CN107045019A - The assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG - Google Patents
The assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG Download PDFInfo
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Abstract
The invention belongs to biological medicine detection technique field; it is related to the assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG; double-strand Fc fragments, IVIG are obtained through digestion and chromatography by IVIG and remove the processing of carbohydrate protective agent, saliva acid dissociation, fluorescent derivatization, high performance liquid chromatography detection, the drafting of standard curve and testing result substitution standard curve calculation procedure, obtain the sialic acid content of IVIG sialic acids total content and Fc fragments；Fab section sialic acid content for=（Sialic acid content × Fc fragments molecules amounts of sialic acid total content × IVIG molecular weight Fc fragments in IVIG）IVIG molecular weight is 150,000 Da in/Fab fragments molecules amounts, formula, and Fc fragments molecules amount is 55,000 Da, and Fab fragments molecules amount is 95,000 Da.Of the invention simple, accurate, sensitivity is high, and repeatability and accuracy are high.
Description
Technical field
The invention belongs to biological medicine detection technique field, and in particular to IgG Fab fragments and Fc fragments in a kind of IVIG
The assay method of sialic acid content.
Background technology
Intravenous human immunoglobulin(HIg) pH4 (intravenous immunoglobulins, IVIG), is a kind of important people
Plasma protein products, main component is Immunoglobulin IgG.
Sialic acid (Sialic acid) is the natural carbohydrate of a class, generally with oligosaccharide, glycolipid or glycoprotein
Form is present, and is the derivative of 9- carbon monose.It is a kind of negatively charged ions that saliva can be made to produce smooth sensation.It not only has
Play the role of " to induce " invasion germ, current cognition is the transmission mediator of gangliosides, and is the part of brain.Saliva
Liquid acid can prevent germ from invading, and be that influenza virus combines the combination in mucilage cell while being also the acceptor of influenza virus
Site.The concentration of sialic acid modification has very important influence on large biological molecule function, obtains and sialic acid is carried out
Quickly and accurately quantitative analysis method in terms of the Clinical practice of medicine, quality testing for having important application value.
IgG is located at its Fab section and Fc sections of N- ends sugar chain terminals as a kind of glycoprotein, wherein sialic acid.Fab section is antigen
Binding fragment (fragment antigen binding, Fab) is complete light by one equivalent to two arms of antibody molecule
VH the and CH1 domains composition of chain and heavy chain；Combined with foreign protein.Fc sections are crystalline fragments (fragment
Crystallizable, Fc), CH2 the and CH3 domains equivalent to IgG.Combined with phagocyte.IgG can in the presence of enzyme
It is degraded to Fab fragments and Fc fragments.
IgG Fc fragments Degree of sialylation can influence the combination of itself and Fc receptor systems in IVIG, and anti-inflammatory is played to IVIG
Curative effect is most important, in IVIg IgG Fc fragment sialic acid contents number be important indicator that it treats various inflammation diseases.
And the Degree of sialylation of Fab section can influence the combination of antigen-antibody, effects of the influence IVIG when treating immune deficiency disorder.
The detection to sialic acid content, is limited to the detection of sample or biological products Total silicic acid content more, there is no specifically designed for Fab at present
Fragment and Fc fragments are separately detected, and the difference of the sialic acid content of both has different meanings in Clinical practice.
According to current pharmacopeia relevant regulations and the AAS delivered, HPLC methods are accurate, sensitivity is higher.But mesh
It is preceding that there is no literature reported on the detection that HPLC- fluorescence methods are applied to sialic acid content in IVIG products.Therefore, it is necessary to set up one kind
For determining IgG Fab fragments and the method for Fc fragment sialic acid contents in IVIG.
The content of the invention
Contain in order to solve the above technical problems, the present invention provides IgG Fab fragments and Fc fragment sialic acids in a kind of IVIG
The assay method of amount, can determine IVIG products IgG Fab fragments and Fc fragment sialic acid contents, and method is simple, accurate, sensitive
Degree is high, and repeatability and accuracy are high.
The assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG of above technical problem is solved,
The assay method is the purification step Jing Guo double-strand Fc fragments, IVIG removes carbohydrate protective agent process step, saliva acid dissociation
Step, fluorescent derivatization step, the plot step of high performance liquid chromatography detection and standard curve, produce sialic acid total content and Fc pieces
The sialic acid content of section；Wherein, Fab section sialic acid content is=(sialic acid total content × IVIG molecular weight-Fc pieces in IVIG
Sialic acid content × Fc fragments molecules amount of section)/Fab fragments molecules amounts, IVIG molecular weight is 150,000Da, Fc fragments in formula
Molecular weight is that 55,000Da, Fab fragments molecules amount is 95,000Da.
Carbohydrate protective agent refers to monose such as glucose, and disaccharide such as sucrose, trehalose, lactose, glycan have glucan etc., all had
There is substantial amounts of free hydroxyl.
In the present invention in a kind of IVIG IgG Fab fragments and Fc fragment sialic acid contents assay method, including following tool
Body step：
Step one：Testing sample is pre-processed：
(1) purifying of double-strand Fc fragments：Double-strand Fc pieces are obtained after taking IVIG, digestion, Protein G chromatography and sieve chromatography
Section；Obtained double-strand Fc fragments solution is wherein chromatographed after measured after protein concentration, dilution or is concentrated, it is 0.5 to obtain protein concentration
~4mg/ml solution is to be measured；The effect of molecular sieve is purified in being the product by Fc from digestion and after Protein G chromatographies
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, carbohydrate components are removed, obtained solution protein concentration after measured is chromatographed
Afterwards, dilute or concentrate, obtain protein concentration to be measured for 0.5~4mg/ml solution；The effect of molecular sieve is by the IgG in IVIG
Separated with carbohydrate protective agent.
Contain the glucides such as a large amount of maltose, sucrose in the sample IVIG detected in the present invention, its composition as IVIG
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 in the reaction of chromonic material or fluorescent material and carbohydrate, sample contained
Glucide even seldom residual also contribute to determine accuracy height.Detection method takes molecule first in the present invention
Sieve chromatography removes the glucide in sample, removes interference, improves detection accuracy.
The IVIG after carbohydrate protective agent will be removed again and double-strand Fc fragments after purification carry out step 2 to step 5 respectively
Processing, obtains the sialic acid total content in IVIG and the sialic acid content of Fc fragments；
Step 2：Saliva acid dissociation：The sialic acid of testing sample is disintegrated down with acid flux material；
Step 3：Fluorescent derivatization, the derivative liquid of benzoic acid dimethylamino ethyl ester (DMB) is added in step 2,50 DEG C of gold
Category bath lucifuge reaction 2.5h；Single sialic acid be can not be detected well by high performance liquid chromatography (HPLC) instrument, it is necessary to
Add after the fluorescent derivatization agent reaction reacted with sialic acid (glucide), then upper HPLC detections.
Step 4：The drafting of high performance liquid chromatography detection and standard curve：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 seat
Mark, standard curve is drawn using linear equation；
Step 5：The calculating of sialic acid content：
The derivative peak area through high performance liquid chromatography detection of testing sample is substituted into standard curve, calculated in sample per milli
Solution sialic acid content is risen, by the result of calculating divided by the protein concentration of sample solution, that is, sialic acid in every mg samples is obtained
Content；
Wherein, Fab section sialic acid content is the=(saliva of sialic acid total content × IVIG molecular weight-Fc fragments in IVIG
Liquid acid content × Fc fragments molecules amount)/Fab fragments molecules amounts；In formula, IVIG molecular weight is 150,000Da, Fc fragments molecules amounts
It is 95,000Da for 55,000Da, Fab fragments molecules amount.
Digestion condition is that EDTA-Na is added in IVIG in the step one2, 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 that Protein G is chromatographed to the fragment containing Fc collected after column chromatography in (1) step in the step one
Component, is splined on the molecular sieve that separating ranges include 5000Da-300000Da, each component in sample is separated according to molecular weight
Come, collect the albumen that molecular weight is 50000Da, as complete double-strand Fc fragments.
Testing sample solution protein concentration is 0.5~4mg/ml in the step one, and optimization concentration is 0.5~2mg/ml,
More optimization concentration is 0.5mg/ml.
The sialic acid dissociation steps are that the 1mg/ml testing samples for taking 300 μ L add 100 μ L 0.1M trifluoroacetic acids, gold
80 DEG C of category bath, 1h, 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 columns,
30 DEG C of column temperature, fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, mobile phase is methanol, the main ultra-pure water of acetonitrile, wherein
Methanol：Acetonitrile：Ultra-pure water=7:8:85, flow velocity is 0.9mL/min.
1mg sialic acid standards add 10mL water to be configured to 0.1mg/mL sialic acid standard in the drafting of the standard curve
Solution, add water and be diluted to 5 respectively, 10,20,30,40ug/mL, take each concentration standards solution of 300 μ L, be separately added into 50 μ L
DMB derives liquid, and 50 DEG C of metal bath lucifuges react 2.5h, 20 μ L sample detections.
The present invention determines IVIG products IgG Fab fragments and Fc fragment sialic acid contents, and method is simple, accurate, sensitivity
Height, repeatability and accuracy are high.
Brief description of the drawings
Fig. 1 is the sialic acid content examination criteria curve of experiment one in the present invention
Fig. 2 is the sialic acid content examination criteria curve of experiment two in the present invention
Fig. 3-Fig. 9 is the chromatogram in experiment one in the present invention
Embodiment
With reference to embodiment, the present invention is described in further detail.
Embodiment 1
The assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG, including step in detail below：
Step one：Testing sample is pre-processed：
(1) purifying of double-strand Fc fragments：Double-strand Fc pieces are obtained after taking IVIG, digestion, Protein G chromatography and sieve chromatography
Section；Obtained double-strand Fc fragments solution is wherein chromatographed after measured after protein concentration, dilution or is concentrated, it is 0.5 to obtain protein concentration
~4mg/ml solution is to be measured；Molecular sieve has two effects, at the beginning of experiment, to IgG desalinations；Or by Fc from digestion and process
It is purified in product after ProteinG chromatographies.
Sieve chromatography is the component that Protein G is chromatographed to the fragment containing Fc collected after column chromatography, is splined on separating ranges bag
Molecular sieve containing 5000Da-300000Da, each component in sample is separated according to molecular weight, is collected molecular weight and is
50000Da albumen, as complete double-strand Fc fragments.
Testing sample solution protein concentration is 0.5~4mg/ml, and optimization concentration is 0.5~2mg/ml, more optimizes concentration and 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, carbohydrate components are removed, obtained solution protein concentration after measured is chromatographed
Afterwards, dilute or concentrate, obtain protein concentration to be measured for 0.5~4mg/ml solution；
Containing glucides such as a large amount of maltose, sucrose in the sample IVIG detected in the present invention, its composition, and saliva
Acid is also a kind of carbohydrate derivative.Whether chromatography or spectrophotometric method in present sialic acid content detection method,
Contained glucide even seldom residual also shadow in reaction based on chromonic material or fluorescent material and carbohydrate, sample
Ring measure accuracy height.Detection method takes sieve chromatography to remove the glucide in sample first in the present invention, goes
Except interference, detection accuracy is improved.
The IVIG after carbohydrate protective agent will be removed again and double-strand Fc fragments after purification carry out step 2 to step 5 respectively
Processing, obtains the sialic acid total content in IVIG and the sialic acid content of Fc fragments；
Step 2：Saliva acid dissociation：The sialic acid of testing sample is disintegrated down with acid flux material；Sialic acid dissociation steps
To take 300 μ L 1mg/ml testing samples 100 μ L 0.1M trifluoroacetic acids of addition, 80 DEG C of metal bath, 1h, product is filtered through 0.22 μm
Membrane filtration.
Step 3：Fluorescent derivatization, derives liquid by DMB and adds in step 2,50 DEG C of metal bath lucifuges react 2.5h；Individually
Sialic acid can not be detected well by HPLC, it is necessary to which the fluorescent derivatization agent added with sialic acid (glucide) reaction is anti-
Ying Hou, then upper HPLC detections.
Step 4：The drafting of high performance liquid chromatography detection and standard curve：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 seat
Mark, standard curve is drawn using linear equation；
Chromatographic column in high performance liquid chromatograph：4.6 × 250 5 μm of Waters Symmetry C18 liquid-phase chromatographic columns, column temperature
30 DEG C, fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, mobile phase is methanol, the wherein main ultra-pure water of acetonitrile, first
Alcohol：Acetonitrile：Ultra-pure water=7:8:85, flow velocity is 0.9mL/min.
In the drafting of standard curve 1mg sialic acid standards add 10mL water be configured to 0.1mg/mL sialic acid standard it is molten
Liquid, adds water and is 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 bath lucifuges react 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 testing sample is substituted into standard curve, calculated in sample per milli
Solution sialic acid content is risen, by the result of calculating divided by the protein concentration of sample solution, that is, sialic acid in every mg samples is obtained
Content；
Wherein, Fab section sialic acid content is the=(saliva of sialic acid total content × IVIG molecular weight-Fc fragments in IVIG
Liquid acid content × Fc fragments molecules amount)/Fab fragments molecules amounts；In formula, IVIG molecular weight is 150,000Da, Fc fragments molecules amounts
It is 95,000Da for 55,000Da, Fab fragments molecules amount.Embodiment 2
The assay method of a kind of IVIG products IgG Fab fragments and Fc sections of sialic acid contents, comprises the following steps：
(1) purifying of Fc fragments
A digestions：
IVIG 1ml are taken, EDTA-Na2, L-cysteine hydrochloride and papain, digestion, 37 DEG C, 4h is added.
B chromatographic purifyings：
A, Protein G chromatography
Protein G (Protein G) chromatography column chromatography is splined on after digestion products are filtered, collects in digestion products and contains Fc
The component of fragment.
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 buffers：1M
Tris-HCl, pH9.0.
2. sample loading pre-treatment
Sample adds 2 times of volume combination buffers, fully mixes, stands overnight.Disposable 0.22um filter mistakes before loading
Filter.
3. column equilibration
Post first is washed with the distillation of 5 times of column volumes, then post is washed with 5 times of volume combination buffers, flow velocity is 1ml/min,
It is stable in 280nm wavelength detectings isobase to conductance, UV absorption UV-detector.
4. loading
Sample pump S2 pipeline sample introductions, 1ml/min.Sampling volume about 20mL.
5. wash
With the combination buffer equilibrium separation post of 5-10 times of column volume, until baseline, i.e., all uncombined materials are all rushed
Wash out pillar (UV-detector is in 280nm wavelength detectings).
6. elute and sample collection
Eluted with the elution buffer of 10 times of column volumes.Eluting peak is collected, neutralization buffer is used after collection immediately
(0.06ml~0.2ml 1M Tris-HCl) is neutralized to neutrality.
B, sieve chromatography
By Protein G chromatograph all fragments containing Fc collected after column chromatography component (including double-strand Fc, the IVIG of non-digestion,
And single-stranded Fc of the part containing hinge area), the molecular sieve that separating ranges include 5000Da-300000Da is splined on, by sample
Each component separated according to molecular weight, collect molecular weight be 50000Da albumen, as complete double-strand Fc fragments.
Sieve chromatography step：
1. column equilibration
With 2 times of column volume ultrapure water HiPrep 26/60Sephacryl S-200High Resolution prepacked columns
Afterwards, balanced with 2 times of physiological saline of column volume 0.9%.
2. sample treatment and loading
Concentrating sample, to concentration 10mg/ml or more.Sampling volume 5ml, flow velocity 1.3ml/min.
3. chromatograph
Physiological saline 1.3ml/min is washed, and about 3 hours are until the peak of all components has all gone out.
4. peak is collected
Between about 90-120min, there is second sample peak, the main component at this peak is the Fc fragments required for us
Peak.
(2) sialic acid content is detected
A testing sample pre-treatments：
Testing sample is the double-strand Fc fragments of IVIG and purifying；
IVIG is splined on being capable of molecular sieve, the carbohydrate components that removal wherein contains；
The protectant IVIG of carbohydrate and double-strand Fc fragments will be removed through diluting or being concentrated by ultrafiltration, acquisition concentration is 0.5mg/ml
Protein sample.
B saliva acid dissociations：
The sialic acid on IVIG or double-strand Fc fragments is disintegrated down using acid condition (TFA, hydrochloric acid, acetic acid).
Taking 300 μ L protein samples (1mg/ml) to add 100 μ L 0.1M trifluoroacetic acids, (7.45mL trifluoroacetic acids are added water to
100mL), 80 DEG C of metal bath, 1h, product is through 0.22 μm of membrane filtration.
C fluorescent derivatizations
Previous step reaction product is taken, DMB is added and derives liquid, 50 DEG C of metal bath lucifuges react 2.5h, 20 μ L sample detections.
D high performance liquid chromatography detections：
Chromatographic column：4.6 × 250 5 μm of Waters Symmetry C18 liquid-phase chromatographic columns；30 DEG C of column temperature；Fluorescence detector
Excitation wavelength 373nm, launch wavelength 448nm；Mobile phase is methanol：Acetonitrile：Ultra-pure water=(7:8:85)；Flow velocity is 0.9mL/
min；
The drafting of e standard curves：
1mg sialic acid standards add 10mL water to be configured to 0.1mg/mL sialic acid standard solutions.Add water and be diluted to respectively
5、10、20、30、40ug/mL.Each concentration standards solution of 300 μ L is taken, 50 μ L DMB is separately added into and derives liquid, 50 DEG C of metal baths
Lucifuge reacts 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, and standard curve is drawn using linear equation.
The calculating of f sialic acid contents：
The derivative peak area through high performance liquid chromatography detection of testing sample is substituted into standard curve, saliva in sample is calculated
Acid content.
The sialic acid content of sialic acid total content and Fc fragments in wherein IVIG is calculated more than and immediately arrived at；Fab section
Sialic acid content obtains for the sialic acid content that the sialic acid total content in IVIG subtracts Fc fragments.
Experiment one：More whether carbohydrate protective agent influence to sample measurement result is removed
If test group and control group, other steps are identical, test group has removal carbohydrate protective agent step, and product solution is A1
And B1；Control group does not remove carbohydrate protective agent step, and product solution is A2 and B2；Comprise the following steps that：
(1) prepared by testing sample：
Using the IVIG products of commercially available A, B Liang Ge companies as detection sample, respectively through chromatography remove it is protectant processing and
Without removing after the protectant processing of carbohydrate, draw mark curve with the method for the invention and detect that its IVIG sialic acid always contains
Amount.
The protectant IVIG sample preparations of carbohydrate are 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 columns, balanced with 2 times of column volume physiological saline；The IVIG products 2ml of commercially available company A is taken to be splined on layer
Column chromatography is analysed, collection molecular weight is 150,000Da (protein liquids；It is dilute to its using physiological saline after measured after protein concentration
Release, obtain the solution A 1 that protein concentration is 0.5~4mg/ml；
Above step is repeated, B companies intravenous human immunoglobulin(HIg) is handled, acquisition protein concentration is 0.5~4mg/
Ml solution B 1, it is to be measured.
The protectant IVIG sample preparations of carbohydrate are not removed in control group
A, B company IVIG products are diluted to 0.5~4mg/ml using physiological saline, protein solution A2, B2 are obtained respectively,
It is to be measured.
(2) sialic acid content is detected
A saliva acid dissociations：
300 μ L protein samples are taken to add 100 μ L 0.1M trifluoroacetic acids, 80 DEG C of metal bath, 1h.
B fluorescent derivatizations：
Previous step reaction product is taken, DMB is added and derives liquid, 50 DEG C of metal bath lucifuges react 2.5h.
C high performance liquid chromatography detections：
Select chromatographic column：Waters Symmetry C18 liquid-phase chromatographic columns；30 DEG C of column temperature；Fluorescence detector excitation wavelength
373nm, launch wavelength 448nm；Mobile phase is methanol：Acetonitrile：Ultra-pure water=(7:8:85)；Setting flow velocity is 0.9mL/min, on
The μ L of sample volume 10, testing sample sample introduction is detected in chromatographic column, and calculates the derivative peak of the maximum of 10~14min of sample introduction appearance
Area.
The drafting of d standard curves：
Using N-acetyl-neuraminate as standard items, dissolved with physiological saline and be diluted to concentration gradient：0、6.25、
12.5、25、50、100、200μg/mL.Using various concentrations standard items as sample, repeat step " b ", " c " obtain each concentration standard
The area at the derivative peak of maximum that product occur in 10~14min of sample introduction.Various criterion product high-efficient liquid phase chromatogram such as Fig. 3-9 institutes
Show, target peak is obvious, it is easy to judge.
Target derives peak all to be occurred at 12.3 minutes, to various concentrations standard items test experience Plays product concentration, color
The related corresponding data of appearance time, peak area of spectral peak, which is arranged, is shown in Table 1：
Table 1：Standard items (standard curve) chromatographic data
(note：Numbering A to G, is corresponded same as below in Fig. 3 to Fig. 9, experiment 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, is obtained preferably as shown in figure 1, can be fitted
Linear equation, square (R of coefficient correlation2) it is more than 0.99.
The calculating of e sialic acid contents：
The derivative peak area through high performance liquid chromatography detection of testing sample is substituted into standard curve, calculated in sample per milli
Solution sialic acid content is risen, by the result of calculating divided by the protein concentration of sample solution, that is, sialic acid on every mg albumen is obtained
Content.
F repeats to test
Above method is repeated, testing sample processed is carried out to repeat experiment, as a result averaged such as table 2 below：
Table 2
From result, it can be seen that test group A1, B1 is less than control group A 2, B2, its reason is：The sample detected in the present invention
Containing glucides such as a large amount of maltose, sucrose in IVIG, its composition, and sialic acid is also a kind of carbohydrate derivative.Chromatogram
Method carries out the reaction based on chromonic material or fluorescent material and carbohydrate during sialic acid content detection method, because control group A 2, B2
The carbohydrate contained in protective agent is not removed, can derive with DMB, cause measurement result bigger than normal, it is inaccurate.Test group sample
A1, B1 are to be free of the protectant protein sample of carbohydrate by what IVIG products A, B were obtained after sieve chromatography, eliminate influence
The factor of detection, makes result more accurate.Therefore, IVIG is divided before the sialic acid content of detection IVIG products in the present invention
Sub- sieve chromatography processing is the effect for needing and having achieved.
Experiment two
Using the IVIG products of commercially available tetra- companies of C, D, E, F as detection sample, detected with the method for the invention.
(1) purifying of Fc fragments
A digestions：
The IVIG product 1ml of commercially available C companies are taken, 100 μ g EDTA-Na are separately added into2, 500 μ g Cys salt
Hydrochlorate and 2mg papains, digestion, 37 DEG C, 4h.
B chromatographic purifyings：
(1) Protein G is chromatographed
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. chromatograph
Flow velocity 1ml/min is set, first post is analysed with the distillation water washing protein G layer of 5 times of column volumes, then combined with 5 times of volumes
Buffer solution washes post；Take the digestion products obtained in step " a " to add 2 times of volume combination buffers, layer is splined on after fully mixing
Post is analysed, then after the combination buffer balance chromatographic column of 5 times of column volumes, is eluted with the elution buffer of 10 times of column volumes,
Eluent is collected, and its pH is adjusted 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 columns, 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 0.5~4mg/ml solution；
C repeats above step, purifies tri- company's Fc fragments of B, C, D, to be measured.
(2) IVIG Image processing
Flow velocity 1ml/min is set, with 2 times of column volume ultrapure water HiPrep 26/60Sephacryl S-200High
After Resolution prepacked columns, balanced with 2 times of column volume physiological saline；The IVIG products 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 is detected
A saliva acid dissociations：
300 μ L protein samples are taken to add 100 μ L 0.1M trifluoroacetic acids, 80 DEG C of metal bath, 1h.
B fluorescent derivatizations：
Previous step reaction product is taken, DMB is added and derives liquid, 50 DEG C of metal bath lucifuges react 2.5h.
C high performance liquid chromatography detections：
Select chromatographic column：Waters Symmetry C18 liquid-phase chromatographic columns；30 DEG C of column temperature；Fluorescence detector excitation wavelength
373nm, launch wavelength 448nm；Mobile phase is methanol：Acetonitrile：Ultra-pure water=(7:8:85)；Setting flow velocity is 0.9mL/min, on
The μ L of sample volume 10, testing sample sample introduction is detected in chromatographic column, and calculates the derivative peak of the maximum of 10~14min of sample introduction appearance
Area.
The drafting of d standard curves：
Using N-acetyl-neuraminate as standard items, dissolved with physiological saline and be diluted to concentration gradient：3.125、
6.25、12.5、25、50、100、200μg/mL.Using various concentrations standard items as sample, repeat step " b ", " c " are obtained each dense
The area at the derivative peak of 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 contents：
The derivative peak area through high performance liquid chromatography detection of testing sample is substituted into standard curve, calculated in sample per milli
Solution sialic acid content is risen, by the result of calculating divided by the protein concentration of sample solution, that is, sialic acid on every mg albumen is obtained
Content.
The sialic acid content of sialic acid total content and Fc fragments in wherein IVIG is calculated more than and immediately arrived at；Fab section
Sialic acid content is：
(sialic acid content × Fc fragments molecules amount of sialic acid total content × IVIG molecular weight-Fc fragments in IVIG)/
Fab fragments molecules amounts
Wherein, IVIG molecular weight is that 150,000Da, Fc fragments molecules amount is that 55,000Da, Fab fragments molecules amount is 95,
000Da。
F repeats to test
Above method is repeated, amounts to and 3 repetition experiments is carried out to testing sample processed, as a result statistics such as table 3 below：
Table 3
It can be drawn more than, the detection method in the present invention is accurate, and repeatability is high.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Claims (8)
1. the assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG, it is characterised in that：The measure side
Method is that purification step, IVIG by IVIG digestions and double-strand Fc fragments remove carbohydrate protective agent process step, saliva acid dissociation
Step, fluorescent derivatization step, high performance liquid chromatography detection step, the drafting of standard curve and testing result substitute into standard curve meter
Step is calculated, the sialic acid content of sialic acid total content and Fc fragments is produced；Wherein, Fab section sialic acid content for=(in IVIG
Sialic acid content × Fc fragments molecules amount of sialic acid total content × IVIG molecular weight-Fc fragments)/Fab fragments molecules amounts, in formula
IVIG molecular weight is that 150,000Da, Fc fragments molecules amount is that 55,000Da, Fab fragments molecules amount is 95,000Da.
2. the assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG according to claim 1, its
It is characterised by：Including step in detail below：
Step one：Testing sample is pre-processed：
(1) purifying of double-strand Fc fragments：Double-strand Fc fragments are obtained after taking IVIG, digestion, Protein G chromatography and sieve chromatography；
(2) IVIG removes the processing of carbohydrate protective agent；IVIG is taken, sieve chromatography removes carbohydrate protective agent；Carbohydrate will be removed again to protect
IVIG after shield agent and double-strand Fc fragments 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 fragments；
Step 2：Saliva acid dissociation：The sialic acid of testing sample is disintegrated down with acid flux material；
Step 3：Fluorescent derivatization, derives liquid by DMB and adds in step 2,50 DEG C of metal bath lucifuges react 2.5h；
Step 4：The drafting of high performance liquid chromatography detection and standard curve：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 of the testing sample through high performance liquid chromatography detection is substituted 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, that is, obtains sialic acid content in every mg samples；
Wherein, Fab section sialic acid content is the=(sialic acid of sialic acid total content × IVIG molecular weight-Fc fragments in IVIG
Content × Fc fragments molecules amount)/Fab fragments molecules amounts；In formula, IVIG molecular weight is that 150,000Da, Fc fragments molecules amounts are
55,000Da, Fab fragments molecules amount are 95,000Da.
3. the assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG according to claim 2, its
It is characterised by：Digestion condition is that EDTA-Na is added in IVIG in the step one2, L-cysteine hydrochloride and pawpaw egg
White enzyme, 37 DEG C, 4h, EDTA-Na2, L-cysteine hydrochloride and papain amount ratio are 1：5：20.
4. the assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG according to claim 2, its
It is characterised by：Sieve chromatography is that Protein G is chromatographed to the fragment containing Fc collected after column chromatography in (1) step in the step one
Component, is splined on the molecular sieve that separating ranges include 5000Da-300000Da, each component in sample is separated according to molecular weight
Come, collect the albumen that molecular weight is 50000Da, as complete double-strand Fc fragments.
5. the assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG according to claim 2, its
It is characterised by：Testing sample solution protein concentration is 0.5~4mg/ml in the step one, and optimization concentration is 0.5~2mg/ml,
More optimization concentration is 0.5mg/ml.
6. the measure side of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG according to claim 1 or 2
Method, it is characterised in that：The sialic acid dissociation steps are that the 1mg/ml testing samples for taking 300 μ L add 100 μ L 0.1M trifluoro second
Acid, 80 DEG C of metal bath, 1h, product is through 0.22 μm of membrane filtration.
7. the measure side of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG according to claim 1 or 2
Method, it is characterised in that：Chromatographic column in the high performance liquid chromatograph：Waters Symmetry C18 liquid-phase chromatographic columns 4.6 ×
250 5 μm, 30 DEG C of column temperature, fluorescence detector excitation wavelength 373nm, launch wavelength 448nm, mobile phase is methanol, acetonitrile master surpasses
Pure water, wherein methanol：Acetonitrile：Ultra-pure water=7:8:85, flow velocity is 0.9mL/min.
8. the measure side of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG according to claim 1 or 2
Method, it is characterised in that：1mg sialic acid standards add 10mL water to be configured to 0.1mg/mL saliva in the drafting of the standard curve
Sour standard liquid, add water and be diluted to 5 respectively, 10,20,30,40ug/mL, take each concentration standards solution of 300 μ L, be separately added into
50 μ L DMB derive liquid, and 50 DEG C of metal bath lucifuges react 2.5h, 20 μ L sample detections.
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