Discussion On Internal Sterility Test Method Of Microspheres For Injection

The sterility test of microspheres for injection should focus on both “outside sterility” and “inside sterility”, and the difficulty of detection is “inside sterility”, that is, the sterility test inside the microsphere. On the basis of investigating the production process of microspheres for injection, this study selected representative varieties, simulated the dissolution process of microspheres in the presence of potential microbial pollutants, and established two internal sterility inspection methods: one is to use dimethyl methylene Sulfone (DMSO) was used to dissolve the microspheres; the second was to use pH 10.0 buffer to dissolve the microspheres. The test solutions for sterility test prepared by the two methods used bacterial tolerant spores as the test bacteria, and the sterility test was carried out by direct inoculation method, and the key technical links such as the preparation of the test solution, the strains used for the test, and the applicability of the method were discussed. scientific and rational. This study is a beneficial exploration of the development and verification strategy of the microsphere sterility test method for injection, and can provide research ideas and data support for the quality control and standard improvement of this type of pharmaceutical preparations.

With the development of the pharmaceutical industry, there are endless drug dosage forms to adapt to the needs of disease treatment or prevention. Drugs and suitable carriers (biodegradable materials) are micron or nanoscale solid, liquid or gaseous drugs prepared by a certain dispersion and embedding technology, including microcapsules, microspheres, microcrystals, liposomes and nanoparticles, etc. It is a microparticle preparation, and microspheres for injection are one of the most widely used microparticle preparations. The approval numbers of more than 30 imported and domestic varieties of microspheres for injection can be found on the website of the State Drug Administration. Representative varieties include leuprolide acetate microspheres for injection, risperidone microspheres for injection, Octreotide Acetate Microspheres for Injection, Perfluorobutane Microspheres for Injection, etc. The quality control and analysis methods of these varieties have attracted much attention, among which the scientific and rationality of the sterility test method is an important content of its attention.

At present, the provisions under the sterile item in the microsphere standard for injection are mainly divided into three categories.

① Point to the general rules, no specific operation method is specified, such as: take this product, add the attached suspending agent to make a suspension, check according to the sterility test method, and it should meet the regulations.

② Point to the general rules and specify the specific operation method, such as: take this product, follow the instructions for each tube, use the attached syringe and needle, add the attached solvent to make the dispersion even, adopt the direct inoculation method, take the full amount of each tube and inoculate it until cultured Base 50 ml, with Staphylococcus aureus as the positive control, check according to the sterility test method, and it should meet the requirements.

③The internal and external sterility inspections of the microspheres should be carried out separately, such as: take this product, dissolve it in 2 ml of the corresponding culture medium, filter, and use Staphylococcus aureus as the positive control bacteria to conduct an external sterility inspection; This product was dissolved in 2 ml of sterile dimethyl sulfoxide (DMSO), filtered, and the filtration volume of each membrane was not more than 10 ml. Staphylococcus aureus was used as the positive control bacteria for internal sterility inspection.

The difficulty of sterility inspection of microspheres for injection focuses on “intrasphere sterility”, that is, the sterility inspection inside the microspheres. Generally speaking, if the product adopts the terminal sterilization process, and the particle size distribution is smaller than the microbial pollutants, and the microorganisms are not easy to exist or are difficult to exist in the preparation during the production process, the external sterility inspection of the microspheres is carried out; the product adopts the aseptic production process, However, the particle size distribution is larger than or similar to microbial contaminants, and when there may be potential contamination risks in the production process, the sterility inspection of the inside and outside of the microspheres should be carried out separately.

According to research, most injection microspheres adopt aseptic production process, with polylactic acid-glycolic acid copolymer (PLGA) as an excipient, mixed with raw materials, and wrapped in a closed system to form a preparation. The production process involves methylene chloride, acetic acid Such conditions are not conducive to the growth and survival of microorganisms, but there is a risk of contamination by resistant spores. For representative varieties (such as octreotide acetate microspheres for injection, etc.), DMSO was used as a solvent to prepare the test solution when performing the internal sterility test of the microspheres, but high concentrations of DMSO have antibacterial effects, and whether it affects the applicability of the sterility test method and effectiveness are yet to be assessed. In addition, in the process of establishing methods for certain varieties (such as risperidone microspheres for injection), risk assessments have been carried out for each link of the production process, and it is believed that the aseptic operations involved are all after the microspheres harden, and potential pollutants It may only exist on the outer surface, so the internal sterility test was canceled when the standard was changed. How the normativeness of the above assessments can be general technically stipulated in drug standards has not yet been discussed in depth.

In this study, leuprolide acetate microspheres for injection, risperidone acetate microspheres for injection, octreotide acetate microspheres for injection and other domestic and foreign representative varieties were selected, focusing on the sterility inspection inside the microspheres, and investigating the preparation of test solutions, The scientificity and rationality of key technical links such as strains and inspection methods used in the test are expected to provide research ideas and data support for the establishment of sterility inspection methods for injection microspheres and the formulation and revision of drug standards.

Part 1 Method And Results

1.1 Preparation Of Test Bacteria Solution

With reference to the ChP 2020 sterility test method (general rule 1101), prepare the test bacteria liquids of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger and Clostridium sporogenes; ChP 2020 Antibiotic Microbiological Testing Method (General Rule 1201), preparation of test bacterial liquid of Bacillus subtilis spores and Bacillus pumilus spores. Use sterile sodium chloride-peptone buffer solution at pH 7.0 to prepare bacterial suspensions containing 108 cfu and no more than 100 cfu per 1 ml.

1.2 Preparation Of Test Solution

DMSO test solution: Take microsphere powder for injection (filling volume is about 30 mg per bottle), add 2 ml of DMSO solution to each bottle, and mix well. Take another sample and add 10% DMSO, 25% DMSO, 50% DMSO, 75% DMSO solution respectively, none of which can be dissolved to form a uniform test solution. According to ChP 2020 general rule 1101, the inoculation ratio of the test solution should not be greater than 10% of the medium volume. In subsequent tests, 100% DMSO (high concentration) and 10% DMSO solution (low concentration) were used to investigate the effect of the solvent on the growth of the test bacteria. Influence.

Water-soluble test solution: Accurately weigh 2.7 g of ammonium chloride, put it in a 500 ml measuring bottle, dissolve it in 450 ml of water, add 6.0 ml of triethylamine into the bottle, mix well, make up to volume with water, and adjust with triethylamine. To pH 10.0±0.1, make pH 10.0 buffer solution, and sterilize. Take the microsphere powder for injection (the filling volume is about 30 mg per bottle), transfer it to a glass bottle containing 25 ml of the above sterilization buffer, seal it, and put it on a constant temperature shaker at 45 °C and 120 r/min until the microsphere completely dissolved.

1.3 Method Suitability Test

The corresponding culture medium was prepared according to the ChP 2020 sterility test method (general rule 1101), and the suitability and sensitivity of the culture medium were checked to meet the pharmacopoeia standards. The setting of the method applicability test is divided into 3 groups, among which the blank control group is used to judge whether the test method system is normal, the solvent group is used to judge whether the solvent interferes with the test method, and the test group is used to judge whether the test method meets the regulations.

Test group: take microspheres for injection, add corresponding solvent (DMSO or pH 10.0 buffer solution), add test bacteria solution with a bacterial content of not more than 100 cfu to the above solution, and mix well; will contain Escherichia coli, aeruginosa Transfer the mixed solution of Pseudomonas, Staphylococcus aureus and Clostridium sporogenes to 50 ml of FTM medium, and culture at 33°C for no less than 5 days, and the mixed solution containing Bacillus subtilis, Candida albicans, Aspergillus niger, subtilis The mixed solution of Bacillus spores and Bacillus pumilus spores was transferred to 50 ml of TSB medium, cultured at 23°C for no less than 5 days, and observed whether the test bacteria grew.

Solvent control group: Take the corresponding solvent (DMSO or pH 10.0 buffer solution), add the test bacteria solution with a bacterial content of not more than 100 cfu, mix well, and operate in the same way as the test group.

Blank control group: take sterilized purified water, add the test bacteria solution with a bacterial content of not more than 100 cfu, mix well, and operate in the same way as the test group.

1.4 Morphological Investigation Of Microspheres For Injection

Take a small amount of microspheres for injection respectively, take out a little powder or powder block with a small medicine spoon, spread it evenly on the quartz glass table, use a disposable carving knife, repeatedly and randomly cut 10 times; Sprinkle it on the conductive glue on the sample stage, blow the surface of the sample gently with the ear cleaning ball, clean the sample that is not firmly adhered, use a sputtering instrument (setting the sputtering power to 20 W, and the gold plating time to 30 s), and then load it into the scanning electron microscope ( SEM) instrument, according to different imaging ratios, set the accelerating voltage to 5 or 10 kV.

1.5 Applicability Test Of Sterility Test Method After Dissolving Microspheres In DMSO

1.5.1 Investigation Of The Growth Inhibitory Effect Of DMSO On The Test Bacteria

Take an appropriate amount of 100% DMSO (high concentration) and 10% DMSO solution (low concentration) under item “2.2” respectively, according to ChP 2020 general rule 1101, add each test bacterial suspension not greater than 100 cfu, and mix well. Transfer the mixed solution containing Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Clostridium sporogenes to 50 ml of FTM medium, and culture it at 33°C for no less than 5 days. The mixed solution of Candida albicans, Aspergillus niger, Bacillus subtilis spores and Bacillus pumilus spores was transferred to 50 ml of TSB medium, and cultured at 23°C for no less than 5 days. The results are shown in Table 1. 100% DMSO has antibacterial effect, and the test bacteria (vegetative bodies) specified in the Pharmacopoeia cannot grow; Bacillus subtilis spores and Bacillus pumilus spores can grow. The 10% DMSO solution has no antibacterial effect, and the test bacteria (vegetative body) and tolerant spores specified in ChP 2020 can grow, but the microspheres cannot be dissolved.

1.5.2 Method Suitability Test

Two representative varieties of leuprolide acetate microspheres for injection and risperidone microspheres for injection were selected respectively, and the applicability test of the method for internal sterility inspection of the microspheres was carried out. Take the above samples, and use 2 ml of DMSO solution to dissolve the microspheres in each bottle as the test solution. Microscopic examination of the test solution after freeze-drying showed that no complete microspheres were seen under the SEM field of view, suggesting that the excipients had been dissolved by the DMSO solution. According to ChP 2020 General Rule 1101, after mixing the microspheres for injection and DMSO solution, add the suspension of each test bacteria not more than 100 cfu, and mix well. Transfer the mixed solution containing Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Clostridium sporogenes to 50 ml of FTM medium, and culture it at 33°C for no less than 5 days. The mixed solution of Candida albicans, Aspergillus niger, Bacillus subtilis spores and Bacillus pumilus spores was transferred to 50 ml of TSB medium, and cultured at 23°C for no less than 5 days as the test group; at the same time, sterilized purified water was used as the blank The control group and 100% DMSO were used as the reagent control group, and operated in the same way.

100% DMSO can dissolve the above two types of microspheres for injection, but the results under “2.5.1” show that 100% DMSO can inhibit the growth of the test bacteria (vegetative bodies) specified in the Pharmacopoeia, so Bacillus subtilis can be selected Bacillus spores and Bacillus pumilus spores were used as the test bacteria for method applicability test. Although the 10% DMSO solution has no growth inhibitory effect on the test bacteria (vegetative body) specified in the Pharmacopoeia, it cannot dissolve the microspheres to make the test solution. The results suggest that the internal sterility inspection of the above two representative varieties of microspheres for injection can use 100% DMSO as the solvent to prepare the test solution, combined with the risk assessment of potential contamination during the production process, and use tolerant spores as the test bacteria , to conduct method applicability tests.

1.6 Applicability Test Of Sterility Test Method After Dissolving Microspheres In Water-Soluble Buffer Solution

According to the application of the injection, the sterility test generally prepares a water-soluble test solution, and conducts a sterility test by membrane filtration. DMSO is an organic solvent and has inhibitory effect on the growth of the test bacteria in the vegetative state. In order to seek a water-soluble “breaking ball” method, the water-soluble test solution was prepared by simulating the in vitro release of microspheres for injection. Since the ester bond of the PLGA excipient is broken to produce acid, adjusting the pH value of the buffer system to alkaline can accelerate the degradation of the excipient.

1.6.1 Investigation Of PH 10.0 Buffer Solution On Growth Inhibition Of Test Bacteria

Take an appropriate amount of pH 10.0 buffer under item “2.2”, add each test bacteria not more than 100 cfu according to ChP2020 general rule 1101, and mix well. Transfer the mixed solution containing Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Clostridium sporogenes to 50 ml of FTM medium, and culture it at 33°C for no less than 5 days; The mixed solution of Candida albicans, Aspergillus niger, Bacillus subtilis spores and Bacillus pumilus spores was transferred to 50 ml of TSB medium, and cultured at 23°C for no less than 5 days. At the same time, in order to confirm the stability of the test strain in the pH 10.0 buffer solution, another appropriate amount of pH 10.0 buffer solution was added to the test bacteria, and incubated in a constant temperature shaker at 45 °C and 120 r/min for 4 to 6 h, and then transferred to To different media, observe the growth of strains. The above-mentioned test group using freshly prepared pH 10.0 buffer solution was used as solvent test group 1, and the test group using pH 10.0 buffer solution incubated for 4 to 6 h was used as solvent test group 2.

1.6.2 Method Suitability Test

Two representative varieties of leuprorelin acetate microspheres for injection and octreotide microspheres for injection were selected to conduct the applicability test of sterility testing methods. Take the above samples, transfer each bottle to a glass bottle containing 25 ml of pH 10.0 buffer solution, incubate at 45 °C, 120 r/min in a sealed sterile state for 4-6 h, and obtain the test solution (simultaneously with pH 4.0, pH 7.0 buffer was used as a control, and the microspheres could not be dissolved under the same conditions, and the accelerated release behavior was consistent with the relevant research results [9]). Microscopic examination of the test solution after freeze-drying showed no complete microsphere morphology under the SEM field of view, suggesting that the excipients can accelerate degradation under these conditions.

According to ChP 2020 general rule 1101, after mixing the microspheres for injection with pH10.0 buffer, add the suspension of each test bacteria not more than 100 cfu, mix well, and incubate under the above conditions until the microspheres are completely dissolved. Transfer the mixed solution containing Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Clostridium sporogenes to 225 ml of FTM medium, and culture it at 33°C for no less than 5 days; The mixed solution of Candida albicans, Aspergillus niger, Bacillus subtilis spores and Bacillus pumilus spores was transferred to 225 ml of TSB medium and cultured at 23°C for no less than 5 days as the test group; at the same time, sterilized purified water was used as the test group. The blank control group and the pH 10.0 buffer as the reagent control group were operated in the same way.

Part 2 Discussion

In this study, on the basis of investigating the production process of injection microspheres and product registration application materials, selected representative varieties such as leuprolide acetate microspheres for injection, and established two sterility testing methods: one is to use DMSO to dissolve microspheres The second is to use pH 10.0 water-soluble buffer to dissolve the microspheres after accelerating the degradation of excipients. The prepared sterility test solution was prepared in accordance with ChP 2020 general rule 1101, with Bacillus subtilis spores or Bacillus pumilus spores as the test bacteria, and the sterility test was carried out by direct inoculation method.

Based on the characteristics of microspheres for injection, the sterility inspection of this preparation mainly focuses on the following technical links.

①On the basis of fully understanding the product production process, quality standards, and key links of microbial contamination in process control, establish a sterility inspection method after method verification and relevant method applicability tests; for example, most of the microspheres for injection are currently The product adopts an aseptic production process, and it is difficult for microbial vegetative bodies to survive inside the microspheres. Bacillus subtilis spores or Bacillus pumilus spores can be used as test bacteria, or other tolerances can be used according to the risk assessment of potential pollutants in the product production process. Spores were used as test bacteria for method validation.

② For varieties that require internal sterility testing, an appropriate solvent should be selected to prepare the test solution to simulate the microsphere dissolution process in the presence of potential microbial pollutants, and investigate whether the solvent affects the growth of the test bacteria; for example, use Dissolving DMSO, or using an alkaline (pH 10.0) buffer system to accelerate the degradation of excipients to prepare the test solution, are both beneficial attempts to establish sterility testing methods for special sterile preparations.

③Due to the different proportions of PLGA monomers, the polymer degradation time, drug viscosity, and drug release behavior are all different when forming preparations [10-11]. Therefore, when establishing sterility testing methods for different varieties, specific test parameters need to be adjusted to verify or confirm. In addition, the visual judgment of the degradation behavior of the microsphere skeleton is not sufficient. For example, some varieties have “erosion” phenomenon in a certain accelerated release medium. “Gradually changed to “honeycomb porous gap spherical surface”. Therefore, if necessary, SEM can be used for morphological observation, content determination and other methods to support the development or verification strategy of sterility inspection methods.

The production process of microspheres for injection is personalized, and compared with common injections, it is still a niche special dosage form. In this study, representative varieties such as Leuprolide Acetate Microspheres for Injection, Risperidone Acetate Microspheres for Injection, and Octreotide Acetate Microspheres for Injection were selected for research, basically covering the currently developed and marketed varieties. In addition to the varieties studied in this study, there are also dosage forms under special production processes, such as perfluorobutane microspheres for injection, which are hollow microspheres. The test solution was prepared after the structure of the microspheres was destroyed by pressurization, and the overall sterility inspection of the exterior and interior was carried out.

According to the relevant regulations of ChP 2020 General Rule 1101, if the traits of the variety are allowed, the membrane filtration method should be preferred for sterility testing. Since DMSO is corrosive to filter membranes for sterility testing made of mixed cellulose, polyvinylidene fluoride, etc., and whether the pH 10.0 buffer solution has an impact on the integrity of the filter membrane still needs to be evaluated, so this study was established when the sterility testing method was established. , using the direct inoculation method. Whether the membrane filtration method can be used for other species in the sterility test still needs to be investigated on the factors affecting the integrity of the filter membrane.