WO2013011178A1 - Análisis rápido y preciso de la sialilación de proteínas - Google Patents
Análisis rápido y preciso de la sialilación de proteínas Download PDFInfo
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- WO2013011178A1 WO2013011178A1 PCT/ES2012/070501 ES2012070501W WO2013011178A1 WO 2013011178 A1 WO2013011178 A1 WO 2013011178A1 ES 2012070501 W ES2012070501 W ES 2012070501W WO 2013011178 A1 WO2013011178 A1 WO 2013011178A1
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- galactose
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- 102000005348 Neuraminidase Human genes 0.000 claims description 9
- 108010006232 Neuraminidase Proteins 0.000 claims description 9
- 239000013642 negative control Substances 0.000 claims description 8
- 102000002464 Galactosidases Human genes 0.000 claims description 7
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- 108091005804 Peptidases Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
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- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
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- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 description 1
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- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/363—Anion-exchange
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
Definitions
- Sialic acids comprise a family of N-linked and N-linked neuraminic acids.
- N-linked sialic acids are formed by linking acetyl or glucosyl moieties to the amino residue of neuraminic acid, forming iV-acet-ilneuraminic acid (Neu5Ac) and iV-glucolylneuraminic acid (Neu5Gc), respectively. If the amino group of the neuraminic is replaced by a hydroxyl moiety, this produces 3-deoxy-D-glyceric-D-galacto-2-nonulosonic acid (KDN).
- KDN 3-deoxy-D-glyceric-D-galacto-2-nonulosonic acid
- O-linked sialic acids are formed by substituting one or more of the hydroxyl groups of Neu5Ac, Neu5Gc or KDN by methyl, acetyl, lactoyl, sulfate or phosphate groups. Therefore, there is a large and diverse population of sialic acids.
- sialylation may be important for the pharmacokinetics and efficacy of protein biotherapeutics.
- several analytical methods have been developed to evaluate the sialic acid content of glycoproteins. For example, they can be used antibody-based assays to identify particular carbohydrate residues.
- the terminal sialic acid residues can be enzymatically separated from the glycoprotein of interest and analyzed by HPLC.
- each of these methods has defects and usually requires pure samples or high concentrations.
- Conventional methods used by the pharmaceutical industry suffer from low precision, high data variability and cannot be used with complex culture media due to matrix interference.
- the enzymatic part of the method involves the release of exposed terminal galactose residues (uncoated) by specific exo-glucosidase, ⁇ - (1-4) -galactosidase ( ⁇ -galactosidase), while terminal sialic acid residues are released by a- (2- 3, 6, 8, 9) -sialidase (OC-sialidase).
- a sample is divided between at least three tubes.
- the first tube, Reaction A is a background sample and comprises only the enzymatic reaction buffers.
- the second tube, Reaction B is reacted with ⁇ -galactosidase which cleaves all galactose residues that are not coated by sialic acids.
- the third tube, Reaction C is co-digested with both neuraminidase and ⁇ -galactosidase.
- the enzyme neuraminidase removes sialic coating acids and allows ⁇ -galactosidase to cleave all exposed galactose residues.
- High-resolution anion exchange chromatography with pulsed amperometric detection (HPAEC PAD) is then used to determine the amount of galactose present in the three samples.
- the proportion of uncoated galactose (i.e. Reaction B) with respect to total galactose (i.e. Reaction C) is used to calculate the percentage of galactose residue coating, while also explaining any free galactose present in the media (Reaction A).
- a method for determining the sialylation content of a protein comprising: (a) preparing a protein for analysis; (b) enzymatically treating the prepared protein, which comprises: dividing the prepared protein into a plurality of protein samples comprising (i) at least one protein sample as a media sample (Reaction A); (ii) add at least ⁇ -galactosidase to at least one protein sample (Reaction B); (iii) add at least ⁇ -galactosidase and OC-sialidase to at least one different protein sample (Reaction C); and incubate the plurality of protein samples; and (c) analyze the plurality of protein samples using HPAEC-PAD chromatography; (d) determine a carbohydrate content for the plurality of protein samples; and (e) calculate a percentage of sialylation for the protein.
- the use is also described which further comprises: (f) analyzing a plurality of positive and negative controls using HPAEC-PAD chromatography; (g) analyze a plurality of patterns using HPAEC-PAD chromatography; and (h) compare the plurality of protein samples with the plurality of patterns and controls.
- a kit is also described for determining the sialylation content of any protein comprising: at least one container comprising a plurality of containers comprising previously measured amounts of a galactosidase and a sialidase; optionally, containers containing at least one buffer composition, a positive control sample, a negative control sample and carbohydrate standards, and instructions describing a method for determining the sialylation content of a protein, comprising descriptions of: (a) prepare a protein for analysis; (b) enzymatically treating the prepared protein, which comprises: dividing the prepared protein into a plurality of protein samples comprising (i) at least one protein sample as a media sample (Reaction A); (ii) add at least ⁇ -galactosidase to at least one protein sample (Reaction B); (iii) add at least ⁇ -galactosidase and OC-sialidase to at least one different protein sample (Reaction C); and incubate the plurality of protein samples; and (c) analyze the
- a kit is also described which further comprises: (f) analyzing a positive and negative control using HPAEC-PAD chromatography; (g) analyze a plurality of patterns using HPAEC-PAD chromatography; and (h) compare the results of the plurality of protein samples with the results of the plurality of standards.
- Figure 1 shows a scheme of Reactions A, B and C, and the data obtained from each respective reaction.
- the percentage of coating by sialylation can be determined from the ratio of the differences in Reactions C and B and Reactions C and A, respectively. See equation 1.
- Figure 2 shows a typical chromatogram for a sample of recAlpha-1 digested in Reaction C. Elution times for galactose vary between approximately 14 and 16 minutes.
- Figure 4 demonstrates that excipients upstream of recAlpha-1 culture media can be detected and can co-elute near the galactose peak.
- an excipient peak is eluted next to the galactose peak in some samples after cleaning the centrifuge filter.
- the impurity peak initially resolves from the galactose peak and does not integrate.
- the The impurity peak of the process is co-eluted with a sample of Reaction C. In this case, the impurity peak must be divided as shown above to prevent it from being integrated with the galactose peak area.
- Figure 5 The specificity of the method was confirmed by analyzing a mixture of neutral monosaccharides and aminomonosaccharides derived from glycoproteins.
- Figure 6. Example of a typical galactose standard curve.
- the recombinant alpha-1 proteinase inhibitor (recAlpha-1) was designed for secretion by the PerC6 cell line with N-linked glycan carbohydrate structures that are partially or fully coated by terminal sialic acids (iV-acetylneuraminic acids). A decrease in the amount of terminal sialic acids has been shown to reduce the half-life of recAlpha-1 in serum. Thus, It is important to know the percentage of galactose residues coated by sialic acids in recAlpha-1 when investigating their function or efficacy as a therapeutic drug.
- Example 1 Preparation of the sample for analysis and enzymatic digestion
- a method for determining the sialylation content of a glycoprotein begins with the preparation of a protein sample for enzymatic hydrolysis of carbohydrate residues. Therefore, the protein must be placed under conditions compatible with enzymatic reactions, including adjusting the protein concentration, eliminating solution components such as dissolved salts, buffers, other proteins, carbohydrates, excipients, etc., which could interfere with the enzyme. or enzymes
- the term "prepared” or the phrase "prepare a protein for analysis” describes the process of removing components of the solution that could interfere with enzymatic hydrolysis and diluting the protein solution to an optimal concentration for the test with deionized water.
- MWCO molecular weight cutoff limit
- the protein was inserted into a dialysis membrane and dialyzed against an excess of deionized water or suitable buffer and / or saline solution for at least 4 hours at 4 ° C.
- a protein sample can be prepared for enzymatic digestion by centrifugal filtration.
- the protein solution was centrifuged against a semipermeable membrane with a specified MWCO.
- the components of the solution with molecular weights below the MWCO pass through the filter during centrifugation, while the protein and the higher molecular weight species are retained.
- the protein solution is concentrated during centrifugal filtration, because water passes through the semipermeable membrane.
- a 10 kDa centrifuge filter according to the manufacturer's instructions, was used to prepare the protein sample.
- reaction A was the background control (to control exogenous galactose). This reaction is composed only of buffers for enzymatic reactions and serves as a control for carbohydrates that may exist in the medium comprising the protein analyte.
- Reaction B contained ⁇ -galactosidase, which hydrolyzed non-sialylated carbohydrate groups, but not those that were “coated” with sialyl groups.
- Reaction C contained both OC-sialidase and ⁇ -galactosidase. In this reaction, OC-sialidase hydrolyzed the coating sialyl moieties, which then allowed ⁇ -galactosidase to hydrolyze all carbohydrate groups.
- High resolution anion exchange chromatography analysis with pulsed amperometric detection was performed in Dionex ICS-3000 ion chromatography systems with single pumps, automatic sample processors with thermostat set at 10 ° C, and electrochemical detection units (Dionex, Sunnyvale, CA).
- Disposable (Au) gold electrodes were used for pulsed amperometric detection (PAD) (Dionex Prod. No. 060139).
- PAD pulsed amperometric detection
- the waveform used for the sample analysis was based on Dionex Data Sheet 21, which describes optimal settings for carbohydrate PADs as shown in Table 2. See Data Sheet 21 from Dionex, Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemical Detector [Dionex (1998)).
- the first mobile phase (A) contained 20 mM NaOH and was used for isocratic separation at a flow rate of 1 ml / min for 30 minutes (initial run time was 28 minutes but extended to 30 minutes during development to leave more time for 100% re-balancing of the mobile phase A).
- the second mobile phase (B) It contained 500 mM NaOH and was used for column elution, column cleaning and electrode cleaning.
- the chromatographic elution method, including ramp washing using mobile phases A and B is shown in table 3.
- the sample injection volume was 20 ⁇ . Data analysis was performed using Chromeleon® 6.8 Chromatography Data Analysis System (Dionex) software.
- Example 3 Patterns, controls, calibration and suitability of the system
- the control reaction was a 1 to 1 mixture of bovine sialylated fetuine and commercially available asialofetuine standard (Sigma F3004 and A4781, respectively). Individually, sialylated fetuine has a coating percentage above 99% and asialofetuine has a coating percentage of 0%. When mixed in equal proportions, the coating ratio (sialylation) for fetuine should be 50% ⁇ 3%.
- the fetuin control was prepared as a large batch that was divided into aliquots, frozen at -70 ° C and an individual sample was thawed and used as a control each time a set of samples was digested and processed using the methods described in the present document.
- the low concentration standard was 8 pmol galactose and the high concentration standard was 1500 pmol galactose. These test standards were intended to ensure that the results of the sample were within the linear range of the lowest and highest concentrations for the assay.
- the 1500 pmol test control was also used as a control of the suitability of the system (for example, high concentration standard). Table 4: Typical sequence of a chromatography series
- the enzyme target was an injection containing the enzymes (ie, ⁇ -galactosidase and OC-sialidase) and buffers without any protein sample (ie, Reaction C without any protein analyte).
- Bovine fetuine (a 1: 1 mixture of sialylated fetuine and asyalofetuine) was used as a positive control for sialylation.
- the area Galactose was divided by the deoxyribose area to give corrected galactose areas.
- the percentage of coating was calculated for each of the injections in duplicate of a sample using the corrected areas.
- the corrected galactose area measured for Reaction B was subtracted from the corrected galactose area determined from Reaction C; This value corresponds to the amount of galactose coated with sialyl.
- the corrected galactose area determined for Reaction A was then subtracted from the corrected galactose area measured for Reaction C; This value corresponds to total galactose (i.e. coated and uncoated).
- the coated galactose (C-B) was divided by total galactose (C-A) and multiplied by 100 to give the percentage of sialyl coating. Representative data are shown in table 5.
- Reaction C The suitability of the system was monitored using a mixture of galactose and deoxyribose that was injected at the beginning of a series and at the end of the series to monitor the performance of the electrode and the column throughout the series.
- the average percentage of coating described was determined from the corrected areas of duplicate injections. If the area of the galactose peak in Reaction B was smaller than the area of the galactose peak in the 8 pmol pattern, then the percentage of coating was described as ">% of [coating]" (ie, "greater than ”) calculated based on the galactose peak area of the 8 pmol pattern in the numerator of the calculation.
- the calculation of the tail factor (called "asymmetry" in Chromeleon® software), theoretical plates and resolution were performed and determined by Dionex data acquisition software. The calculations for the remaining system suitability criteria were determined manually. Representative system suitability parameters are shown in table 6.
- the enzyme ⁇ -galactosidase is supplied by the manufacturer at an activity of> 3 Units / ml (specific activity> 6 Units / mg), while the enzyme GC-sialidase has an activity of 5 Units / ml (specific activity at 135 Units / mg)
- the amount of each enzyme was kept constant at 4 ⁇ each, corresponding to 0.012 Units of ⁇ -galactosidase and 0.02 Units of GC-sialidase in the reaction, while the amount of protein varied from 540 to 2160 pmol.
- the samples analyzed were a recAlpha-1 exchanged with buffer and filtered in cell culture supernatant at a concentration of 1.4 mg / ml. Samples were prepared as shown in table 8.
- the stoichiometry of the reaction was also examined by comparing the coating results for a sample of upstream recAlpha-1 (RAD-0637) prepared on Day 1 using 4 ⁇ of enzyme in the reaction and on Day 2 (14 days later), using 2 ⁇ of enzyme.
- the results of the experiment show the same coating value for enzymatic amounts of 2 ⁇ and 4 ⁇ , indicating that 2 ⁇ of enzyme were sufficient for the reaction to continue until complete, which was consistent with the previous observation (Table 10 ).
- an internal standard was used to normalize the galactose peak area due to the inherent variability of amperometric detection at each injection.
- two internal standards were tested: galactosamine and deoxyribose. Both internal standards functioned properly and both eluted at times sufficiently different from galactose and did not interfere with quantification. Although galactosamine behaved properly, some inconsistencies were observed in the peak areas that would require broader acceptance criteria to monitor system performance. Therefore, deoxyribose was selected as internal standard.
- deoxyribose is commonly used as a standard in the industry for amperometric detection methods. The areas for the deoxyribose peak showed less variability during the long injection sequences and could be used with narrower acceptance criteria.
- Glyko-Prozyme was also eliminated as an option when the galactose peak areas were equal for samples treated with ⁇ -galactosidase to those samples treated with both ⁇ -galactosidase and a-sialidase in two different experiments (i.e. the activity of - sialidase was undetectable).
- Direct comparative experiments were prepared comparing the New England BioLabs and QA Bio enzymes and were performed the same day. The results were also compared with data from previous days with the same samples digested with QA Bio enzymes.
- Example 8 Type and preparation of the sample
- the upstream protein samples may contain 5 mg / ml of galactose from cell culture media
- a sample preparation method was developed to remove most of the excess galactose from the media, as well as other potentially interfering excipients. This cleaning alone was not enough to remove all impurities from the process, so additional cleaning should be performed as part of the preparation for the coating method.
- Two quick cleaning methods were evaluated: dialysis against deionized water and filtration in a 10 kDa centrifuge filter. The experiment used a sample of recAlpha-1 exchanged with buffer. In this experiment, a sample was dialyzed against deionized water for 4 hours while another sample was cleaned simultaneously using a 10 kDa centrifuge filter. Both samples were then analyzed by the coating method.
- Specificity is the ability of the method to evaluate the analyte in the presence of components that can be expected to be present, such as impurities, degradation products, matrices, etc.
- the specificity of the method was determined by preparing a mixture of neutral monosaccharides and commercially available aminomonosaccharides and analyzing the mixture by HPAEC-PAD.
- the sugars evaluated were fucose, galactosamine, glucosamine and galactose. Sugars were analyzed individually to confirm retention times and then analyzed as a mixture to determine specificity (Figure 5).
- the monosaccharide separation was comparable to that observed in Dionex Data Sheet 20, where galactose elutes after the other three monosaccharides.
- the linearity of the HPAEC PAD sialylation test is its ability to obtain test results that are directly proportional to the concentration or content of analyte at a given interval.
- an interval derived from the linearity study was used to confirm the acceptable degree of linearity, accuracy and precision attainable by the procedure.
- the linearity for the method was evaluated by preparing a standard calibration curve for galactose with an optimal range of 8 pmol to 1.5 nmol ( Figure 6).
- the coefficient of determination was 0.99 or greater for the range of 8 pmol to 1.5 nmol. Regression residuals were also analyzed and showed not to be biased in that interval.
- the quantification limit (LDC) of the HPAEC PAD sialylation assay indicates the lowest amount of analyte in a sample that can be quantitatively determined with adequate precision and accuracy.
- LDC quantification limit
- the LDC was also calculated in a different way. Based on the values in table 14 and specifically on the standard deviation of the ordinate at the origin (0.0577) divided by the average slope (0.0283) and multiplied by 10, the LDC was calculated to be 20 pmol . The two methods of calculating the quantification limit suggest that LDC was approximately 8 to 20 pmol.
- the accuracy of the method was determined by concordance between a known standard and the experimentally measured results. Since there is no "gold standard" that serves as a reference, the accuracy of the method was determined using an equal part mixture of sialylated fetuin and asialofetuin standards. available in the market.
- the sialylated bovine fetuin pattern and the asialofetuin pattern were prepared at equal concentrations as determined by UV absorbance at 280 nm (i.e., A280). The patterns were analyzed individually, then prepared in a 1: 1 ratio and analyzed.
- the sialylated fetuin pattern had a 99.4% coating percentage while the asyalofetuin pattern had a 1.2% coating percentage, because the amount of galactose in Reaction B is slightly higher than Reaction C
- the expected coating percentage for the control fetuin mixture would be approximately 50%. It was established that the actual result for the fetuin mixture was 49.3% based on the average of multiple series. The results for the experiment are summarized in Table 16. Although the derivation of exact variability or accuracy cannot be made from the A280 measurement, these results indicate that this method was accurate within a few percentage points.
- the repeatability of the assay was evaluated for the consistency of the results obtained from the method for a short period of time under the prescribed conditions.
- the repeatability of the method was determined using a sample of cell culture supernatant of recAlpha-1 in six duplicate injections.
- the background sample i.e. Reaction A
- the relative standard deviation was determined for the galactosamine area, the galactose area and the percentage of coating in the six duplicate injections.
- the results are summarized in Table 17 and are shown both corrected by the galactosamine area and without correction. The data shows that the repeatability is approximately 0.20%.
- Table 17 Reproducibility of the method
- the intermediate precision analysis incorporated several additional factors: different days, different instrument settings and different sample preparation.
- the intermediate precision of the method was investigated by preparing a sample of downstream process development recAlpha-1 (RAD-5904) for coating analysis on three different days and at three different concentrations.
- different Dionex ICS-3000 chromatography systems, disposable electrodes, AminoTrap columns, precolumns and analytical columns were used.
- the results show that the relative standard deviation (DER) of the sample is 0.25%.
- the coating percentages of the fetuin control prepared in the three days of analysis were also compared.
- the fetuin control DER was 2.99%.
- Table 18 and Table 19 The results were averaged from duplicate series and were not corrected.
- the robustness of the test is a measure of its ability not to be affected by small, but deliberate, variations in method parameters or sample handling. Several different factors were deliberately modified in a few sets of experiments, such as automatic sample processor stability, enzyme reaction time, enzyme volume and matrix interference.
- the sample was then prepared using the usual exchange wash procedure with 20 mM phosphate buffer, pH 7 followed by filtration by centrifugation at 10 kDa. He Coating percentage of the Alpha-1 PD added to the culture media was compared with the coating results for Alpha-1 PD that had not been added to the cell culture media (see Table 22). The results show a 99.4% coverage percentage for PD Alpha-1 added to the media and 99.2% (average) for PD Alpha-1 not added to the media, which was within the associated intermediate accuracy to this method These results indicate that cell culture media do not interfere in the assay after the samples have undergone the appropriate cleaning steps.
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AU2012285696A AU2012285696B2 (en) | 2011-07-14 | 2012-07-05 | Rapid and accurate analysis of protein sialylation |
US14/232,783 US9260743B2 (en) | 2011-07-14 | 2012-07-05 | Rapid and accurate analysis of protein sialylation |
CN201280033128.7A CN103635590A (zh) | 2011-07-14 | 2012-07-05 | 蛋白质唾液酸化的快速和准确分析 |
BR112013033883-0A BR112013033883B1 (pt) | 2011-07-14 | 2012-07-05 | método para determinar o teor de sialilação de uma proteína, e, uso de cromatografia hpaec-pad |
EP12815322.8A EP2733218B1 (en) | 2011-07-14 | 2012-07-05 | Rapid and accurate analysis of protein sialylation |
CA2837981A CA2837981C (en) | 2011-07-14 | 2012-07-05 | Rapid and accurate analysis of protein sialylation |
NZ618889A NZ618889B2 (en) | 2011-07-14 | 2012-07-05 | Rapid and accurate analysis of protein sialylation |
KR1020147003822A KR101864468B1 (ko) | 2011-07-14 | 2012-07-05 | 단백질 시알릴화의 신속하고 정확한 분석방법 |
RU2013154278/10A RU2605900C2 (ru) | 2011-07-14 | 2012-07-05 | Быстрый и точный анализ сиалирования белков |
JP2014519588A JP2014525742A (ja) | 2011-07-14 | 2012-07-05 | タンパク質のシアリル化の、迅速且つ正確な分析 |
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MX2014000378A MX344407B (es) | 2011-07-14 | 2012-07-05 | Analisis rapdo y preciso de la sialilacion de proteinas. |
ES12815322.8T ES2675511T3 (es) | 2011-07-14 | 2012-07-05 | Análisis rápido y preciso de la sialilación de proteínas |
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