Method for detecting poloxamer residual quantity in recombinant human urokinase raw material for injection
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A detection method, poloxamer technology, applied in the field of detection and analysis, can solve problems such as increasing the difficulty of optimization and difficult separation
Active Publication Date: 2020-11-24
TASLY BIOPHARMACEUTICALS CO LTD
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In addition, both recombinant human prourokinase and poloxamer 188 are macromolecular substances, which are ...
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Embodiment 1
[0089] Example 1: A method for detecting the residual amount of poloxamer in the raw material of recombinant human urokinase for injection
[0090] Sample pretreatment method: Add 200 μl of original sample to 300 μl of acetonitrile that has been frozen overnight, shake for 30 seconds, sonicate for 3 minutes, shake again for 30 seconds, centrifuge at 9500 rpm for 1 minute, and take the supernatant.
[0091] Standard solution preparation method: Take poloxamer standard, weigh and prepare standard solution, the concentration is 2mg / ml, 1mg / ml, 0.5mg / ml, 0.25mg / ml, 0.1mg / ml, 0.04mg / ml, 0.02mg / ml, 0.01mg / ml, 0.005mg / ml.
[0092] Chromatographic conditions (HPLC-CAD): column Acclaim TM Surfactant Plus column, 3μm, 30mm×150mm; injection volume 10μL; column temperature 40℃; flow rate 0.7mL / min; CAD detector: sampling frequency 5 Hz, peak filtration time parameter 3.6 seconds, nebulization temperature 50℃; mobile phase and gradient elution are shown in Table 1.
[0093]Detection: ...
Embodiment 2
[0094] Embodiment 2: verification of the method
[0095] 1. Preparation of samples and solutions:
[0096] 1.1 Standards and samples
[0097] Standard product: F68 (STD), Sigma, batch number SLBP3447V;
[0098] Sample 1: recombinant human urokinase stock solution for injection (SPL1), Shanghai Tasly, batch number T20161219B;
[0099] Sample 2: Recombinant human pro-urokinase stock solution for injection (SPL2), Shanghai Tasly, batch number V20170526B.
[0100] Blank sample: a sample not containing F68 and recombinant human prourokinase, 60% acetonitrile (Blank).
[0101] 1.2 Preparation of sample solution
[0102] Stock#1 solution (2mg / mL): Accurately weigh 10.84mg of F68 into a 5mL volumetric flask, add 3mL of diluent, dilute to the mark with diluent, and mix well;
[0103] Stock#2 solution (2mg / mL): Accurately weigh 10.38mg of F68 into a 5mL volumetric flask, add 3mL of diluent, dilute to the mark with diluent, and mix well;
[0104] L8 solution (2mg / mL): take stock#2 ...
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Abstract
The invention relates to a method for detecting poloxamer residual quantity in a recombinant human urokinase raw material for injection. The method comprises the following steps: (1) a sample solutionpreparation method: adding overnight frozen acetonitrile into a raw material sample, oscillating and ultrasonically treating for 2-4 minutes, oscillating for 20-40 seconds, centrifuging at 8000-9800rpm for 1-3 minutes, and taking a supernatant, wherein the ratio of the volume of the original sample to the volume of the acetonitrile is within 1-3; 2) preparing a standard substance solution: taking, weighing and preparing a poloxamer standard substance into standard substance solutions, wherein the concentrations of the standard substance solutions are 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, 0.04 mg/ml, 0.02 mg/ml, 0.01 mg/ml and 0.005 mg/ml in sequence; and 3) detection: respectively taking a standard solution and a sample solution, performing injecting into a high performanceliquid chromatograph, recording a chromatogram detected by CAD, drawing a standard curve by taking the concentration of the standard substance solution as an abscissa and the peak area as an ordinate,calculating the content of poloxamer in the sample solution by using an external standard method, and converting to obtain the content of poloxamer in the recombinant human urokinase raw material forinjection.
Description
technical field [0001] The invention belongs to detection and analysis technology, and in particular relates to a method for determining the residual amount of poloxamer in the raw material of recombinant human urokinase for injection. Background technique [0002] Recombinant human prourokinase for injection (trade name, Puyouke) is used for thrombolytic therapy of acute ST-segment elevation myocardial infarction. [0003] The raw material of recombinant human pro-urokinase for injection is recombinant human pro-urokinase (pro-UK), that is, the stock solution of recombinant human pro-urokinase for injection, and its preparation method is described in 200410058006.0 (publication number is CN1730098A). [0004] The original production process of recombinant human pro-urokinase for injection (Pro-UK) raw material is 20L fermentation scale. In order to meet the booming market demand, the process is now enlarged to 300L fermentation scale. The 300L new process will produce a lo...
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