit
Mark draws standard curve using linear equation；
Chromatographic column in high performance liquid chromatograph: 4.6 × 250 5 μm of Waters Symmetry C18 liquid-phase chromatographic column, column temperature
30 DEG C, fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, mobile phase is methanol, the main ultrapure water of acetonitrile, wherein first
Alcohol: acetonitrile: ultrapure water=7:8:85, flow velocity 0.9mL/min.
The sialic acid standard that 1mg sialic acid standard adds 10mL water to be configured to 0.1mg/mL in the drafting of standard curve is molten
Liquid adds water to be diluted to 5,10,20,30,40 μ g/mL respectively, takes each concentration standards solution of 300 μ L, is separately added into 50 μ L DMB
Derivative liquid, 50 DEG C of metal baths are protected from light 2.5h, 20 μ L sample detections.
Step 5: the calculating of sialic acid content:
The derivative peak area through high performance liquid chromatography detection of sample to be tested is substituted into standard curve, calculates every milli in sample
Solution sialic acid content is risen, by the result of calculating divided by the protein concentration of sample solution to get the sialic acid into every mg sample
Content；
Wherein, Fab section sialic acid content is=(sialic acid total content × IVIG molecular weight-Fc segment saliva in IVIG
Liquid acid content × Fc fragments molecules amount)/Fab fragments molecules amount；In formula, IVIG molecular weight is 150,000Da, Fc fragments molecules amount
It is 95,000Da for 55,000Da, Fab fragments molecules amount.Embodiment 2
A kind of measuring method of IVIG product IgG Fab segment and Fc sections of sialic acid contents, comprising the following steps:
(1) purifying of Fc segment
A digestion:
It takes IVIG 1ml, is added EDTA-Na2, L-cysteine hydrochloride and papain, digestion, 37 DEG C, 4h.
B chromatographic purifying:
A, Protein G chromatographs
It is splined on Protein G (Protein G) chromatographic column chromatography after digestion products are filtered, collects in digestion products and contains Fc
The component of segment.
Protein G chromatographic step:
1. fliud flushing is prepared:
Combination buffer: 20mM phosphoric acid is received, pH7.0.Elution buffer: 100mM glycine, pH2.7 neutralization buffer: 1M
Tris-HCl, pH9.0.
2. sample loading pre-treatment
2 times of volume combination buffers are added in sample, mix well, stand overnight.Disposable 0.22um filter mistake before loading
Filter.
3. column equilibration
Column first to be washed with the distillation of 5 times of column volumes, then washes column with 5 times of volume combination buffers, flow velocity is 1ml/min,
Stablize to conductance, UV absorption UV detector in 280nm wavelength detecting isobase.
4. loading
Sample pumps S2 pipeline sample introduction, 1ml/min.Sampling volume about 20mL.
5. washing
With the combination buffer equilibrium separation column of 5-10 times of column volume, until baseline, i.e., all unbonded materials are all rushed
It washes out pillar (UV detector is in 280nm wavelength detecting).
6. elution and sample collection
It is eluted with the elution buffer of 10 times of column volumes.Eluting peak is collected, uses neutralization buffer after collection immediately
(0.06ml~0.2ml 1M Tris-HCl) is neutralized to neutrality.
B, sieve chromatography
By Protein G chromatographic column chromatography after collect all segments containing Fc component (IVIG including double-strand Fc, non-digestion,
And single-stranded Fc of the part containing hinge area), it is splined on the molecular sieve that separating ranges include 5000Da-300000Da, it will be in sample
Each component separated according to molecular weight, collect molecular weight be 50000Da albumen, as complete double-strand Fc segment.
Sieve chromatography step:
1. column equilibration
With 2 times of column volume ultrapure water HiPrep 26/60Sephacryl S-200High Resolution prepacked columns
Afterwards, it is balanced with 2 times of 0.9% physiological saline of column volume.
2. sample treatment and loading
Concentrating sample, until concentration 10mg/ml or more.Sampling volume 5ml, flow velocity 1.3ml/min.
3. chromatographing
Physiological saline 1.3ml/min washing, about 3 hours are until the peak of all components is all complete out.
4. peak is collected
Between about 90-120min, there is second sample peak, the main component at this peak i.e. Fc segment required for us
Peak.
(2) sialic acid content detects
A sample to be tested pre-treatment:
Sample to be tested is the double-strand Fc segment of IVIG and purifying；
By IVIG be splined on can molecular sieve, remove the carbohydrate components that wherein contain；
The protectant IVIG of carbohydrate will be removed and double-strand Fc segment is diluted or is concentrated by ultrafiltration, acquisition concentration is 0.5mg/ml
Protein sample.
B saliva acid dissociation:
The sialic acid on IVIG or double-strand Fc segment is disintegrated down using acid condition (TFA, hydrochloric acid, acetic acid).
Taking 300 μ L protein samples (1mg/ml) that 100 μ L 0.1M trifluoroacetic acids are added, (7.45mL trifluoroacetic acid adds water to
100mL), 80 DEG C of metal bath, 1h, product is through 0.22 μm of membrane filtration.
C fluorescent derivatization
Previous step reaction product is taken, the derivative liquid of DMB is added, 50 DEG C of metal baths are protected from light 2.5h, 20 μ L sample detections.
D high performance liquid chromatography detection:
Chromatographic column: 4.6 × 250 5 μm of Waters Symmetry C18 liquid-phase chromatographic column；30 DEG C of column temperature；Fluorescence detector
Excitation wavelength 373nm, launch wavelength 448nm；Mobile phase is methanol: acetonitrile: ultrapure water=(7:8:85)；Flow velocity is 0.9mL/
min；
The drafting of e standard curve:
1mg sialic acid standard adds 10mL water to be configured to the sialic acid standard solutions of 0.1mg/mL.Water is added to be diluted to respectively
5,10,20,30,40ug/mL.Each concentration standards solution of 300 μ L is taken, the derivative liquid of 50 μ L DMB, 50 DEG C of metal baths are separately added into
It is protected from light 2.5h, 20 μ L sample detections.
Using standard curve sample gradient concentration as abscissa, derivative peak of the standard curve sample through high performance liquid chromatography detection
Area is ordinate, draws standard curve using linear equation.
The calculating of f sialic acid content:
The derivative peak area through high performance liquid chromatography detection of sample to be tested is substituted into standard curve, calculates saliva in sample
Acid content.
Wherein the sialic acid total content in IVIG and the sialic acid content of Fc segment are immediately arrived at through the above calculating；Fab section
Sialic acid content obtains for the sialic acid content that the sialic acid total content in IVIG subtracts Fc segment.
Test one: influence of the carbohydrate protective agent to sample measurement result more whether is removed
If test group and control group, other steps are identical, and test group has removal carbohydrate protective agent step, product solution A1
And B1；Control group does not remove carbohydrate protective agent step, and product solution is A2 and B2；Specific step is as follows:
(1) prepared by sample to be tested:
Using commercially available A, B Liang Ge company IVIG product as test sample, respectively through chromatography removal it is protectant processing and
After the removal protectant processing of carbohydrate, mark curve is drawn with the method for the invention and detects its IVIG sialic acid and always contains
Amount.
The protectant IVIG sample preparation of carbohydrate is removed in test group
Flow velocity 1ml/min is set, with 2 times of column volume ultrapure water HiPrep 26/60Sephacryl S-200High
After Resolution prepacked column, balanced with 2 times of column volume physiological saline；The IVIG product 2ml of commercially available company A is taken to be splined on layer
Column chromatography is analysed, collection molecular weight is 150,000Da (protein liquid；It is dilute to its using physiological saline after measured after protein concentration
It releases, obtains the solution A 1 that protein concentration is 0.5~4mg/ml；
Above step is repeated, B company intravenous human immunoglobulin(HIg) is handled, acquisition protein concentration is 0.5~4mg/
The solution B 1 of ml, it is to be measured.
The protectant IVIG sample preparation of carbohydrate is not removed in control group
A, B company IVIG product are diluted to 0.5~4mg/ml using physiological saline, obtain protein solution A2, B2 respectively,
It is to be measured.
(2) sialic acid content detects
A saliva acid dissociation:
Take 300 μ L protein samples be added 100 μ L 0.1M trifluoroacetic acids, 80 DEG C of metal bath, 1h.
B fluorescent derivatization:
Previous step reaction product is taken, the derivative liquid of DMB is added, 50 DEG C of metal baths are protected from light 2.5h.
C high performance liquid chromatography detection:
Select chromatographic column: Waters Symmetry C18 liquid-phase chromatographic column；30 DEG C of column temperature；Fluorescence detector excitation wavelength
373nm, launch wavelength 448nm；Mobile phase is methanol: acetonitrile: ultrapure water=(7:8:85)；Setting flow velocity is 0.9mL/min, on
10 μ L of sample volume detects sample to be tested sample introduction in chromatographic column, and the derivative peak of the maximum for calculating 10~14min of sample introduction appearance
Area.
The drafting of d standard curve:
6.25 using N-acetyl-neuraminate as standard items, concentration gradient is dissolved and is diluted to physiological saline: 0,
12.5,25,50,100,200μg/mL.Using various concentration standard items as sample, step " b ", " c " are repeated, each concentration standard is obtained
The area at the derivative peak of the maximum that product occur in 10~14min of sample introduction.Various criterion product high-efficient liquid phase chromatogram such as Fig. 3-9 institute
Show, target peak is obvious, is easy to judge.
The derivative peak of target all occurred at 12.3 minutes, to various concentration standard items test experience Plays product concentration, color
The appearance time of spectral peak, the arrangement of peak area correlation corresponding data are shown in Table 1:
Table 1: standard items (standard curve) chromatographic data
(note: number A to G is corresponded same as below in Fig. 3 to Fig. 9, test one respectively).
Using standard curve sample concentration as ordinate, derivative peak area of the standard curve sample through high performance liquid chromatography detection
For abscissa, standard curve is drawn using linear equation, and obtains curvilinear equation, as shown in Figure 1, can be fitted to obtain preferable
Linear equation, square (R of related coefficient2) it is greater than 0.99.
The calculating of e sialic acid content:
The derivative peak area through high performance liquid chromatography detection of sample to be tested is substituted into standard curve, calculates every milli in sample
Solution sialic acid content is risen, by the result of calculating divided by the protein concentration of sample solution to get to sialic acid on every mg albumen
Content.
F repeats to test
Above method is repeated, sample to be tested processed is carried out to repeat test, is as a result averaged such as the following table 2:
Table 2
From result, it can be seen that test group A1, B1 is less than control group A 2, B2, the reason for this is that: the sample detected in the present invention
IVIG, containing glucides such as a large amount of maltose, sucrose in ingredient, and sialic acid is also a kind of carbohydrate derivative.Chromatography
Reacting based on chromonic material or fluorescent material and carbohydrate when method carries out sialic acid content detection method, because of control group A 2, B2
The carbohydrate contained in protective agent is not removed, can derive with DMB, cause measurement result bigger than normal, inaccuracy.Test group sample
A1, B1 are obtained IVIG product A, B after sieve chromatography without the protectant protein sample of carbohydrate, and influence is eliminated
The factor of detection keeps result more acurrate.Therefore, IVIG is divided before the sialic acid content of detection IVIG product in the present invention
Sub- sieve chromatography processing is the effect for needing and having achieved.
Test two
Using the IVIG product of tetra- companies of commercially available C, D, E, F as test sample, detected with the method for the invention.
(1) purifying of Fc segment
A digestion:
The IVIG product 1ml for taking commercially available C company is separately added into 100 μ g EDTA-Na2, 500 μ g L-cysteine salt
Hydrochlorate and 2mg papain, digestion, 37 DEG C, 4h.
B chromatographic purifying:
(1) Protein G chromatographs
1. buffer:
Combination buffer: 20mM phosphoric acid is received, pH7.0.Elution buffer: 100mM glycine, pH2.7, neutralization buffer:
1M Tris-HCl, pH9.0
2. chromatographing
Flow velocity 1ml/min is set, first analyses column with the distillation water washing protein G layer of 5 times of column volumes, then combined with 5 times of volumes
Buffer washes column；It takes the digestion products obtained in step " a " that 2 times of volume combination buffers are added, is splined on layer after mixing well
Column is analysed, then after the combination buffer of 5 times of column volumes balance chromatographic column, is eluted with the elution buffer of 10 times of column volumes,
Eluent is collected, and adjusts its pH to neutrality with neutralization buffer.
(2) sieve chromatography
Flow velocity 1ml/min is set, with 2 times of column volume ultrapure water HiPrep 26/60Sephacryl S-200High
After Resolution prepacked column, balanced with 2 times of column volume physiological saline；The digestion products obtained in step " a " are taken to be splined on layer
Column chromatography is analysed, the protein liquid that molecular weight is 50000~60000Da is collected.
(3) the obtained solution of chromatography utilizes the concentration of albumen ultra-filtration centrifuge tube, obtains protein concentration after measured after protein concentration
For the solution of 0.5~4mg/ml；
C repeats above step, purifies tri- company's Fc segments of B, C, D, to be measured.
(2) Image processing of IVIG
Flow velocity 1ml/min is set, with 2 times of column volume ultrapure water HiPrep 26/60Sephacryl S-200High
After Resolution prepacked column, balanced with 2 times of column volume physiological saline；The IVIG product 2ml of commercially available company A is taken to be splined on layer
Column chromatography is analysed, the protein liquid that molecular weight is 150000Da is collected；After measured after protein concentration, it is diluted using physiological saline,
Obtain the solution that protein concentration is 0.5~4mg/ml；
Above step is repeated, tri- company's intravenous human immunoglobulin(HIg)s of D, E, F are handled, it is to be measured.
(3) sialic acid content detects
A saliva acid dissociation:
Take 300 μ L protein samples be added 100 μ L 0.1M trifluoroacetic acids, 80 DEG C of metal bath, 1h.
B fluorescent derivatization:
Previous step reaction product is taken, the derivative liquid of DMB is added, 50 DEG C of metal baths are protected from light 2.5h.
C high performance liquid chromatography detection:
Select chromatographic column: Waters Symmetry C18 liquid-phase chromatographic column；30 DEG C of column temperature；Fluorescence detector excitation wavelength
373nm, launch wavelength 448nm；Mobile phase is methanol: acetonitrile: ultrapure water=(7:8:85)；Setting flow velocity is 0.9mL/min, on
10 μ L of sample volume detects sample to be tested sample introduction in chromatographic column, and the derivative peak of the maximum for calculating 10~14min of sample introduction appearance
Area.
The drafting of d standard curve:
Using N-acetyl-neuraminate as standard items, concentration gradient is dissolved and is diluted to physiological saline: 3.125,
6.25,12.5,25,50,100,200μg/mL.Using various concentration standard items as sample, step " b ", " c " are repeated, is obtained each dense
The area at the derivative peak of the maximum that degree standard items occur in 10~14min of sample introduction.
Using standard curve sample concentration as ordinate, derivative peak area of the standard curve sample through high performance liquid chromatography detection
For abscissa, standard curve is drawn using linear equation, and obtains curvilinear equation, as shown in Figure 2:
The calculating of e sialic acid content:
The derivative peak area through high performance liquid chromatography detection of sample to be tested is substituted into standard curve, calculates every milli in sample
Solution sialic acid content is risen, by the result of calculating divided by the protein concentration of sample solution to get to sialic acid on every mg albumen
Content.
Wherein the sialic acid total content in IVIG and the sialic acid content of Fc segment are immediately arrived at through the above calculating；Fab section
Sialic acid content are as follows:
(sialic acid total content × IVIG molecular weight-Fc segment sialic acid content × Fc fragments molecules amount in IVIG)/
Fab fragments molecules amount
Wherein, it be 55,000Da, Fab fragments molecules amount is 95 that IVIG molecular weight, which is 150,000Da, Fc fragments molecules amount,
000Da。
F repeats to test
Above method is repeated, amounts to and 3 repetition tests is carried out to sample to be tested processed, as a result statistics such as the following table 3:
Table 3
It can be concluded that, the detection method in the present invention is accurate, and repeatability is high from above.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (7)
1. the measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG, it is characterised in that: including following
Specific steps:
Step 1: sample to be tested pretreatment:
(1) purifying of double-strand Fc segment: IVIG is taken, obtains double-strand Fc segment after digestion, Protein G chromatography and sieve chromatography；Enzyme
Tangent condition is that EDTA-Na is added in IVIG2, L-cysteine hydrochloride and papain, 37 DEG C, 4h, EDTA-Na2、L-
Cysteine hydrochloride and papain amount ratio are 1:5:20；Sieve chromatography is that will collect after Protein G chromatographic column chromatography
The segment containing Fc component, be splined on separating ranges include 5000Da-300000Da molecular sieve, each component in sample is pressed
It is separated according to molecular weight, collects the albumen that molecular weight is 50000Da, as complete double-strand Fc segment；
(2) IVIG removes the processing of carbohydrate protective agent；IVIG is taken, sieve chromatography removes carbohydrate protective agent；Removal carbohydrate is protected again
IVIG after shield agent and double-strand Fc segment after purification carry out step 2 to step 5 processing respectively, and the sialic acid obtained in IVIG is total
The sialic acid content of content and Fc segment；
Step 2: saliva acid dissociation: the sialic acid of sample to be tested is disintegrated down with acid flux material；
Step 3: the derivative liquid of DMB is added in step 2 fluorescent derivatization, and 50 DEG C of metal baths are protected from light 2.5h；
Step 4: the drafting of high performance liquid chromatography detection and standard curve: being detected with high performance liquid chromatograph, then with standard
Curve sample gradient concentration is abscissa, and derivative peak area of the standard curve sample through high performance liquid chromatography detection is ordinate,
Standard curve is drawn using linear equation；
Step 5: the calculating of sialic acid content:
Derivative peak area by sample to be tested through high performance liquid chromatography detection substitutes into standard curve, calculate in sample every milliliter it is molten
Liquid sialic acid content, by the result of calculating divided by the protein concentration of sample solution to get the sialic acid content into every mg sample；
Wherein, Fab section sialic acid content be=(sialic acid total content × IVIG molecular weight-Fc segment sialic acid in IVIG contains
Amount × Fc fragments molecules amount)/Fab fragments molecules amount；In formula, IVIG molecular weight is 150,000 Da, and Fc fragments molecules amount is
55,000 Da, Fab fragments molecules amount are 95,000 Da.
2. the measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG according to claim 1,
Be characterized in that: testing sample solution protein concentration is 0.5 ~ 4mg/ml in the step 1.
3. the measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG according to claim 2,
It is characterized in that: 0.5 ~ 2mg/ml of testing sample solution protein concentration in the step 1.
4. the measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG according to claim 3,
Be characterized in that: testing sample solution protein concentration is 0.5mg/ml in the step 1.
5. the measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG according to claim 1,
Be characterized in that: the sialic acid dissociation steps are to take the 1mg/ml sample to be tested of 300 μ L that 100 μ L 0.1M trifluoroacetic acids are added,
80 DEG C of metal bath, 1h, product is through 0.22 μm of membrane filtration.
6. the measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG according to claim 1,
It is characterized in that: chromatographic column in the high performance liquid chromatograph: 4.6 × 250 5 μ of Waters Symmetry C18 liquid-phase chromatographic column
M, 30 DEG C of column temperature, fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, mobile phase is methanol, acetonitrile and ultrapure water,
Wherein methanol: acetonitrile: ultrapure water=7:8:85, flow velocity 0.9mL/min.
7. the measuring method of IgG Fab segment and Fc segment sialic acid content in a kind of IVIG according to claim 1,
It is characterized in that: the saliva acidity scale that 1mg sialic acid standard adds 10mL water to be configured to 0.1mg/mL in the drafting of the standard curve
Quasi- solution adds water to be diluted to 5,10,20,30,40ug/mL respectively, takes each concentration standards solution of 300 μ L, be separately added into 50 μ L
DMB derives liquid, and 50 DEG C of metal baths are protected from light 2.5h, 20 μ L sample detections.
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