Method Suitability and Sterility Test Validation of Ciprofloxacin Lactate Solution for Infusion: Overcoming Antimicrobial Activity via Membrane Filtration with Magnesium Sulfate

1. INTRODUCTION

Parenteral pharmaceutical products must meet sterility requirements to ensure patient safety, as they bypass the body’s natural defense system [1, 2]. Ciprofloxacin lactate is an injection used to treat systemic infections. It must meet sterility as a quality attribute as such contamination can cause complications, including sepsis and infections in vulnerable populations [3].

Sterility testing is an important quality control step for injectable formulations. Pharmacopoeial standards, such as the United States Pharmacopeia and European Pharmacopeia (European Directorate for the Quality of Medicines), recommend two main approaches: direct inoculation and membrane filtration [2, 4]. Membrane filtration is preferred for aqueous solutions and products with antimicrobial properties, as it removes inhibitory substances by concentrating specific microbial contaminants on a filter that can be incubated in suitable growth media [5].

Residual antibacterial activity in the ciprofloxacin formulation can affect sterility test results by inhibiting microbial growth, resulting in a false-negative outcome [6, 7]. This supports validation of the sterility testing method, including suitability for effective neutralization or removal of any antimicrobial agent and the ability to recover low levels of microorganisms, as per the EDQM and USP [2, 4].

This study focused on validating the membrane filtration method for sterility testing of ciprofloxacin lactate solution for infusion. Validation helps evaluate key parameters such as microbial recovery, method suitability, and the effectiveness of the rinsing procedure in eliminating antibacterial activity, based on pharmacopeia guidelines and international standards such as the International Council for Harmonization [2, 8]. This study investigated whether adding the neutralizing agent Mg2+ during sterility testing can eliminate the test sample’s antibacterial activity, as Mg2+ forms complexes with quinolone drugs. Establishing a reliable and validated sterility testing method is essential to ensure compliance with regulatory standards and guarantee the microbiological safety of the final product [9].

2. MATERIALS AND METHODS

2.1. Reagents and Instrumentations

In this study, the following reagents and instruments were used: Analytical balance Explorer OHAUS, Euroclone Top safe microbiology safety cabinet, Petri plates Isolab, IKA MS 3 Basic Vortex, Tuttnauer model 3850 MLV, INE 800 Memmert incubator, UM 800 Memmert incubator, pH meter Ultrabasic UB-10, Denver Instrument, Millipore Steritest Compact Pump (Merck), Steritest Devices for antibiotics (Merck Millipore).

Three batches of ciprofloxacin solution for infusion Cat no. 2404; 2502; 2505 was manufactured in Profarma Sh.a), Trypticasein Soy Agar EP/USP/ISO (medium Cat No.1068 by Condalab, Spain), Sabouraud Dextrose Agar 4% medium (Cat. No.1024 by Condalab, Spain); Fluid A (Cat. No. 638042) and Fluid D (Cat. no. 638043), Thioglycolate Medium (FTM, Cat. No. 638040 and Soybean-Casein Digest Medium (SBCDM, Cat. No. 622787) were purchased from Merck Millipore, USA); Lyophilized bacterial cultures (containing 10-100 CFU/ml) of Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 9027), Clostridium sporogenes (ATCC 19404), and fungal strains Candida albicans (ATCC 10231) and Aspergillus brasiliensis (ATCC 16404) were obtained as EZ- Accu Shot ™ quantitative microorganism preparation (Microbiologics, Inc., St. Cloud, MN, USA) and were prepared according to the manufacturer’s instructions to obtain the challenge inoculum. The microorganism Micrococcus luteus was an environmental isolate collected from the cleanrooms to organize the tests.

2.2. Sterilization

All sterility tests were performed aseptically in ISO Class 5 Biological Safety Cabinet (BSC), providing a controlled environment to minimize the risk of environmental contamination during sample handling and filtration [2, 4, 10, 11]. Sterilization of Trypticasein Soy Agar and Sabouraud Dextrose Agar 4% was performed at 121˚C for 15 minutes using a steam autoclave. Sterility testing of ciprofloxacin lactate solution for infusion was performed using the membrane filtration method in accordance with the requirements of the United States Pharmacopeia and the European Pharmacopeia (European Directorate for the Quality of Medicines and HealthCare [2, 4]. The procedure was carried out under aseptic conditions using sterile membrane filters with a nominal pore size of 0.45 micrometer. A defined volume of sample was filtered, followed by thorough rinsing of the membrane with sterile diluent to remove any residual antimicrobial activity. The membrane was transferred into specific culture media, that is, fluid thioglycolate medium and soybean casein medium, incubated at a specific temperature for at least 14 days, and the media were examined for microbial growth. Appropriate positive controls, negative controls, and growth promotion controls were included to confirm the validity of the test and the suitability of the procedure. The suitability, sterility, and sensitivity tests of the culture media were conducted in accordance with Ph. Eur. 2.6.1.

2.3. Growth Media Control

Sterility testing was performed using compendial culture media suitable for detecting a broad range of microorganisms. Before validation of the sterility test method, growth medium control was used to verify the suitability of the culture media. Tryptic Soy Agar (TSA) and Sabouraud Dextrose Agar 4% (SDA) are solid media used for growth promotion, colony isolation, and confirming the viability of challenge organisms used in validation. They were examined for sterility and growth-promoting properties. Each batch of medium was visually inspected for its appearance, pH, color, and incubated under appropriate conditions to confirm sterility.

Growth promotion testing was carried out using appropriate reference microorganisms to demonstrate TSA’s ability to support aerobic bacterial growth for up to 3 days at 35 °C and yeast and mold growth for up to 5 days at 25 °C. Fluid Thioglycolate Medium (FTM) was used for the cultivation of anaerobic and aerobic bacteria. It was incubated at 30 to 35 °C, whereas Soybean-Casein Digest Medium (SCDM) was used to detect aerobic bacteria and fungi and was incubated at 20 to 25 °C. Both media were purchased from Merck, an approved supplier. They underwent growth promotion testing before using to confirm their ability to support the growth of specified indicator microorganisms. Media sterility controls were included to verify the absence of contamination before testing and ensure the validity and reliability of the sterility test results.

Tables 1 and 2 represent the detail of media and microbial cultures used in the validation method. These procedures included the measurement of pH checks to ensure that the media pH was within specifications. We assigned a batch number and shelf life to all media and maintained batch manufacturing documentation.

2.4. Tested Samples

Sixty ampoules from three different batches (20 samples per batch) contained ciprofloxacin lactate solution for infusion (100 mg/10mL) and were selected to ensure representativeness in accordance with pharmacopeial requirements [2, 4]. Before testing, a QC analyst visually inspected the ciprofloxacin lactate ampoules to confirm container-closure integrity and the absence of visible particulate matter. Before testing, the ampoules were inspected for integrity and the absence of visible particulates. The ampoules were properly disinfected, and all manipulations, including opening and transferring the contents, were performed in a laminar airflow cabinet using sterile equipment and aseptic techniques. These precautions helped ensure reliable sterility test results.

2.5. Method Suitability

Method suitability testing was performed to demonstrate that ciprofloxacin lactate (i.e., the infusion solution) had no bacteriostatic or fungistatic effects on the sterility test. The product was inoculated separately with low levels (10-100 CFU/ml) of particular compound-sensitive microorganisms, including anaerobic bacteria, aerobic bacteria, and fungi, and subjected to sterility test procedures. Membrane filtration is followed by validated rinsing steps to minimize antimicrobial interference. The validated rinsing steps included pre-wetting with 50 mL of Fluid A, filtration of 100 mL of ciprofloxacin lactate, and addition of 200 mL of 50 mL of 1 M magnesium sulfate. The final step included rinsing with 200 mL of Fluid D and 100 mL of Fluid A. Thus, a total volume of 500mL was used for each membrane. A total of 20 ampules from each batch were selected for sterility testing, and half of each 5 mL ampule was filtered through each membrane. We compared the growth and recovery of the challenging organism in the presence of the product with that of the positive controls. We used a polyvinylidene difluoride (PVDF) 0.45 µm low-binding filter to effectively trap microorganisms without allowing ciprofloxacin to bind to the filter membrane, which might hinder microorganism recovery. The membrane was pre-wetted with Fluid A before the experiment to condition the membrane, ensure even fluid flow, and reduce product binding. The membrane was then rinsed with Fluid D and Fluid A to eliminate any remaining drugs and inactivating agents.

2.6. Validation Parameters

The sterility testing method was validated using a series of critical parameters to ensure its reliability and regulatory compliance [12–14]. Method suitability testing involved inoculating the test sample with known compendial microorganisms representing aerobic bacteria, anaerobic bacteria, and fungi to confirm the method’s ability to detect low levels of contamination in the presence of the product. We demonstrated neutralization of antibiotic activity by incorporating validated inactivation and rinsing steps and comparing microbial recovery in the presence of the product with that in positive control samples, confirming the absence of bacteriostatic or fungistatic effects [15–17]. Membrane integrity and sterility controls were included to verify the physical integrity of the filtration membrane and ensure that the filtration system, media, and diluents remained sterile throughout the test procedure. In addition, growth promotion testing of the culture media was conducted before use to confirm that the media supported the growth of the specified indicator microorganisms, ensuring the media’s suitability for sterility testing. This traditional approach aligned with the qualitative nature of compendial sterility testing and its validation, where successful recovery of the specified challenge microorganisms was the primary acceptance criterion.

3. RESULTS

3.1. Growth Promotion Testing of TSA and SDA

Growth promotion testing confirmed that Tryptic Soy Agar (TSA) and Sabouraud Dextrose Agar (SDA) supported growth of the specified challenge microorganisms. Each microorganism was inoculated at a concentration of 10-100 cfu/ml. All test microorganisms showed visible growth after incubation, with colony morphology consistent with each organism’s expected characteristics (Tables 3 and 4). No microbial growth was detected in the uninoculated control media (negative control, NC), confirming the sterility of the prepared media. These findings show that both TSA and SDA met the acceptance criteria for growth promotion testing and confirm the viability of the challenge organisms used in the validation.

3.2. Results of Bacteriostasis and Fungistasis Studies

Bacteriostasis and fungistasis Testings demonstrated the successful recovery of all specified bacterial and fungal test organisms in the presence of the product following membrane filtration and appropriate neutralization. Visible bacterial growth was observed within the prescribed incubation period in Fluid Thioglycolate Medium (Table 5), whereas characteristic fungal growth was detected in Soybean-Casein Digest Medium (Table 6). No inhibition of microbial growth was observed compared to the positive controls, and no growth was detected in the negative controls. These results confirm the absence of bacteriostatic and fungistatic effects and validate the sterility testing method for the reliable detection of microbial contamination.

Visible microbial growth was observed in all inoculated test samples (PPC) and positive controls (PC) on the second day of bacterial incubation. Positive Product Control (PPC) showed identical visual turbidity (Fig. 1) to the Positive Control (PC) within 5 days, while no growth was detected in negative controls, demonstrating the absence of inhibitory effects and confirming the suitability of the membrane filtration method for routine sterility testing of ciprofloxacin lactate solution for infusion.