Description:Field of the invention
The invention leads to reduction of estimation time of microbial contamination in pharmaceutical product by optimizing condition and factors using compendial method.
Background of the Invention
References which are cited in the present disclosure are not necessarily prior art and therefore their citation does not constitute an admission that such references are prior art in any jurisdiction. All publications, patents and patent applications herein are incorporated by reference to the same extent as if each individual or patent application was specifically and individually indicated to be incorporated by reference.
Several patents have been issued for microbial contamination but none of these are related to the present invention. For example, US10961593B2 relates to an analysis system for detecting the source of fecal bacteria contamination in an environmental sample, comprising probes capable of detecting at least 10% of the source-specific taxa set forth in Table 1 (bird), Table 2 (grazer), Table 3 (sewage), or a combination thereof, wherein each of said probes is about 16 to about 35 nucleotides in length, and wherein the analysis system consists of about 105 to about 108 of said probes.
Another patent, US20220143232A1 relates to a method, process and apparatus for disinfecting and sterilizing all types of surfaces contaminated with microorganisms and toxic substances to render both inactive. Furthermore, this invention relates to both a method and apparatus for disinfecting and/or sterilizing breathable air and then using this air to protect a confined space from external contamination. The apparatus consists of a new ultra-violet (NUV) source that is more effective than mercury based 254 nm light for destroying DNA of virus, bacteria, spores and cists. It is most effective in breaking chemical bonds in toxic gases and Biotoxins that are useful to terrorists. It is combined with other apparatus that remove particulates and byproducts sometimes produced by the NUV source and maintains positive pressure of the confined space so as to prevent the influx of air from outside the protected zone.
Another patent, WO2005010207A2 provides methods and compositions for the detection and/or quantification of a microbial contaminant, for example, a bacterial endotoxin or a glucan, in a sample. In particular, the invention provides a test cartridge useful in the practice of hemocyte lysate-based assays for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides methods of making and using such cartridges. In addition, the invention provides a rapid, sensitive, multi-step kinetic hemocyte lysate-based assay for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides a glucan-specific lysate that can be used in a variety of assay formats, including, for example, a test cartridge, optionally configured to perform a kinetic assay.
Another patent, WO2006099359A2 disclosed method of delaying the onset of an infection or preventing an infection caused by a microbial organism in an internal cavity of a subject, the method comprising: contacting at least a portion of the interior surface of an opening leading to the internal cavity with an antimicrobial composition; and subsequently at least partially inserting an instrument into the opening, wherein the antimicrobial composition comprises: an effective amount of an antimicrobial component comprising an antiseptic, an antibiotic, or combinations thereof; and a surfactant component distinct from the antimicrobial component, wherein the surfactant component is present in an amount of at least 0.5 wt-% and/or the surfactant component comprises an anionic surfactant, zwitterionic surfactant, poloxamer surfactant, amine oxide surfactant, or combinations thereof; with the proviso that when the internal cavity comprises a nasal passage, vagina, or oral cavity, the antimicrobial component does not include iodine or chlorhexidine.
Another patent, WO2009099419A2 provides antimicrobial compositions comprising one or more acid and one or more organic diol. In one embodiment, the invention's compositions have an acidic pH. The compositions may optionally further contain one or more oxidizing agent (including stabilized oxidizing agent and/or unstabilized oxidizing agent), and/or one or more surfactant. In particular embodiments, the acid lacks one or both of -NH group and-NH2 group. The invention's compositions are useful in all stages of handling agricultural products, in hospitals, and in commercial and household applications.
Another patent, US7326775B2 relates to method of preparing an ultra-cleansed lactoferrin preparation, termed treatment for contaminant reduction (TCR) is provided which includes the steps of treating commercial lactoferrin preparation with at least one each of surfactants, antioxidants and polyphenols to form purified lactoferrin (LF-TCR) and drying the LF-TCR. Additionally, a therapeutic lactoferrin composition is provided which contains LF-TCR and optionally surfactants, antioxidants, polyphenols, tissue/membrane diffusion facilitating agents and anionic compounds. The therapeutic lactoferrin composition can additionally contain bioactive agents, dietary supplements, nutraceuticals/functional foods, prophylactic agents, therapeutic agents and probiotic lactic acid bacteria.
Compendial methods for estimation of Microbial contamination are time taking process. Therefore, pharmaceutical products regulators and manufacturers keep focusing related to methods which may help in the reduction of estimation time of microbial contamination which are suitable for identification and confirmation test with proving suitability, sensitivity and authenticity with respect of concurrent rules and guidelines.
According to the claims, determination of microbial contamination in healthcare products is always critical aspect for regulators and manufacturers. We explored the alternate methods by optimizing conditions and factors to overcome the constraint of present method. Initial screening, characterization, conditions and factors were optimized for one of the known methods i.e., test for specified microorganism (targeted Microbes). In this study, we have covered most of the routinely used raw materials in pharmaceuticals dosage form and finished products.
The primary object of the present invention is reduction of estimation time of microbial contamination in pharmaceutical product by optimizing condition and factors using compendial method.
Another object of the present invention is reduction of estimation time of microbial contamination.
Another object of the present invention is optimizing condition and factors using compendial method.
These and other objects and advantages of the present invention will become readily apparent from the following detailed description.
Summary of Invention
This summary is not a comprehensive overview of the disclosure and does not reflect the main/essential features of the establishment or specify the scope of the establishment. Its sole purpose is to present some of the concepts presented here in a simpler way as a precursor to more detailed.
The primary object of the present invention is reduction of estimation time of microbial contamination in pharmaceutical product by optimizing condition and factors using compendial method.
In some embodiments of the present invention, according to the claims, determination of microbial contamination in healthcare products is always critical aspect for regulators and manufacturers. We explored the alternate methods by optimizing conditions and factors to overcome the constraint of present method.
In some embodiments of the present invention, initial screening, characterization, conditions and factors were optimized for one of the known methods i.e., test for specified microorganism (targeted Microbes). In this study, we have covered most of the routinely used raw materials in pharmaceuticals dosage form and finished products.
In some embodiments of the present invention, transfer a loop full of E. coli culture (Glycerol Stock) in to a 50 ml flask containing 25 ml of SCDM and incubated with agitation at 200 RPM inside the Bacteriological Incubator at 32.5ºC±2.5 ºC. At reaching multiplication of growth OD approx. 0.50 was obtained (It takes 02 to 05 hours, for getting desired OD). The absorbance of the culture was measured at A600 using spectrophotometer (UV-1800).
These and other aspects of the embodiments herein will be better appreciated and understood when considered in concurrence with the following explanation and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.
Brief summary of the figures
Figure-1: Sample In incubation Condition
Figure-2: Plate –I 10-5 Dilution E. Coli, Plate –II 10-5 Dilution E. Coli
Brief summary of the figures
Table 1: Sample arrangement and incubation
Table 2: Incubation Condition
Table 3: Results of Standard Vs Optimized Condition:
Detailed Description
These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In some embodiments of the present invention, according to the claims, determination of microbial contamination in healthcare products is always critical aspect for regulators and manufacturers. We explored the alternate methods by optimizing conditions and factors to overcome the constraint of present method.
In some embodiments of the present invention, initial screening, characterization, conditions and factors were optimized for one of the known methods i.e., test for specified microorganism (targeted Microbes). In this study, we have covered most of the routinely used raw materials in pharmaceuticals dosage form and finished products.
In some embodiments of the present invention, transfer a loop full of E. coli culture (Glycerol Stock) in to a 50 ml flask containing 25 ml of SCDM and incubated with agitation at 200 RPM inside the Bacteriological Incubator at 32.5ºC±2.5 ºC. At reaching multiplication of growth OD approx. 0.50 was obtained (It takes 02 to 05 hours, for getting desired OD). The absorbance of the culture was measured at A600 using spectrophotometer (UV-1800).
In some embodiments of the present invention, Growth Promotion test (GPT) for media was conducted and the media qualify the test which was utilized for further study. Take a loopful of culture (Glycerol Stock) and inoculate it into 25 mL of Casein soyabean digest broth and incubate with agitation (200 RPM) at 30° to 35° C for approx. 02 to 05 hours. At reaching of OD at around 0.5, Pipette out 1.0 mL from it and inoculate it into 9 ml of Buffered sodium chloride-peptone solution pH 7.0 (10-1). And prepare final serial dilution till 10-10 dilution.
In some embodiments of the present invention, prepare duplicate plates of each dilution (10-1 to 10-10) of SCDA & SDA & incubate SCDA plates at 30° to 35° C & SDA plates at 20° to 25° C. After the incubation period, count the number of colonies and then select dilution which are having cells Not more than 100 cfu/mL.
A reduction of estimation time of microbial contamination in pharmaceutical product by optimizing condition and factors using compendial method comprising the steps of:
taking a loopful of culture (Glycerol Stock) and inoculate it into 25 mL of Casein soyabean digest broth;
incubating with agitation (200 RPM) at 30° to 35° C for approx. 02 to 05 hours;
reaching of OD at around 0.5, pipette out 1.0 mL from it;
inoculating it into 9 ml of Buffered sodium chloride-peptone solution pH 7.0 (10-1);
preparing final serial dilution till 10-10 dilution;
prepare duplicate plates of each dilution (10-1 to 10-10) of SCDA & SDA;
incubating SCDA plates at 30° to 35° C & SDA plates at 20° to 25° C;
after the incubation period, count the number of colonies; and
selecting dilution which are having cells Not more than 100 cfu/mL.
EXAMPLE 1
Material and Methods
This present study was conducted in Ved Lifesavers Pvt. Ltd. (A Pharmaceutical company) and Uttaranchal University Dehradun UK.
INOCULAM PREPRATION
Growth Promotion test (GPT) for media was conducted and the media qualify the test which was utilized for further study.

Sterilization and preincubation 24 hours
Take a loopful of culture (Glycerol Stock) and inoculate it into 25 mL of Casein soyabean digest broth and incubate with agitation (200 RPM) at 30° to 35° C for approx. 02 to 05 hours.

Measure the OD (A600) at 01-hour interval

At reaching of OD at around 0.5, Pipette out 1.0 mL from it and inoculate it into 9 ml of Buffered sodium chloride-peptone solution pH 7.0 (10-1 ). And prepare final serial dilution till 10-10 dilution.

Prepare duplicate plates of each dilution (10-1 to 10-10) of SCDA & SDA & incubate SCDA plates at 30° to 35° C & SDA plates at 20° to 25° C.

After the incubation period, count the number of colonies and then select dilution which are having cells Not more than 100 cfu/mL.

TEST FOR SPECIFIED MICROORGANISM (E. coli)
SAMPLE PREPARATION

PRETREATMENT OF SAMPLE

Sample arrangement and incubation as per below table.
S. No. Sample Incubation Condition Incubation Temperature
1 Amlodipine Besilate & Atenolol Tablets
Product Control Standard condition 42- 44ºC
2 Amlodipine Besilate & Atenolol Tablets
Product Control Optimized Condition 42- 44ºC
3 Amlodipine Besilate & Atenolol Tablets
Product Positive Control Standard condition 42- 44ºC
4 Amlodipine Besilate & Atenolol Tablets
Product Positive Control Optimized condition 42- 44ºC
5 MCB Positive Control Standard condition 42- 44ºC
6 MCB Positive Control Optimized condition 42- 44ºC
7 MCB Negative Control Standard condition 42- 44ºC
8. MCB Negative Control Optimized Condition 42- 44ºC

The incubated plates were continuously monitored for growth at interval of every two hours until the growth appeared (in form of turbidity) was measured at A600 nm.

RESULTS AND DISCUSSION
OPTIMIZATION FOR INOCULUM PREPARATION
Preparation of Inoculum
Transfer a loop full of E.coli culture (Glycerol Stock) in to a 50 ml flask containing 25 ml of SCDM and incubated with agitation at 200 RPM inside the Bacteriological Incubator at 32.5ºC±2.5 ºC. At reaching multiplication of growth OD approx. 0.50 was obtained (It takes 02 to 05 hours, for getting desired OD). The absorbance of the culture was measured at A600 using spectrophotometer (UV-1800).
Serial Dilution:
Prepare (10-1 – 10-10) dilution after getting desired inoculum (OD approx. 0.50), each tube contained 9.0 mL of SCDM. Ascetically pipette out 1.0 mL of inoculum from the stock solution and inoculate it into 9.0 ml of SCDM tube or 10-1dilutions. Transfer 1.0 mL from10-1 dilution to next 10-2dilution test tube containing 9.0 ml of SCDM till 10-10 dilution by gently vortexing.
Add 1.0 mL suspension from each prepared dilutions (10-1 – 10-10) into sterile petriplates in duplicates for each dilution. Pour sterile molten Soyabean Casein Digest Agar (SCDA) in all the inoculated Petri plates. Incubate all inoculated plates at 32.5ºC±2.5 ºC for 48 hrs. Preserve all the culture dilutions tubes in the refrigerator at 2°C to 8°C until the last observation of above inoculated plates. After the completion of incubation period, count the number of colonies (CFU) and then select the dilution NMT 100 cfu/mL. Store the selected dilution in refrigerator at 2°C to 8°C and discard all the other dilutions.
Selected 10-5dilutions which have 95 cfu/ml.
OPTIMIZATION OF TEST FOR SPECIFIED MICROORGANISM (E.coli)
Pretreatment of Sample
Dissolve 10 g of the sample to be examined in buffer sodium chloride peptone solution pH-7.0 under the condition of test and adjust the volume to 100 mL with required, further dilutions are prepared with same diluent.
Product Control: Take 1 mL Pretreated sample and transfer the quantity of the sample to 100 mL of MCB.
Product Positive Control (Product + Inoculums): Take 1 mL Pretreated sample corresponding to 1 g or 1mL and transfer the quantity of the sample to 100 mL of MCB and add 1 ml of culture suspension containing 95 cfu/ml of E.coli.(10-5)E.coli.
Negative control: Take1 ml of buffered peptone water without adding sample into 100 ml of Sterile MCB.
Positive Control: Take100 ml of Sterile MCB and add 1 ml of culture suspension containing 95 cfu/ml of E.coli. (10-5)of E.coli.
S. No. Sample Incubation Condition Incubation Temperature
1 Amlodipine Besilate & Atenolol Tablets
Product Control Standard condition 42- 44ºC
2 Amlodipine Besilate & Atenolol Tablets
Product Control Optimized Condition 42- 44ºC
3 Amlodipine Besilate & Atenolol Tablets
Product Positive Control Standard Condition 42- 44ºC
4 Amlodipine Besilate & Atenolol Tablets
Product Positive Control Optimized condition 42- 44ºC
5 MCB Positive Control Standard Condition 42- 44ºC
6 MCB Positive Control Optimized condition 42- 44ºC
7 MCB Negative Control Standard Condition 42- 44ºC
8. MCB Negative Control Optimized Condition 42- 44ºC

• The Aabsorbance of each sample was measured bbefore iinoculation and after inoculation, at wavelength A600 using spectrophotometer UV-1800 (Initially).
• At a time interval of every one hour the samples tubes of each sample were observed visually (for detection of turbidity).
• After visually inspect (the growth appeared in form of turbidity) withdraw each of the sample in eppendorf tube and take the OD of all samples at A600 using spectrophotometer (UV-1800).
• OD was measured at a interval of 02 hours upto the 24 hours.
Results of Standard Vs Optimized Condition:

TEST

STANDARD
CONDITION
OPTIMIZED CONDITION
TOTAL REDUCED TIME (Hr)
Inoculum Preparation 36 – 48 Hours 14 – 23 Hours 22 - 25 Hours
(38.89 % – 47.92 %)
Test For Specified Microorganism 42 – 72 Hours 10 – 17 Hours 32 - 55 Hours
(23.81 % -23.61 %)

DISCUSSION
Determination of microbial contamination in healthcare products is always critical aspect for regulators and manufacturers. Compendial methods for estimation of Microbial contamination are time taking process. Therefore, pharmaceutical products regulators and manufacturers keep focusing related to methods which may help in the reduction of estimation time of microbial contamination which are suitable for identification and confirmation test with proving suitability, sensitivity and authenticity with respect of concurrent rules and guidelines. We explored the alternate methods by optimizing conditions and factors to overcome the constraint of present method. Initials screening, characterization, conditions and factors were optimized for one of the known methods i.e., test for specified microorganism (targeted Microbes). Optimization of conditions and various factors for the preparation of reference working culture (microbial inoculums) was also performed to help in reducing estimation time for aerobic microbial contamination. In this study, we have covered most of the routinely used raw materials (Lactose monohydrate, microcrystalline cellulose, maize starch) in pharmaceuticals dosage form and finished products. In comparison to compendial methods, proposed optimized method for inoculum preparation can reduce 22-25 hours, and Test for specified microorganism can reduce 32-55 hours of the estimation time. Saving of remarkable time for determination of the absence or presence of limited occurrence of targeted microbials may help manufacturing release or take appropriate action for distribution and consumption up to end user of pharmaceutical product.
ADVANTAGES OF THE INVENTION: By this invention,
1. The optimized method for Inoculum Preparation reduced time duration 22-25 Hours (38.89 % – 47.92 %) compared to Standard method (Compendial) requiring (36 – 48 Hours).
2. The optimized method for Specified Microorganism reduced incubation time 32 –55 Hours (23.81 % -23.61 %) as compared to Standard method requiring (42 - 72 Hours) for detection of specific microorganism.
3. Total incubation time reduced 54 - 80 Hours (62 - 71%) for Test of Specified Microorganism in non-sterile pharmaceutical products.
4. Proposed Validated method may play immense application in microbial analysis in pharmaceutical industries during manufacturing release or take appropriate action for distribution of pharmaceutical product.
, Claims:
1. A reduction of estimation time of microbial contamination in pharmaceutical product by optimizing condition and factors using compendial method comprising the steps of:
i. taking a loopful of culture (Glycerol Stock) and inoculate it into 25 mL of Casein soyabean digest broth;
ii. incubating with agitation (200 RPM) at 30° to 35° C for approx. 02 to 05 hours;
iii. reaching of OD at around 0.5, pipette out 1.0 mL from it;
iv. inoculating it into 9 ml of Buffered sodium chloride-peptone solution pH 7.0 (10-1);
v. preparing final serial dilution till 10-10 dilution;
vi. prepare duplicate plates of each dilution (10-1 to 10-10) of SCDA & SDA;
vii. incubating SCDA plates at 30° to 35° C & SDA plates at 20° to 25° C;
viii. after the incubation period, count the number of colonies; and
ix. selecting dilution which are having cells Not more than 100 cfu/mL.