Testing of Purified Water, Raw Materials, In-Process Samples and Finished Non-Sterile Products

Introduction

To start with the end in mind, the current USP Chapter <1111>¹ “Microbiological Examination of Non-sterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use” has recommended microbial limits for aqueous non-sterile products of less than 100 Colony-Forming Units (CFU)/milliliter (mL) of bacteria, less than 10 CFU/mL of fungi and the absence of Escherichia coli in 1 gram (g) or mL. Therefore, when using raw materials (including the water) that are susceptible to microbial contamination, it is critical to evaluate them for microbial content. This would include the number of microorganisms present as well as a lack of potential presence of objectionable organisms, following USP Chapters <60>² “Microbiological Examination of Non-sterile Products Tests for Burkholderia cepacia Complex”, <61>.³ “Microbiological Examination of Non-Sterile Products: Microbial Enumeration Test”, and <62>,⁴ “Microbiological Examination of Non-Sterile Products: Tests for Specified Microorganisms” respectively. Non-aqueous preparations for oral use have a Total Aerobic Microbial Count limit of 10³ CFU/g or mL and 10² CFU/g or mL Total Combined Yeasts/Molds and the absence of Escherichia coli in 1 g or mL.

Purified Water System

The most important raw material used to manufacture non-sterile products is water. Water is the primary ingredient in manufacturing of aqueous non-sterile product formulations, preparation of disinfectant use-dilutions, and is also used for equipment cleaning and sanitization. The source water for purified water preparation starts with either a municipal potable water system or well water that complies with potable or drinking water standards. This source water is a major contributor of microbial contamination in purified water systems. Water, which is suitable for use in the form of purified water (PW), may be produced by distillation, ion exchange, filtration or Reverse Osmosis. It is critical that the system used to produce this PW is designed properly (no dead legs*, proper 2 inch air gaps to drains, etc.), maintained adequately (sanitization on a periodic basis) to prevent biofilms and tested routinely for bio-burden (i.e., E. coli) per USP <61>³ “Microbiological Examination of Non-Sterile Products: Microbial Enumeration Test”, <643>⁵ “Total Organic Carbon (TOC)” and <645>⁶ “Water Conductivity”. Microbial biofilms form when bacteria adhere to surfaces in aqueous environments; they begin to excrete a slimy, glue-like substance that can anchor cells to material and form a protective matrix of extracellular polymeric substances. This matrix contributes to resistance to anti-microbial treatments as compared to separate, individual cells. The recommended bio-burden methodology for conducting microbial water testing is membrane filtration (USP <61>³) by using 100 mL aliquot samples (although other acceptable testing methods are pour plate, spread plate and most probable number), which should be tested per the current USP within two (2) hours or refrigerated for less than 48 hours after sampling. Based on history, there should be process control levels (suggested at less than 100 CFU/mL), beyond which, investigation and Corrective/Preventive Actions (CAPAs) are performed. Levels may include alert, action and a specification as required in the current USP.

USP Chapter <1231>⁷ – “Water for Pharmaceutical Purposes” recommends the usage of sampling and testing methods in “Standard Methods for Examination of Water and Wastewater”.⁸ If the potable or drinking water supply for a purified water is from a well, it is recommended that it be periodically tested for the presence of coliform bacteria for confirmation of compliance to drinking water standards. Usage ports must be sanitized prior to taking purified water samples and the line should be flushed for the same time as when used in production (1-3 minutes) to test for the presence of microbial contamination. The USP <1231>⁷ recommends a test volume of 1 mL aliquot for the pour plate method and 100 mL for a membrane filtration method, in order to get a statistically valid microbial count. Microbial limits should be set based on system capabilities rather than the suggestions from the USP. If action levels are exceeded, identify recovered microorganisms to the genus/species level and perform an investigation. The presence of Bacillus species and Gram-positive cocci isolation could signify poor sampling methods, inadequate flushing or laboratory contamination. Multiple repetitive alert levels should be deemed an action level and this must be defined in SOPs. Companies may wish to repeat sampling, but they should also determine any possible adverse impact to the microbial quality of finished products. Adverse trends (an increase in counts from below alert levels to above action levels over a period-of-time) could show deterioration of your purified water system. Microbial alert and action levels will need to be re-evaluated at least on an annual or other periodic basis if there are changes in the components of purified water system itself, if there is a change in the microbial growth media or the technology that is used to detect microorganism changes in the purified water system.

It is recommended that a low nutrient microbial growth agar be used to recover microorganisms from purified water samples, since these isolates are stressed due to the low nutrient conditions that are present in purified water systems and are not able to grow on high nutrient microbial growth agars. R2A is usually used for conducting microbial counts of purified water system samples because it is a low nutrient recovery agar. Recovery R2A pour or membrane filtration plates are normally incubated at 20-25°C for 5-7 days. If it is not possible to use R2A in a testing laboratory to perform a microbial count on purified water samples, M-HPC or Tryptone Yeast Extract Glucose Agar can be used in place and are incubated at 30-35°C for 48-72 hours. In the past, many opinions have been expressed for or against the usage of selective/differential microbial growth media in the testing of purified water samples for detecting the presence of objectionable microorganisms such as Escherichia coli, coliform bacteria and fluorescent Pseudomonas species such as Pseudomonas aeruginosa, Pseudomonas flourescens and Pseudomonas putida based on a Risk Assessment (RA) done for what would be objectionable in the final finished product.¹⁰ Instead of using selective/differential microbial growth media to recover these isolates and to have a presumptive identification of the isolate, it is recommended that representative recovered colonies of the pour or membrane filtration plate be sub-cultured onto either Plate Count Agar or Soybean-Casein Digest Agar Medium for Gram staining and identification to the genus/species level.

In the last few years, there has been a focus on Burkholderia cepacia (T. Cundell, 201811 and 201912 and L. Torbeck12). T. Cundell does not believe that routine monitoring of PW for objectionable organisms is needed except in the case for B. cepacia. B. cepacia has been the cause of many recalls, is resistant to many antibiotics and in fact has caused high mortality in immunosuppressed patients with Cystic Fibrosis, so it is critical to discern if this particular complex of microorganisms are present in your purified water system. A new chapter in the USP (<60>)² – “Microbiological Examination of Non-sterile Products Tests for B. Cepacia Complex” became effective in December 2019, explaining how to test for the presence of this complex of microorganisms. Some microbiologists are in agreement with T. Cundell in that they feel that there is no need to test for any other objectionable organisms in purified water other than B. cepacia. Note that the determination of whether an organism is objectionable is related to its pathogenicity in the subject patient population as well as its ability to affect the chemical, physical and therapeutic traits of a product.¹⁰

Methods for Control/Sanitization of PW Systems

1. Remove hoses from sample ports and allow to drain by hanging.
2. Use of ozone up to 0.1 parts per million (PPM) or usage of circulating 80°C hot purified water (periodic sanitization of the water system).
3. Routine Preventive Maintenance.

Raw Material Testing

Use compendial monograph specifications, where provided. If not, use harmonized microbial test methods for raw materials that are susceptible to microbial contamination (all the way through to finished product). Use the USP <61>³ “Microbiological Examination of Non-Sterile Products: Microbial Enumeration Test” and USP <62>⁴ “Microbiological Examination of Non-Sterile Products: Tests for Specified Microorganisms”, which have been harmonized with EP and JP. Automated rapid microbial test methods may be used as long as they have been qualified as being equivalent to the manual or traditional microbial test methods. This particularly applies to organism identification.

Bulk and Finished Product Testing: Reference USP <1115>¹⁴ “Bioburden Control of Non-Sterile Drug Substances and Products”

Testing methods of bulk (in-process samples) and finished product must be qualified. The frequency of testing and acceptance criteria should be related to the Risk Assessment for the finished product. This includes issues such as:

1. The patient population (immuno-suppressed patients have the highest risk of disease)
2. Product attributes such as preservative types, pH extremes (e.g., <3.0 and >10.0), absence of nutrients, presence of surfactants and the water activity of the product (lower water activity is less likely to have survival or proliferation of microorganisms). Water activity is a measure of unbound (free) water in a material that is available for chemical and biological reactions. The scale runs from Zero (0) to 1.0 for Pure Water. Most bacteria require water activity (A_w) of 0.93 A_w for microbial proliferation. A water activity (A_w) level below 0.6 reduces the chance for microbial growth, which includes tablets, non-aqueous liquid products, ointments and rectal suppositories.
3. The route of administration – e.g., oral, nasal, rectal, cutaneous, etc.
4. Conditions in the plant and manufacturing to prevent ingress or proliferation in drug substances, excipients and packaging components.

Qualification of laboratory methods is known as method or system suitability, which can be done by neutralizing the antimicrobial properties (Table 1) or removing them before routine testing (Ref: L. Clontz 2009).¹⁵ By inoculating with <100 CFU of USP specified ATCC strains and EM isolates, look for 50-200% recovery of the original inoculum in comparing the recovery in the enumeration methods with sample in diluent and diluent itself. For enrichment, inoculate with <100 CFU for each of the USP specified ATCC strains of the sample in enrichment broth in order to detect their presence after incubation by using selective/differential microbial growth agars. Bulk testing is called bio-burden testing - it shows the degree of microbial contamination or load.

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Non-sterile finished products are allowed to have low levels of microbial contamination per USP <1111>.¹ The procedure in USP Chapter <61>³ requires 10 g of material for tests, but if the batch size is small, you may use 10 units of 1 g each. Test 10 g or 10 mL material (raw material, bulk or finished product) dissolved or suspended (using mortar and pestle) in pH 7.0 PBS (Phosphate Buffered Saline) or TSB (Trypticase Soy Broth). A 1:10 dilution or greater may be needed for the pour plate method or membrane filtration (using 1-3 x 100 mL rinse fluid). The media to be used- for TAMC (Total Aerobic Microbial Count) using TSA (same as Soybean Casein Digest Agar Medium) should be incubated at 30-35°C for 3-5 days. For TYMC (Total Combined Yeast and Mold Count), use Sabouraud Dextrose Agar or Potato Dextrose Agar at 20-25 degrees for 5-7 days. Usually, an analyst cannot count more than 50 mold colonies. It is recommended to periodically examine the microbial count plates during incubation to detect the presence of spreaders and avoid overgrowth. As a usual requirement, growth promotion testing should have been performed prior to use of microbial growth agars and enrichment broths. If media was prepared in-house and not terminally sterilized (i.e., gamma irradiation), pre-incubation of a sample from each batch is recommended.

Acceptance Criteria for Finished Product

Acceptance criteria for TAMC and TYMC are provided in Table 2.

Per USP <61>³ and <1111>¹, if the limit is 10 CFU, the acceptable highest count is 2 fold (200%) = 20. If the limit is 100 CFU, the acceptable highest count is 2 fold (200%) = 200. Is this a reasonable interpretation? It is one thing to allow this for a Growth Promotion Test in comparing results with a positive control (standardized inoculum). It is something else (and to me unacceptable) to allow this for a product specification.

Tests for Specified Organisms (USP Chapters <60>² and <62>⁴)

Soybean-Casein Digest Medium [Tryptic or Trypticase Soy Broth] (TSB) with preservative neutralizers can be used as an initial enrichment broth for detecting the presence of Ps. aeruginosa, S. aureus, E. coli, Salmonella spp. and B. cepacia complex in a test sample. Use of either direct dilution or membrane filtration methods can be applied to a test sample by using TSB as an enrichment broth. By sub-culturing of an aliquot of TSB enrichment onto Cetrimide Agar, it can be used to isolate Ps. aeruginosa. By sub-culturing an aliquot of TSB enrichment onto Mannitol Salt Agar, it can be used to isolate S. aureus. By sub-culturing an aliquot of TSB enrichment into MacConkey Broth and incubating it, MacConkey Agar can be used to isolate E. coli from a test sample. By inoculating an aliquot of TSB enrichment into Rappaport Vassiliadis Salmonella Enrichment Broth for enrichment, Xylose Lysine Deoxycholate media can be used to isolate Salmonella species. B. cepacia Selective Agar (BSCA) can be used to isolate B. cepacia complex organisms from TSB enrichment of aqueous product formulations.

Identification Methods

• Gram Staining – Cocci and Bacilli
  • Gram Positive, e.g., Staphylococcus aureus
  • Gram Negative, e.g., Escherichia coli
• Phenotypic methods
  • Biochemical assays based on growth reactions
  • Fatty acid profiles
  • Mass Spectrometry (Maldi-TOF)
  • Immunological Methods (ELISA; flow cytometry; immunofluorescence)
• Genotypic techniques
  • Nucleic acid amplification techniques

Retesting Guidelines

An out of specification result is referred to as a Microbial Data Deviation (MDD). If the original MDD can be invalidated, retests are allowed but all data must be reported and must be considered when determining product disposition. The investigation can be done by the Fishbone method, Kepner Tragoe and the 5 Whys. If the original MDD cannot be invalidated, a retest may be used as simply as a confirmatory test and product should be rejected.

Causes of Recalls

Examples:

• Incorrect performance of Quality Control microbial release testing (Failure to detect high levels of microorganisms or the presence of a microbial pathogen or an objectionable microorganism, and incorrect microbial identification of a recovered isolate).
• Failure to conduct suitability testing of plate count and enrichment microbial test methods.
• Improper evaluation of a recovered microbial isolate as being objectionable.
• Inadequate preserved product formulations due to the usage of low preservative concentrations, incorrect order of raw ingredient addition that neutralized the antimicrobial activity of preservatives, and the usage of incompatible raw ingredients that neutralized the antimicrobial activity of preservatives.
• Equipment cleaning and sanitization procedures for manufacturing equipment not validated.
• Improper equipment cleaning and sanitization procedures.

Evaluate the following issues as possible root causes:

1. Water issues.
   1. Wrong grade of water used for cleaning.
   2. Wrong grade of water (e.g. potable water) used to rinse equipment surfaces of sanitizers/disinfectant residues.
   3. Incorrect grade of water used to prepare use-dilutions of equipment sanitizers/disinfectants.
   4. Lack or improper sanitization of manufacturing equipment or storage.
   5. Poor water system design or maintenance.
2. Inadequate microbiological testing.
3. Usage of microbially contaminated Raw materials.
4. Inadequate preserved product formulations due to the usage of low preservative concentrations or use of contaminated raw ingredients in the formula.
5. Insufficient environmental monitoring of air, surfaces and equipment to detect contamination.

An investigation must determine most probable root cause and this should lead to development of Corrective and Preventive Actions (CAPAs). Efficacy of any CAPAs implemented must be determined at some point in time.

Conclusion

Testing of water, in-process and finished product samples by microbial enumeration and determination of objectionable organisms (which includes E. coli and others such as B. cepacia) must be proposed by the company based on history of recoveries, dosage form, route of administration and patient population. Risk assessment is a valuable tool in this regard.

References

1. USP <1111> Microbiological Examination of Non-Sterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use.
2. USP <60> Microbiological Examination of Non-Sterile Products Tests for Burkholderia Cepacia Complex.
3. USP <61> Microbiological Examination of Non-Sterile Products: Microbial Enumeration Test.
4. USP <62> Microbiological Examination of Non-Sterile Products: Tests for Specified Microorganisms.
5. USP <643>Total Organic Carbon.
6. USP <645> Water Conductivity.
7. USP <1231> Water for Pharmaceutical Purposes.
8. Standard Methods for Examination of Water and Waste water 2016.
9. PDA Technical Report 13 Fundamentals of an Environmental Monitoring Program Revised 2013.
10. PDA Technical Report 67 Exclusion of Objectionable Microorganisms in Non-Sterile Pharmaceuticals, Medical Devices and Cosmetics, 2014.
11. T. Cundell, “Microbial Monitoring of Pharmaceutical Grade Water Systems – Common Misconceptions”; American Pharmaceutical Review, Oct. 11, 2018.
12. T. Cundell,” Excluding Burkolderia cepacia complex from Aqueous, Non-Sterile Drug Products”; American Pharmaceutical Review, Feb. 12, 2019.
13. L. Torbeck, D. Raccasi, D. Guilfoyle, Burkholderia cepacia: This Decision is Overdue”, PDA J. Pharm. Sci. and Tech. 2011, 65, 535-543.
14. USP <1115> Bioburden Control of Non-Sterile Drug Substances and Products.
15. L. Clontz, “Method Validation and Media Suitability Testing”, Microbial Limit and Bioburden Tests, 2009.

*Dead leg: A dead leg is any area in a piping system where water becomes stagnant and water is not moving even during flushing. Bacteria in dead legs pipe lengths are protected during flushing and sanitization and if they break loose from a biofilm, they can contaminate the whole piping system.

*Objectionable organisms: Microorganisms which, due to their numbers and pathogenicity can cause disease in patients; and can proliferate in a product to cause impact to chemical, physical and therapeutic characteristics.

Author Biographies

Dr. Randy Hutt has 40 years of experience in the Pharmaceutical and Biological industries currently working at American Regent, Inc. (formerly called Luitpold Pharmaceuticals, Inc.). Dr. Hutt is Head of Sterility Assurance (SA) providing an oversight function for aseptic conditions and microbial control. She has over 20 years of QA/QC experience including that at other companies (Wyeth-Lederle and Schering Plough) and has 10 years of aseptic production management experience (Burroughs Wellcome Co.) and several years of consulting. Her Ph.D. is in Microbiology, specializing in virology (Penn. State University at Hershey, Pa.).

Donald J. English has over 40 years of experience in the Research and Development and Q.C. Microbiology Laboratories at Avon Products, Inc., GlaxoSmithKline Consumer Healthcare and Merck and Co. He has a B.S. in Biology from the University of Charleston and M.S. in Microbiology from N.C. State University in Microbiology. He is currently a consultant at Donald J. English Microbiological Quality Consulting LLC to cosmetic/personal care, dietary supplement and pharmaceutical industries.