Introduction to the Microbial World

Addressing how the Pharmaceutical Microbiology Lab can establish best practices to account for the wide range of microbes that they may encounter.

• Bacteria, fungi, mycoplasmas and viruses are the most common types of microbes encountered in the Pharm Microbiology Lab
• Comparison of their structure, composition, replication methods, nutritional requirements, growth conditions, and host associations. 
• Investigate how these characteristics will affect:
• where we will find them
• when we will find them
• how often we will encounter them
• how we will detect them

FDA Submission Requirements for CMC Microbiology

There are 3 microbiology groups within FDA/CDER that evaluate the CMC microbiology section of NDAs, ANDAs and BLAs. These groups are the New Drug Microbiology Staff in OPS/IO (NDAs), the Microbiology Review Group in OPS/OGD (ANDAs) and the Biotech Manufacturing Assessment Branch (BMAB) in OC/OMPQ (BLAs). The Biotech Manufacturing Assessment Branch reviews the CMC microbiology section of BLAs for therapeutic biological products such as enzymes and monoclonal antibodies and performs pre-license and pre-approval inspections of drug substance and drug product manufacturing facilities.

The CMC microbiology review of NDAs and ANDAs consists of the sterility assurance review of sterile drug products and includes sterilization and depyrogenation studies, hold times, terminal sterilization processes, aseptic fill processes, endotoxin, sterility and container-closure integrity testing. In addition, microbial limits are reviewed for non-sterile drug products. The CMC microbiology review of BLAs submitted to CDER evaluates the microbial control of the drug substance manufacturing process, such as monitoring of bioburden and bacterial endotoxins during fermentation, purification and bulk filling operations. The sterility assurance information for the aseptic fill manufacturing process and microbial attributes are assessed for the drug product. Establishments involved in the production of a biological product must meet CGMP requirements as described in 21 CFR 601.2(d). BMAB leads the pre-approval and pre-license inspections. As a pharmaceutical microbiologist, you need to know and understand how the microbiological studies you perform are integrated and submitted to the FDA in an application, NDA, ANDA or BLA. This presentation will provide you with the knowledge to enhance your study reports by knowing what CDER regulatory reviewers are evaluating in an application regarding the following:

• Bioburden, endotoxin and sterility testing
• Hold time studies
• Microbial limits
• Media fill data
• Sterilization and depyrogenation data
• Container-closure integrity studies
• Sterile filtration

Mid-Morning Refreshment Break

Bioburden Control

• Why do we need to control bioburden?
  • Number and resistance
  • Calculation of sterility assurance (SAL)
• What level of bioburden control is needed?
  • Control requirements
  • Requirements for terminally sterilized and aseptic products
• How do you control bioburden?
• Materials, equipment, procedures, facility/environment, people QA/QC
• How would one perform bioburden control testing for excipients, for in process samples, for hold time studies etc.
• What would acceptance criteria be for different sample types
• Sample matrix evaluations for microbial risk assessments
• How rapid methods could be helpful

Sterility Testing:

• 211.194 And 211.165. USP <71> “Sterility Tests”
• Sampling and Incubation
• Sterility failure investigations - investigation of sterility positives

Lunch

• Current trends in Cosmetic Microbiology

• Antimicrobial Effectiveness Test

• Cosmetics Preservatives and Microbial Resistance

Cosmetics microbiologists faces new challenges, such as the need to develop formulations that are less aggressive to consumers but also well-protected against microbial contamination. Current trends in the field include research on new molecules with biocide power and good toxicological compatibility, analysis of the synergisms and antagonisms of preservative blends, and the search for fast, reliable methods to detect microbial contamination and to test preservative efficiency in each formulation. There is a need for specialists in cosmetics development, preservation, plant hygiene and sanitation, good manufacturing practices, toxicology, etc., to solve problems involving cosmetics contamination and to ensure consumer safety.

Review of Some of the Typical Microbiological Methods

• Microbial Limit Test - The Difference Between “Absence of Objectionable Microorganisms” and “Absence of Specified Microorganisms
• Antimicrobial Effectiveness Testing / Preservative Effectiveness Testing - USP <51>
• Bioburden Test
• MIC/MBC
• Microbial Identification for Aerobes, anaerobes, yeast, fungi, Mycobacterium, Actinomyces
• Microbial limit tests (viable counts, absence of specified microorganism)
• Total Aerobic Microbial Count
• Salmonella & Escherichia Coli
• Staphylococcus Aureus& Pseudomonas Aeruginosa
• Total Combined Mold & Yeast
• Antibiotic assay, Vitamin assay
• Preservative Effectiveness Test

Afternoon Refreshment Break

Risk Management: Developing a Rational Program for Assessing Organisms in Upstream Biopharmaceutical Processes

Risk assessments are becoming common in a number of areas associated with microbiology, including but not limited to environmental monitoring, aseptic processing, and assessing the impact of certain microbial species on the final product.

In the area of environmental monitoring, risk assessments can justify the locations of samples and may be employed in the reduction of sampling locations. In aseptic processing, risk assessment can be used to predict and subsequently correct areas of contamination risk for the process. In assessing microbial identity in final products or processes, risk assessments can support an investigation and provide a rational approach for supporting product release or rejection. This session will provide examples of the use of risk assessments in microbiology QC Laboratory.

Container Closure Integrity Testing-Microbiology and Physical Issues:

Sterility is a stability-indicating parameter for parenterals. Microbiological testing has historically been used for evaluating integrity of glass vials sealed with elastomeric stoppers during shelf-life studies. The Sterility Test is still used today during stability shelf-life studies despite its lack of statistical and biological relevance in evaluating aseptically-filled vials. The microbiological challenge-intrusion test is also used despite its potential for false negative results. Physics has provided more challenging conditions to leak testing technology and methods as well as constant improvements in efficiency, sensitivity and consistency. A more robust approach to container closure integrity testing during shelf-life studies will be discussed along with examples of the issues relating to microbiological tests and the lack of standardization of some current test methods.

No Drugs for Bad Bugs: The Trials of Antibacterial Discovery

Antibiotic discovery is not in vogue these days. This is paradoxical, since the resistance-mediated decline in efficacy of marketplace antibiotics should stimulate a continuous demand (and commercial opportunity) for new products. With the various bacterial threats to the public health (multiply resistant strains, emerging pathogens and biothreat organisms), the medical need is certainly high. Nonetheless, most biopharmaceutical companies have left the area. Many factors contributed to this exodus, but the fact remains that a better return on investment can be made in other disease areas (at least based on commercial analysis and forecasting). Strict and sometimes uncertain regulatory requirements as well as the competitive commercial environment figure predominantly in the calculus, especially for public companies which have obligations to their shareholders. Less well recognized is just how difficult it is technically and how much time it takes, to make a new antibiotic. Lead molecules of novel structure are difficult to find, and then the trouble starts. Converting an early prospect into a medicine that can be used in people is a profound scientific challenge. And this is the case when there is a 60 year history of producing small molecule antibiotics designed to kill bacteria - never mind the development complications of other modalities like anti-virulence approaches, monoclonal antibodies, host-based strategies and the like. In critiquing antibacterial research in large pharma, I will (try to) interpret what our experiences have taught us about the discovery process, and will suggest how those lessons may inform a strategy for the future.

Question & Answer Session

Conclusion of Day One

Basic Understanding of Microbial Identification Methods and Systems

• Review the Microbial Identification Process, including initial characterization, preliminary classification, and final identification to the genus & species level (and even down to the strain level)
• Describe the various conventional / manual and automated laboratory methods and systems commercially available for microbial identification
• Discussion of the issues that should be considered when selecting the appropriate ID methods & systems for you lab

Validation on of Microbiology Test Methods

• Overview of microbiological test methods
• The importance of validation of microbial test methods.
• What is required to validate microbiological test methods?
• Case studies:  Different approaches to microbial method validation

Mid-Morning Refreshment Break

Efficacy of Antimicrobial Preservation - USP, EP, JP, PCPC:

This presentation will compare and discuss the various types of preservative challenge test methods (e.g. USP, EP, JP, PCPC, AOAC, ISO and In-House) that are used by cosmetic and pharmaceutical companies to determine the preservative adequacy of product formulations against microbial contamination that may be introduced into a formulation during consumer usage. The following topics will be covered: types of microorganisms that are used in challenge testing, sampling frequency after inoculation, length of various the challenge test methods, and challenge test criteria.

The Specification and Validation of Rapid Microbial Methods for Non-Sterile Products:

One of the issues faced when using rapid methods is the fact that their output is generally not consistent with the traditional method specified in the monographs. This means that considerable thought has to be given to both the validation of the rapid method to assure equivalency in terms of sensitivity and accuracy as well as the means of defining the output of the rapid method in such a way that it is consistent and compatible with the reference methods. Equally there is a need to make sure that the specifications for the rapid methods are easy to use and understandable by the manufacturing operation in order to assure effective implementation. This presentation will try and address both the means and methods of validating rapid methods to assure regulatory compliance, discussing the options for defining and using the new specifications in an operating environment.

Lunch

Rapid Microbial Methods and the Need for Sample Preparation

Rapid detection technologies offer the advantages of speed, sensitivity and automation for microbial detection. However, these technologies remain under utilized. Often the feasibility of a rapid method depends on compatibility of the sample matrix with that approach. Sample preparation is often the key element in adoption of rapid method technologies for microbiological testing. Some sample preparation methods for complex sample matrices will be reviewed and discussed.

Tracking the Untrackable Microbe: The Use of SLST and MLST in Environmental Monitoring Programs

• By combining an accurate method of genotypic identification, such as 16S sequence-based analysis, with multi or single-locus sequence typing (M/SLST), it is possible to resolve some of the most difficult organisms to trend and track in the pharmaceutical, medical device, dietary supplement and nutraceutical industries
• MLST and SLST are well-established, highly accurate sequence-based methods that can be used to distinguish closely-related microorganisms to the subspecies or strain level by analyzing essential protein coding or housekeeping genes which are hypervariable
• MLST and SLST can determine gene combinations that give high level of variability to differentiate to the strain or subspecies level even in situations where the organisms are closely related

Preservative Systems:

The inclusion of a preservative or preservative system into a product formulation will prevent the growth of microorganisms that are introduced during consumer usage.  This presentation will cover the considerations that need to be performed when selecting the use of a preservative or preservative system for a product formulation such as regulatory, formulation factors such as pH, water activity, preservative solubility, and compatibility with raw ingredients, spectrum of antimicrobial activity, order of addition during processing, packaging, irritation/sensitivity concerns and cost.

Afternoon Refreshment Break

The Determination of Objectionable Microorganisms in Non-Sterile Pharmaceutical Products: A Science-Based Risk Assessment Approach

FDA 483 Citations in the Microbiology Lab

FDA Form 483 citations are scrutinized by the pharmaceutical industry to understand FDA's current thinking and expectations. Citations also provide a snapshot of the thinking and focus of FDA investigators when they are conducting facility inspections, whether these inspections are pre-approval, pre-license or surveillance inspections. This presentation will provide an overview of the Laboratory Controls System as part of the 6 systems' FDA inspection program. In addition, recent 483 citations in the microbiology lab will be presented and their impact discussed.

Question & Answer Session

Conclusion of the Program