Nicholas Pryor, ARL Bio Pharma Associate Lab Supervisor, Microbiology

Understanding USP 71 Method Suitability 

Sterility testing by USP 71 is performed in conjunction with other sterility assurance procedures to help ensure that sterile preparations are free of microbial contamination and safe for patient use. Before sterility testing can be reliably performed on a product, a method suitability test must first be completed for the specific formulation. 

What is USP 71 Method Suitability? 

USP 71 focuses on sterility testing, and method suitability is a critical requirement within this framework. Method suitability demonstrates that the sterility test method used is capable of reliably detecting viable microorganisms in a specific pharmaceutical formulation. 

In simple terms, method suitability answers the question: 

“Can this sterility test reliably detect microorganisms in this specific product formulation?” 

To verify this, known microorganisms are intentionally introduced into the product under controlled conditions. The test method must then successfully recover and support the growth of these microorganisms. If recovery cannot be demonstrated, the method is not considered suitable for that formulation. 

Method suitability is formulation-specific because inhibitory properties can vary significantly between products. Active ingredients, inactive ingredients, preservatives, and vehicles may all interfere with microbial recovery. 

Why Does Method Suitability Matter? 

Pharmaceutical formulations can interfere with sterility testing in multiple ways. Without proper method suitability validation, there is a risk of false-negative sterility results, where a contaminated product may incorrectly appear sterile. 

Common formulation challenges include:

• Antimicrobial preservatives that inhibit microbial growth 
• Antibiotics or active ingredients with antimicrobial activity 
• Viscous or oily formulations that trap microorganisms 
• Suspensions or poorly soluble products that are difficult to filter
• Extreme pH or excipient interactions that suppress microbial recovery 

Method suitability ensures that these formulation-specific challenges are identified and addressed before routine sterility testing is performed. 

How is Method Suitability Demonstrated? 

A sterility test method is selected based on a detailed evaluation of the product formulation and any applicable sub-formulations. The two primary sterility testing approaches described in USP 71 are closed-membrane filtration and direct inoculation. 

Closed-Membrane Filtration 

Closed-membrane filtration is the preferred sterility testing method because it separates the product from potential microbial contaminants, helping reduce or eliminate inhibitory effects from the formulation. 

During the test: 

• Product is passed through sterile closed-membrane filtration systems.
• Microorganisms are retained on the membrane filters while the product is directed to waste. 
• The membranes are rinsed using validated rinse fluid to remove residual product inhibition.
• Following rinsing, growth media is aseptically added directly into the closed-membrane filtration systems, including:
• Fluid Thioglycollate Medium (FTM/FTG) 
• Tryptic Soy Broth (TSB) 
  

The closed systems containing the membrane filters and growth media are then incubated and monitored for evidence of microbial growth. 

Direct Inoculation 

Direct inoculation is typically used for products that cannot be filtered, such as certain oils, suspensions, or highly viscous formulations. In this method, the product is directly diluted into the growth media to reduce inhibitory effects. Additional neutralization strategies may also be required. 

Compendial Test Microorganisms 

USP 71 method suitability studies utilize six compendial challenge microorganisms that represent a range of bacterial and fungal microorganisms commonly associated with manufacturing environments and contamination risks. 

The method suitability test is performed by inoculating the selected media with less than 100 colony forming units (CFU), and confirming successful recovery and growth. 

What Happens if Recovery Fails? 

If all challenge microorganisms are not successfully recovered, the method must be modified and re-evaluated. Additional neutralization strategies may include: 

• Increasing product dilution 
• Adding validated neutralizers 
• Incorporating pre-filtration dilution steps 
• Performing additional membrane rinses 
• Selecting alternative test methodologies 

Increasingly conservative approaches may be required until all six challenge microorganisms can be adequately recovered. 

Regulatory Importance 

Demonstrating method suitability is a regulatory expectation and a critical component of sterility assurance programs. Regulatory agencies, including the FDA and international guidance documents such as ICH, expect manufacturers and testing laboratories to demonstrate that sterility testing methods are effective for each specific formulation. 

Failure to establish method suitability may: 

• Invalidate sterility test results 
• Delay product release or approval 
• Trigger regulatory observations or compliance concerns 
• Increase patient safety risk 

Importantly, the validated method remains applicable only as long as the formulation remains unchanged. Changes to active ingredients, preservative systems, vehicles, suppliers, concentrations, or sub-formulations may require method suitability to be repeated. 

Key Takeaway 

USP 71 method suitability is essential for: 

• Ensuring sterility test accuracy 
• Detecting formulation-specific inhibition 
• Adapting sterility methods to challenging products 
• Supporting regulatory compliance 
• Protecting patient safety 

By confirming that sterility testing methods can reliably recover viable microorganisms in a specific formulation, pharmaceutical manufacturers and testing laboratories can maintain confidence that sterility results are meaningful, accurate, and protective of patient health. 

For more information on method suitability, contact ARL today and start the process today to obtain meaningful, accurate sterility results that prioritize patient health.

Amber Gilbert, ARL Bio Pharma Technical Sales Representative

API Vendor Qualification is a critical component of quality assurance for compounding pharmacies and outsourcing facilities. A structured, risk‑based qualification program helps ensure vendor reliability, regulatory compliance, and most importantly, patient safety across the supply chain.

Why API Vendor Qualification Matters in Pharmaceutical Compounding

In pharmaceutical compounding, product quality starts with the supply chain. The identity, purity, and consistency of active pharmaceutical ingredients (APIs), excipients, and packaging components directly affect the safety and performance of the final preparation. A strong vendor qualification program helps control supply chain risk, support regulatory compliance, and protect patient safety.

Vendor qualification is a formal, documented process used to evaluate, approve, and monitor suppliers and manufacturers of critical materials against defined quality and regulatory expectations. Not all materials and not all vendors carry the same level of risk. As a result, vendors should be selected and managed through established quality system controls using a risk-based approach.

Key considerations in API vendor qualification often include: 

• Material Quality and Conformance: Identity, purity, and lot-to-lot consistency
• Patient Safety Risk: Control of impurities, contaminants, and suitability for intended use
• Supply Reliability: Production/Distribution capacity, lead times, and historical quality performance

Vendors that cannot consistently meet these expectations may increase the likelihood of deviations, impact formulation stability, or supply chain disruptions.

Meeting Regulatory Expectations

A robust vendor qualification program also supports compliance with expectations of regulatory authorities and quality standards organizations, including the U.S. Food and Drug Administration (FDA), the United States Pharmacopeia (USP), the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), and the International Organization for Standardization (ISO). 

Under 21 CFR 211, supplier qualification is addressed through validation and quality system activities and supported by documented criteria such as supplier assessments or checklists. USP 1083 outlines core principles for identifying, approving, and monitoring suppliers of materials, packaging components, and services. ICH Q10 further integrates supplier management into the pharmaceutical Quality Management System (QMS) framework based on ISO 9001 concepts.

For API suppliers, it’s important to distinguish between the manufacturer and the distributor and understand expectations for each. Under ICH Q7 and FDA Q7, both must be qualified. Regulators also expect any entity performing GMP-critical activities in the API supply chain to be qualified and audited, including distributors that act as contract manufacturers/packagers or that store, handle, or transport API.

While regulations do not prescribe a single method for qualifying vendors, regulatory authorities expect pharmaceutical manufacturers, distributors, and compounding pharmacies to demonstrate due diligence in evaluating and maintaining their supply chains. Inadequate oversight can lead to FDA Form 483 observations, warning letters, product recalls, regulatory delays, and loss of consumer trust.

Essential Qualification Activities

An effective vendor qualification program typically includes several key activities:

• Vendor Selection: The process begins with identifying potential suppliers and assessing their capabilities, certifications, and regulatory compliance history.
• Vendor Audit & Risk Assessment: Each vendor should be evaluated based on material criticality and supplier risk to determine the appropriate level of qualification and oversight. Evaluation may include quality system assessments, documentation review, regulatory history, on‑site or remote audits, and laboratory testing of representative samples.
• Documentation & Compliance: Qualified vendors should provide key documents such as quality agreements, Certificates of Analysis (CoAs), stability data, and safety data sheets (SDS). When a CoA is issued by or for a repacker/reprocessor, agent, or broker, it should identify the testing laboratory (name, address, and phone number) and reference the original manufacturer and original batch CoA; a copy of the original batch CoA should be attached. Approval or disqualification decisions must be documented, and records retained per quality system requirements.
• Ongoing Monitoring and Requalification: For qualified vendors, written quality agreements establish expectations for documentation review, change control, communication, and performance. Ongoing monitoring may include periodic audits, questionnaires, interviews, and routine analytical testing of incoming materials against predefined acceptance criteria.

Vendor qualification is not a one‑time event, but a continuous quality and compliance control designed to ensure upstream suppliers consistently meet established requirements. By maintaining effective supplier oversight, compounding pharmacies can better manage risk while meeting regulatory expectations and continue to deliver safe, high-quality preparations to patients.

How ARL Can Help

ARL Bio Pharma supports vendor qualification programs through comprehensive testing, documentation review, and quality‑focused expertise. To learn more about how ARL can support your supplier qualification and material testing needs, contact info@arlok.com or call (800) 393‑1595.

Resources

• 21 CFR 211 – Current Good Manufacturing Practice for Finished Pharmaceuticals
• United States Pharmacopeia 1083 Supplier Qualification
• FDA Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients, Guidance for Industry
• ICH Harmonised Tripartite Guideline Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients Q7
• ICH Harmonised Tripartite Guideline Pharmaceutical Quality System Q10
• ISO 9001:2015 Quality Management Systems – Requirements

Tiffany Corbin, ARL Bio Pharma Associate Lab Supervisor, Microbiology

Compounding pharmacies and outsourcing facilities must ensure that sterile preparations are compounded in controlled environments to minimize the risk of microbial contamination. Environmental monitoring (EM) is a process required by the United States Pharmacopeia (USP) and the Food and Drug Administration (FDA) to verify that aseptic processing areas consistently maintain a state of control during compounding. Pharmacies and facilities are required to document their EM procedures, including the selection of sampling sites, the frequency of sampling, acceptance criteria for monitoring particulates and microorganisms, and the investigative and corrective actions taken when necessary.

Sampling Sites

When selecting sampling sites, it is important to consider both routine operations and worst-case scenarios. The selection of these sites should be based on a documented risk assessment that accounts for airflow patterns, room classifications, personnel activities, equipment placement, and proximity to sterile preparation areas.

Common sampling locations typically include:

• Critical sites within ISO environments
• Equipment surfaces and high-touch areas
• Cleanroom floors, walls, and pass-throughs
• Personnel gloves and gowns following aseptic manipulations

Sampling should occur under dynamic conditions, with operations actively ongoing, to yield relevant data regarding routine processing conditions. Relying solely on static conditions for sampling does not adequately demonstrate environmental control.

Worst-case scenario conditions may include periods of maximum personnel occupancy, high batch throughput, upper/lower limits of temperature or humidity, and differential pressure excursions (within acceptable action limits).

Sampling Frequency

The FDA and USP specify sampling intervals based on the ISO classification of the area and the category of compounded sterile preparations. More frequent monitoring is required during initial qualifications, after any significant changes, or when adverse trends or excursions are detected. Additionally, it is important to consider when samples are collected, particularly during peak activity periods and at the conclusion of operations.

Sample Collection

Various methods are used to collect EM samples, each serving a specific purpose in assessing environmental conditions. Using a combination of these sampling methods allows for a comprehensive evaluation of the environment:

• Active air sampling draws a measured volume of air over a growth medium to quantify viable airborne microorganisms and is a requirement in classified areas.
• Passive air sampling (using settle plates) serves as a supplementary method in ISO environments.
• Surface sampling involves contact plates (e.g., RODAC plates) or sterile swabs to evaluate microbial contamination on surfaces and is essential for routine monitoring.

All sampling media must promote microbial growth and have successfully passed growth-promotion testing prior to use, in accordance with USP requirements.

Incubation and Observation

After collecting EM samples, they must be incubated under controlled conditions to allow for microbial growth. USP and FDA recommend a two-stage incubation approach using temperature ranges of 20-25°C and 30-35°C to ensure recovery of both environmental and human-associated microorganisms, including slow-growing microorganisms.

Once the incubation is complete, the samples are examined for microbial growth. Pharmacies and facilities should compare the results against established acceptance criteria, including alert and action levels specific to the ISO environment. It is essential to establish alert and action levels based on regulatory guidance, historical data, and risk assessment. These alert and action levels serve as indicators to identify potential issues or prompt further investigations, rather than simply categorizing results as pass or fail.

Interpreting Results and Investigations

It is important to trend and review EM results. USP emphasizes that trends observed over time are often more meaningful than individual data points. Gradual increases in microbial recovery or changes in microorganism types can signal a potential loss of control.

When results exceed established limits or show adverse trends, pharmacies and facilities must initiate an investigation that includes:

• Identifying the recovered microorganism down to the genus and, when applicable, species level.
• Assessing potential sources of contamination, including personnel practices, cleaning procedures, or equipment issues.
• Reviewing recent actions or interventions that may have contributed to the contamination.
• Evaluating environmental monitoring trends to determine whether the issue is an isolated incident or part of a broader pattern.

The goal of EM goes beyond simple detection; it aims to promote continuous improvement and maintain control in aseptic processing environments. Ongoing evaluation, trending, and refinement of the EM program are vital for safeguarding the safety, quality, and sterility of compounded preparations.

ARL Bio Pharma Testing Services

ARL Bio Pharma provides incubation and enumeration testing for EM samples. Pharmacies and facilities can send plates to ARL for incubation, colony-forming unit (CFU) counting, and a Certificate of Analysis to document the results for EM programs. To demonstrate microorganism growth capabilities, ARL offers growth promotion testing of media used in EM programs, media fills, and personnel qualifications. Additionally, microbial identification testing is available to identify microbial contaminants and support investigative and corrective actions to improve compounding processes.

For more information on ARL testing services, contact info@arlok.com or 800-393-1595. 

Resources:

• USP 797 Pharmaceutical Compounding—Sterile Preparations
• USP 1116 Microbiological Control and Monitoring of Aseptic Processing Environments
• FDA Guidance for Industry – Current Good Manufacturing Practice – Guidance for Human Drug Compounding Outsourcing Facilities Under Section 503B of the FD&C Act
• FDA Guidance for Industry – Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice

Bailey Rubin, ARL Bio Pharma Technical Sales Representative

Analytical testing plays a critical role in ensuring that compounded preparations meet established quality, safety, and regulatory standards throughout the preparation’s lifecycle, including development, stability testing, and final release. One primary way this is achieved is by using validated analytical methods, which are formulation-specific and demonstrate that the test procedure produces reliable, accurate, and reproducible results for its intended purpose.

Organizations such as the U.S. Food and Drug Administration (FDA), the United States Pharmacopeia (USP), and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) publish guidance and standards that define expectations for pharmaceutical testing. According to ICH Q2(R2) Guideline on Validated Analytical Procedures, the analytical performance characteristics to be evaluated depend on the type of test procedure and its intended purpose. The typical validation parameters for analytical methods used to measure specific attributes in release, stability or impurity testing include:

• Accuracy
• Precision
• Linearity and Range
• Specificity
• System Suitability, if measurements are susceptible to various analytical conditions
• Detection Limit
• Quantitation Limit
• Robustness

Together, these characteristics confirm that the method can accurately measure the active ingredient in a formulation while accounting for other components such as excipients, impurities, or potential degradation products.

Validated analytical methods are critical for supporting release, stability, and impurity testing of compounded preparations. For example, potency assays used for quality control release testing must accurately measure the active pharmaceutical ingredient (API) in the finished dosage form, while stability studies require stability-indicating methods capable of separating the API from excipients and potential degradation products to monitor changes over time.

Regulatory expectations for validated methods vary depending on the type of compounding facility. 503B outsourcing facilities, which operate under current good manufacturing practice (cGMP) regulations (21 CFR Parts 11, 210–211), are generally expected to use validated analytical methods for all routine release and stability testing. 503A compounding pharmacies, which operate under a different regulatory framework, are not strictly held to cGMP requirements; however, analytical methods used to support extended beyond-use dating (BUD) or stability claims should still be demonstrated to be suitable for their intended purpose (USP 795 Pharmaceutical Compounding—Nonsterile Preparations; USP 797 Pharmaceutical Compounding—Sterile Preparations).

At ARL, our analytical team supports both 503A compounding pharmacies and 503B outsourcing facilities by developing and applying validated, stability-indicating methods to generate reliable data for stability studies, cGMP release testing, and impurity analysis, supporting the quality and stability of compounded preparations.

For questions regarding obtaining or validating an analytical method for quality control testing, please contact ARL at info@arlok.com or call 800-393-1595.

Resources:

• FDA Guidance for Industry – Current Good Manufacturing Practice – Guidance for Human Drug Compounding Outsourcing Facilities Under Section 503B of the FD&C Act
• ICH Q2(R2) – Validation of Analytical Procedures
• USP 795 Pharmaceutical Compounding – Nonsterile Preparations
• USP 797 Pharmaceutical Compounding – Sterile Preparations
• USP Formulation and Stability Reference Document for Pharmaceutical Compounding
• USP 1225 Validation of Compendial Procedures

Michael Darnaby, ARL Bio Pharma Technical Sales Representative

Visual inspection is an essential quality-control test that helps ensure patient safety and is critical to the release of finished compounded sterile preparations (CSP). Visual inspection is expected for all sterile injectables and should include evaluation of particulates, container defects, color, and clarity. Visible particulates can originate from many sources in the compounding process, including personnel and equipment. It is imperative that visual inspection results are used not only for CSP release, but also to evaluate the drug compounding process itself.

USP 797 states that 100% of the produced batch must be visually inspected. USP 1790 states that visual inspections should be performed immediately after compounding and before labeling to easily detect defects. This inspection can be performed manually or using automation. Once the 100% inspection is complete, acceptance sampling and testing should be conducted on a statistically valid sample taken from the accepted units via manual inspection. This acceptance sampling and testing should be conducted via manual inspection means only by trained personnel and under tightly controlled conditions:

• Use of contrasting backgrounds (white/black)
• Controlled light amount (2000-3750 lux)
• No magnification
• Sufficient inspection time
• Limiting inspection sessions to reduce inspector fatigue

Acceptance limits, test flow, and sampling procedures should be risk-based.

USP 1790 – Figure 1: Typical Process Flow Chart. This chart shows a simplified process flow where solid boxes indicate required process operations, and those with dotted lines (supplemental testing) may be required for difficult-to-inspect products.

For more information on establishing these parameters, please refer to USP 1790.

Visual inspection is not merely a regulatory requirement—it is critical to patient safety control. Data obtained during inspection can also help pharmacies identify process improvements, such as:

• Enhanced filtration techniques
• Improved material handling practices
• Identification of recurring contamination sources
• Refinement of aseptic technique training

Trending visual inspection failures can serve as an early warning indicator of process drift or environmental control issues.

For compounding pharmacists, visual inspection is a critical step in mitigating risk before sterile preparations reach patients. USP 790 establishes the non-negotiable expectation that injectable CSPs must be essentially free of visible particulates, while USP 1790 provides the operational framework for building reliable, consistent, and defensible inspection programs. By implementing structured inspection procedures, investing in personnel training, and incorporating ongoing performance monitoring, compounding pharmacies can significantly reduce the risk of contamination and reinforce their commitment to patient safety and CSP quality.

Resources:

• USP 1 Injections and Implanted Drug Products (Parenterals) -Product Quality Tests
• USP 790 Visible Particulates in Injections
• USP 797 Pharmaceutical Compounding – Sterile Preparations
• USP 1790 Visual Inspection of Injections
• ISO 2859-1: Sampling Procedures for Inspection
• ANSI/ASQ Z1.4: Sampling Procedures and Tables for Inspection by Attributes
• FDA Guidance for Industry – Inspections of Injectable Products for Visible Particulates

Kayla Lipcaman, ARL Bio Pharma Associate Microbiology Supervisor

Compounding pharmacies and outsourcing facilities must ensure that sterile compounded preparations are free from microbial contamination. To achieve this, they use aseptic processing. According to the United States Pharmacopeia (USP), aseptic processing is a method by which separate components (e.g., drugs, containers, or closures) are brought together under conditions that prevent microbial contamination.  

A media fill, also known as a process simulation, is performed to evaluate the effectiveness of the aseptic process and the personnel’s aseptic technique in preventing contamination. 

The Food and Drug Administration (FDA) has established guidelines for performing media fills to validate aseptic operations. Media fills should be conducted during the initial qualification and at least twice a year, or as required, to maintain aseptic process control. Additionally, if there are significant changes to the facility, equipment, processes, or test methods, a new media fill must be performed to re-validate aseptic processing.

USP has also established standards for performing media fills as part of a competency test to assess personnel’s aseptic technique. A media fill is required before beginning to compound Category 1, 2, or 3 CSPs, and at predetermined intervals for each drug product category.

Media Fill Design

During a media fill, a microbiological growth medium that has previously or concurrently passed growth promotion testing is used in place of a drug product. The growth medium is exposed to the same contact surfaces and process conditions encountered during routine aseptic production, including:

• Equipment
• Container closure systems
• Critical environments
• Aseptic process manipulations

Media fills must closely simulate routine aseptic operations and be performed under worst-case, most challenging conditions, as required by USP and FDA guidance. The simulation should reflect actual production practices and batch sizes. Critical factors to consider include:

• Line speed
• Batch size
• Number and qualifications of personnel
• Routine and non-routine operational practices

Growth Media

The most common growth medium for media fills is Soybean Casein Digest Medium (SCDM), also known as Trypticase Soy Broth (TSB). Growth media may be obtained from a qualified commercial supplier or prepared in-house:

• If using a commercial supplier, a certificate of analysis (COA) must be obtained, according to the FDA and USP, to confirm that the media lot supports microbial growth. 503A pharmacies and 503B outsourcing facilities are required to qualify their suppliers.
• If preparing growth media in-house, each batch must undergo documented growth promotion testing in accordance with USP 71 Sterility Tests, prior to use in a media fill.

Incubation and Observation

After completion of the aseptic process simulation using growth media, the final sealed containers are incubated and observed under controlled conditions to detect potential microbial contamination.

The FDA recommends that incubation may be performed at:

• A single temperature range of either 20–25°C or 30–35°C; or,
• A two-stage incubation approach may be used, consisting of incubation at 20–25°C for a minimum of 7 days, followed by incubation at 30–35°C for an additional minimum of 7 days.

USP recommends incubation at 20–25°C for a minimum of 7 days, followed by incubation at 30–35°C for an additional minimum of 7 days.

Interpreting Test Results

If microbial growth is observed during or after the incubation period, the contaminated unit must be investigated. A microbial identification may be performed, as appropriate, to identify the organism to the genus and species levels and to support the investigation.

Compounding pharmacies and outsourcing facilities should interpret results to assess the risk of a CSP unit becoming contaminated during actual operations (e.g., start-up, sterile ingredient additions, aseptic connections, filling, and closing). 

According to the FDA, the recommended criteria for assessing the state of aseptic processing control are as follows:

• When filling fewer than 5,000 units, no contaminated units should be detected. One contaminated unit is considered a cause for revalidation, following an investigation.
• When filling 5,000 to 10,000 units, one contaminated unit should result in an investigation, including consideration of a repeat media fill. Two contaminated units are considered a cause for revalidation, following investigation.
• When filling more than 10,000 units, one contaminated unit should result in an investigation. Two contaminated units are considered a cause for revalidation, following an investigation. 

According to USP, the recommended criteria for assessing the state of personnel competency in aseptic technique are as follows:

• No contaminated units should be detected.
• Media fill failure constitutes an overall failure of aseptic manipulation competency.

Contact ARL Bio Pharma today for more information on media fills at info@arlok.com or 800-393-1595.

Resources:

• USP 797 Pharmaceutical Compounding—Sterile Preparations
• USP 1116 Microbiological Control and Monitoring of Aseptic Processing Environments
• USP 1211 Sterility Assurance
• FDA Guidance for Industry – Current Good Manufacturing Practice – Guidance for Human Drug Compounding Outsourcing Facilities Under Section 503B of the FD&C Act
• FDA Guidance for Industry – Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice

Possible Causes and Investigation

A sterility test detects microbial contamination and provides data to determine whether a product is ready for release. A “Sterile” result indicates no contaminating microorganism is found in the sample. A “Not Sterile” result indicates microbial growth, and the product examined does not comply with the test for sterility, unless it is demonstrated that the test is invalid for causes unrelated to the product examined.

Sterility Test and Microbial Growth

Trained microbiologists examine the sterility test sample for microbial growth using the following conditions:

Once evidence of microbial growth is found, the test is out-of-specification (OOS), and an investigation is required to determine the cause of the test failure.

Possible Causes of Sterility Test Failure

• Compromised container integrity
• Improper aseptic processing and facilities
• Invalid sterilization processes
• Laboratory error

ARL Bio Pharma Sterility OOS Investigation Process

An OOS investigation is conducted for every sterility test failure. ARL Bio Pharma’s investigation includes a detailed examination of:

• Sterility test method
• Environmental monitoring (EM) data
• Sanitization logs for all ISO environments
• DNA sequencing reports, if applicable
• Deviation reports, if applicable
• Negative control data
• Potential retest results, if the test meets invalidation criteria

Criteria for Invalidating a Sterility Test Failure 

A traditional sterility or Celsis rapid sterility test may be considered invalid only if one or more of the following conditions are fulfilled:

• A species-level microbial identification match between EM isolate(s) collected during the applicable testing shift and the microorganism isolate from the sterility test.
• Evidence that the testing procedure used during the test in question reveals a fault.

A ScanRDI rapid sterility test may be considered invalid only if both of the conditions are fulfilled.

If the test is declared invalid, it is repeated with the same lot and number of samples as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the sterility test. If microbial growth is found in the repeat test, the product examined does not comply with the sterility test.

Next Steps

While a species-level match meets a portion of the USP 71 invalidation requirements, a sterility failure should prompt an investigation at the facility where the product was prepared. This investigation should take place at the same time as the laboratory investigation and include a review of:

• Compounding sterility assurance practices
• Aseptic processing practices
• Equipment qualifications
• Sterilization validations
• Environmental monitoring
• Personnel training

Pharmacies and outsourcing facilities should review all the data, including ARL’s findings, to determine how to proceed after a sterility test fails.

For more information, see USP chapters:

• USP 71 Sterility Tests
• USP 1207 Package Integrity Evaluation – Sterile Products
• USP 1211 Sterility Assurance
• USP 1229 Sterilization of Compendial Articles

Bailey Rubin, Technical Sales Representative

What happens to your compounded preparations after they leave your pharmacy? During transit, compounding preparations can experience shipping delays, which may result in temperature changes. Even the most carefully packaged preparations can be exposed to unexpected environmental changes. Without supporting data to demonstrate that a product remains stable under these conditions, a pharmacy could face compliance consequences, and patients could receive compromised medications.

Understanding Stability Studies and Temperature Excursions

Stability studies are required to demonstrate the stability of compounded preparations. They test how a preparation’s physical and chemical integrity performs under its intended storage condition(s) over time and provide data needed to support a Beyond-Use-Date (BUD). During the stability study, samples are stored in stability chambers that must be maintained within specifications that allow only very small changes in temperature and humidity. As stability samples are held under defined and controlled environmental conditions, stability studies are not intended to, nor can they, demonstrate the stability of compounded preparations under temperature excursions or conditions not included in the validated study parameters.

Temperature excursion studies are designed to evaluate how a compounded preparation responds when exposed to short-term temperature or humidity fluctuations outside its labeled storage range, such as 40°C ± 2°C / 75% RH ± 5% for 24 or 48 hours. After exposure to the higher temperature and humidity, the compounded preparations are then tested for stability using a stability-indicating method.

Why Temperature Excursion Studies Matter

Some compounds, such as GLP-1 (semaglutide, tirzepatide) and trimix formulations, can be highly susceptible to degradation when exposed to temperatures higher than those for which they are intended to be stored. Even just a few hours at room temperature or higher can lead to degradation, potentially compromising product quality. Regulatory authorities and quality standard organizations like the Food and Drug Administration (FDA), United States Pharmacopeia (USP), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) expect supporting data demonstrating that compounded preparations remain stable during a deviation from their labeled storage condition(s).

The FDA has repeatedly issued 483s and warning letters for failing to investigate temperature fluctuations. Examples of these observations include:

Firm failed to thoroughly investigate any unexplained discrepancy or failure of a batch or any of its components to meet any of its specifications, whether or not the batch has already been distributed (21 CFR 211.192).

• “During the inspection, firm personnel indicated that this lot had experienced high temperature excursions while in storage.”
• “You had prolonged problems assuring maintenance of the required temperatures of the refrigerated storage unit in which this API was stored.”
  

Firm failed to establish a written testing program designed to assess the stability characteristics of drug products and determine appropriate storage conditions and expiration dates. Firm also failed to have buildings used in the manufacture, processing, packing, or holding of drug products with adequate space for the orderly placement of equipment and materials to prevent mix-ups and contamination (21 CFR 211.166(a) and 21 CFR 211.42(b)).

• Inadequate Material Storage – “Bulk totes of ethanol hand sanitizer gel were stored outdoors, and you informed our investigator that these totes had been stored outdoors since April 2020. Labeling for the bulk ethanol hand sanitizer gel lists a temperature storage range of (b)(4) F. The totes are exposed to temperature excursions that often exceed the specified temperature storage range, which could compromise the quality of the drug product therein.”
  

Firm failed to review and investigate production and QC laboratory deviations.

• “The inspection documented that, despite the fact that your firm has an uninterrupted power supply used by the QC laboratories, power failures have impacted the QC stability chambers. However, in each case, no investigation was conducted to determine the impact of the power loss on the samples kept within the chambers.”
  

Firm’s quality unit is not involved in quality related matters; the unit fails to review deviations from established specifications or procedures and does not adequately assess the need for corrective actions for deviations it is made aware of. 

• “No follow-up or investigations were conducted for the excursions … to determine the root cause and potential impacts on the products and stability studies.”
  

Written procedures are lacking, which describe in sufficient detail the storage of components, drug product containers, and closures. 

• “Specifically, there is no temperature monitoring in the warehouse where raw material and blended drug products are stored.”

Be Proactive

ARL Bio Pharma offers complete stability and temperature excursion testing that meets USP, FDA, and ICH standards. Our studies show how preparations hold up under different temperature and humidity conditions and provide the data needed to support beyond-use dates and quality claims.

Contact ARL Bio Pharma at 800-393-1595 or info@arlok.com today to see how a temperature excursion study can strengthen your stability program, protect patients, and keep your pharmacy compliant.

References:

• Food and Drug Administration
• ICH Stability Testing of New Drug Substances and Products Q1A(R2)
• USP 659 – Packaging and Storage Requirements
• USP 797 – Pharmaceutical Compounding – Sterile Preparations
• USP 1079 – Risks and Mitigation Strategies for the Storage and Transportation of Finished Drug Products
• USP 1149 – Guidelines for Assessing and Controlling the Physical Stability of Chemical and Biological Pharmaceutical Raw Materials, Intermediates and Dosage Forms

James Zellner, Technical Sales Representative

The FDA defines photostability as the ability of a drug substance or product to resist chemical changes when exposed to light and considers photostability testing as an “integral part of stress testing”. Much of the FDA’s standing on photostability and the associated studies to perform comes from the ICH Q1B Guidance for Industry Photostability Testing of New Drug Substances and Products. The FDA has officially adopted this document as guidance, so it should be used to design photostability studies.  

ICH recommends a systematic approach to photostability testing, which, as appropriate, may include testing of the drug substance, exposed drug product (out of its primary packaging), drug product in any intermediate packaging, and, finally, drug product in its final packaging. Later, if there are changes to either the formulation or packaging, ICH states these studies should be repeated to demonstrate that no change to product stability occurs.

When conducting a photostability study, Q1B is specific in what types of light, wavelength, and exposure should be used. For visible light, this consists of not less than 1.2 million lux hours and a near UV exposure of 200-watt hours/ square meter. Exposure should be measured via chemical actinometric systems and/or calibrated radiometers/ lux meters. Throughout the exposure period, the temperature of the photostability chamber must be maintained to avoid additional stressors on the product that may lead to inaccurate photostability conclusions. Sample sizes should be adequate to demonstrate that no variability exists between individual product units. While there isn’t a specific number used in Q1B, 20 units are cited as an example for a solid dosage form. “Dark control” samples are included for comparison. During light exposures, the test samples are arranged in the chamber to ensure uniform exposure of the product to the light source and continue until the appropriate amount of lux/ watt hours is achieved. 

At the conclusion of the exposure period, many tests may need to be performed to assess photostability. The drug substance or product should be assayed for stability of the API (active pharmaceutical ingredient) using a formulation-specific, stability-indicating analytical method. As with other guidance, FDA expects this to be done via a chromatographic method that is reliable, meaningful, uses a reference standard, and is highly specific. The assay method should have, during its development, been created using both visible light and UV stressors to ensure the potential degradants created during this exposure are separated from the API. Testing to assess photostability should not be limited to the assay. Appearance, color and clarity of solution (in the case of a liquid product), and dissolution/disintegration (in the case of a solid product) are examples of other tests that may need to be assessed to fully understand the photostability of the drug product. The appropriate tests to include depends on the drug substance or product being tested. Dark control samples are tested using the same scheme as the primary samples to compare.

Evaluation of the test results should include a determination of whether changes observed, if any, are acceptable. The results from stability studies should be considered when evaluating photostability data. If the substance or product is not stable when exposed to light, an assessment of the product or packaging systems is needed. Special labeling or packaging may be required to mitigate the effects of light exposure.  

Contact ARL today to learn more about Photostability Studies and Testing. 800-393-1595 or info@arlok.com.

Gary Rhodes, Analytical Associate Laboratory Supervisor

Total Organic Carbon (TOC) testing measures the amount of carbon present in a water sample and is conducted based on USP 643 requirements. Chemical impurities can enter water from various sources, including:

• Source water used for pharmaceuticals (such as injectables)
• Packaging for sterile and non-sterile water
• Purification systems
• Processing equipment

This test serves as a key quality indicator, as organic carbon in water can promote microbial growth and signal potential contamination, posing risks to patient safety.

While a connection exists between TOC levels and microbial activity, a direct numerical correlation does not exist. Therefore, TOC measurements should not be used as a substitute for endotoxin or microbiological control testing.

TOC Test Method

ARL utilizes a Shimadzu TOC-LCSH, High-Sensitivity Analyzer to conduct the TOC test method. TOC is measured by injecting a volume of the sample into a combustion tube containing an oxidation catalyst heated to 680°C. The sample is burned in the tube, converting it into carbon dioxide, which is then measured by the non-dispersive infrared (NDIR) gas analyzer. This measurement is compared to a standard solution prepared from Sucrose at the specified limit to determine the amount of carbon in mg/L.

While this measurement quantifies the amount of carbon in the sample, it does not identify the specific contaminant. USP has established acceptance criteria for carbon content, ensuring that materials meeting these specifications are deemed suitable for their intended pharmaceutical application. If it does not meet requirements, further analysis and/or investigation is required to determine the source and identity of the contamination.

Definition and Limits

USP 643 sets limits for Bulk Water and Sterile Water. Bulk Water is defined as Purified Water, Water for Injections, Water for Hemodialysis, and the condensate of Pure Steam. The limit for carbon contained in samples of these waters is 0.5 mg/L or 500ppb of carbon.

Sterile Water is defined as Sterile Water for Injection, Sterile Purified Water, Sterile Water for Irrigation, Sterile Water for Inhalation, and any specific monograph that references Sterile Water. These waters are derived from Purified Water or Water for Injections; therefore, they have been determined to be compliant with the Bulk Water requirements before being stored and sterilized in their container. The limits for carbon contained in samples of these waters are dependent on the nominal container volume, as stated below.

USP 643 – Table 1. TOC Limit Based on Container Volume

Other Applications for TOC Testing

In addition to testing water, TOC is used for cleaning validations. It is suitable for testing direct surfaces and rinse water. However, TOC does not identify which compounds contain oxidizable carbon. Any carbon detected is attributed to the target compound(s) and compared to a set limit. If testing for a specific compound, contact ARL to discuss a residual drug cleaning validation study.

For more information on TOC, contact ARL Bio Pharma at 800-393-1595 or info@arlok.com.

Resources:

• USP643 Total Organic Carbon
• Shimadzu TOC-L Series Manual
• FDA Guidance for Q&A on Current Good Manufacturing Practice Requirements – Equipment
• Total Organic Carbon Testing – Back to Basics: American Pharmaceutical Review