Rapid Methods & Technologies

Transcription

1 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science Rapid Methods & Technologies Finding the Guilty Party Faster! James Spedding MT (ASCP) EMD Millipore Lab Solutions - BioMonitoring

2 Where are we and why? Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S) have stated that compendial sterility testing suffers from statistical deficiencies Many compendial methods offer low probability of detection except with gross contamination RMMs provide the ability to move microbial detection and identification operations from routine multi-day activities to in-line or in-process activities. RMMs have shown equivalency to compendial methods RMMs can identify microbial risks faster than traditional methods, monitor critical control points in real time, contribute to improved manufacturing processes, enhance safety profile of products and decrease overall costs of quality programs.

3 RMM Misconceptions Are not accepted by regulatory authorities FDA will never buy into this It will take forever to gain regulatory approvals Rapid methods do not support QbD or PAT Will never replace pharmacopoeia tests Little validation guidance Offers no return on investment We will exceed our specifications and action levels, which will translate to an increase in batch rejections Changing acceptance levels will not be allowed There s just not enough information out there It costs too much 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

4 RMMs Are Not Accepted By the Regulators The authorities not only understand, but also embrace and encourage the use of RMMs U.S. Food and Drug Administration (FDA) European Medicines Agency (EMA) Australian Therapeutic Goods Administration (TGA) Japanese Pharmaceuticals 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

5 Limitations of Traditional Testing Small sample sizes Long incubation periods Membrane filter incompatibility Turbidity used as detection endpoint Multiple incubation and plating procedures Reduced testing Increased costs due to failure further into product manufacturing need to commit raw materials in production cycle prior to test results

6 RMMs Advantages RMMs can reduce: Product loss Product release time Costs (Testing & Labor) RMMs can increase product safety: Higher sample throughput Real-time data analysis Through earlier contamination notification ( In-Process)

7 Barriers To Change Perception that the regulatory approval process is very difficult This argument is getting increasingly weaker based on recent FDA observations Management may not support the change due to comfort with existing working methodologies, hence no incentive to adopt new assays Fear that methods with lower detection levels than less robust current assays may uncover microbiological concerns Validation costs may be too high Return on Investment may be too long

8 Regulatory Status of RMMs Regulations have not discouraged exploration of RMM as an alternative 21CFR<610.9> - prescribes providing increased assurance that a product meets microbiological specifications while describing equivalent methods and processes. French regulatory agency(afssaps), German Health agencies, and FDA have all published evaluations of a number of rapid detection platforms Additionally the USP and EP have published relevent chapters: USP <1223> Validation of Alternative Microbiological Methods Ph.EUR Alternative Methods for Control of Microbiological Quality Ph.EUR Microbiological Control of Cellular Products FDA published (2008) Guidance for Industry defining validation criteria for growth-based RMM

9 There s Not Enough Validation Guidance PDA Technical Report #33, Evaluation, Validation and Implementation of New Microbiological Testing Methods USP <1223>, Validation of alternative microbiological methods Ph. Eur , Alternative methods for control of microbiological quality All are under revision to reflect current perspective 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

10 FDA PAT Initiative A desired goal of the PAT framework is to design and develop well understood processes that will consistently ensure a predefined quality at the end of the manufacturing process. Such procedures would be consistent with the basic tenet of quality by design and could reduce risks to quality and regulatory concerns while improving efficiency. Gains in quality, safety and/or efficiency will vary depending on the process and the product, and are likely to come from: Reducing production cycle times by using on-, in-, and/or at-line measurements and controls Preventing rejects, scrap, and re-processing Real time release Increasing automation to improve operator safety and reduce human errors Improving energy and material use and increasing capacity Facilitating continuous processing to improve efficiency and manage variability * Guidance for Industry PAT A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance (September 2004)

11 USP <1223> The purpose of USP <1223> is to provide guidance for validating methods for use as alternatives to the official compendial microbiological methods. Describes in detail the requirements for validation of both Qualitative and Quantitative Tests Qualitative Tests: Specificity Limit of Detection Ruggedness Robustness Quantitative Tests: Accuracy Precision Specificity Limit of Quantification Linearity Operational Range

12 CBER Guidance on Sterility Testing Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products Published February 2008 Demonstrates that an alternative, growth-based ATP based RMM is equivalent to the conventional sterility test method 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

13 FDA Approval Process Include RMMs in an NDA, ANDA, IND for new products Existing products may require a prior-approval supplement in the relevant CMC sections Utilize a comparability protocol approach For in-process tests and methods that are not in a regulatory dossier, a formal submission may not be necessary Discuss your strategy with the Agency 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

14 EMEA PAT Initiative Exact same initiative as the FDA PAT Initiative. Recognizes that: Quality cannot be tested into products. Quality has to be built by design. This means designing a good manufacturing processes but also developing good real time quality control processes as well.

15 Regulatory Take Away The regulatory authorities get it There is no excuse not to move forward Many contamination issues we have seen in recent years could have been prevented with early detection technologies In the future, and in the context of product quality, continuous process improvement and patient safety, RMMs will be the expectation, not simply nice to have 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

16 Benefits of switching to a Rapid Microbiology Method 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

17 Why would Rapid Microbiology Methods Benefit You? Reduction of Risk Improved and faster processing controls resulting in faster product release Quicker Corrective/Preventative Action implementation More sensitive detection for microbial contaminations Reduce staff training requirements Reduction in repeat testing and waste Keeping up with Industry and Regulatory Standards

18 Development of User Requirements for a Rapid Microbiological Method 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

19 Steps to Implement a Rapid Microbiology System Select your microbiological application Feasibility study Determine potential sample effects Determine if a rapid system will work for your application Know the limitations of the technology Purchase system On-Site Service and Training, Installation, Qualification and Validation

20 Understand your Current Method What is the volume/weight of product that you sample? Does your product have inhibitory properties? What is your typical bioburden? What are the organisms of concern? What type of media are used? How many samples per day do you process/throughput?

21 Understand your Current Method What is your current sample preparation time? What is your current sample incubation time? What are your validation requirements? What is your time-frame for adoption of the new method?

22 Develop a User Requirement Specification (URS) The purpose of the URS Is to specify the requirements and deliverables for a rapid system based on the your needs. Because every requirement may not be met by one product, categorize requirements as Mandatory or Desirable. The URS can be the predecessor for: Vendor Selection & Qualification Risk Analysis Report Functional Specification Hardware & Software Design Specification (if applicable) Validation/Performance Qualification of system

23 Develop a URS The URS should contain: Introduction Objective Purpose Background information Main functionality requirements of the system - must be able to obtain fast and reliable enumeration of organisms - must be able to store the obtained results as data access to altering parameters, properties and results must be restricted to the appropriate personnel. Software must be 21CFR Part 11 compliant - must be able to reduce incubation time by X amount of time - must be able to meet a throughput demands - sample preparation must be based upon Membrane Filtration - Health and Safety of operators must not be changed with the addition of this new method - the vendor will continue to support the product for a minimum period of 5 7 years after purchase

24 Evaluate Potential Vendors Support Availability Knowledge Training Response time Product Life Cycle Repair & Preventative Maintenance Service Programs Calibration Validation Protocols/documentation Execution of IQ, OQ, PQ

25 Ask the Vendors What technologies are the system based upon? What is the level of knowledge/skill required to operate the instrument? What type and frequency of maintenance/service is required? Do you have any references?

26 Rapid Microbiology Testing Methods 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

27 Rapid Microbiological Methods New technologies that provide microbial detection, quantification and identification results much faster than conventional methods Increased accuracy, reproducibility and sensitivity Automated, miniaturized and high-throughput processing Enhanced sampling, data handling and trend analysis Based on a variety of core scientific principles Those based on compendial methodologies reduce implementation process 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

28 RMMs Candidates Raw material testing Media components Buffers components Pharmaceutical ingredients Purified water / WFI In process sample testing Bioburden prior sterilization Cell culture/fermentation Media for fermentation Buffers for manufacturing Rapid endotoxin testing Rapid Mycoplasma testing Real-time environmental monitoring Final product testing 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

29 Qualitative Microbial Detection Impedance: measures ionic changes from metabolic byproducts due to growth CO2 Detection: measures carbon dioxide produced during growth Headspace Pressure: measures pressure change, positive or negative, due to production or consumption of gases Nucleic Acid-based Technologies: commonly PCR based Flow Cytometry: first applied to eukaryotic cells can also detect viable bacteria Endotoxin: LAL test for bacterial endotoxin ATP Bioluminescence: through surface swabs, membrane filtration, etc

30 Quantitative Microbial Detection Direct Laser Scanning: detects microcolonies ATP Bioluminescence: detects microcolonies Autofluorescence: detects autofluorescence from viable organisms Nucleic Acid-based Technologies: commonly real-time PCR

31 Microbial Identification Phenotypic and Biochemical Methods: classical phenotypic/biochemical methods. May require preliminary characterization such as Gram Stain Genotypic Methods: most involve comparative DNA sequencing of the 16S rrna gene in bacteria or 26S rrna gene in fungi. Can reduce identification to an overnight procedure Mass Spectrometry: MALDI-TOF methodology, can be available in minutes rather than days

32 Polymerase Chain Reaction (PCR) A powerful method to synthesize DNA in a test tube Many identical DNA molecules are created from just a few. Sample integrity and matrix is critical Inhibitions must be considered

33 DNA template PCR works like this: 2 n n = PCR cycles 1. Annealing of DNA primer 2. DNA synthesis with DNA Polymerase In 30 cycles, 1 template generates 1 billion copies!

34 Transcription Mediated Amplification (TMA) A powerful method to synthesize RNA in a test tube Many identical RNA molecules are created from just a few. More RNA copies present in cellular matrix than DNA Filtration based method RNA degrades faster than DNA which lends to live cell detection only

35 ATP Bioluminescence Bioluminescence is a known chemical reaction within an organism that has been established for microbial detection. The oxidation of luciferin catalyzed by the enzyme luciferase results in oxyluciferin and light. Adenisine triphosphate (ATP) provides the energy to produce the luciferin. Luciferins allow for the production of 'cold light' as little to no heat is given off during these reactions.

36 Fluorescence The principle of the fluorescence detection is based on an routine enzymatic reactions within the microbial cells. A fluorogenic substrate is a non fluorescent viability marker that is cleaved by nonspecific intracellular enzymes resulting in a fluorescent microcolony. Accumulation of fluorescence inside cells is an indicator of microbial metabolism activity and membrane integrity. The fluorescent stain is permeable to the microbial cell membrane thus stain introduction into cells.

37 Principle of Fluorescence Microorganism before staining Membrane is incubated on a pad pre-soaked with 2ml of fluorescence reagent Marker is non-fluorescent when outside cells Only viable microorganisms are stained (viability marker) Fluorescence accumulates inside cells after cleavage by the bacterial metabolism (enzyme cleavage) Natural amplification of the signal by accumulation of reagent inside the microorganisms Stained micro-organisms are then exposed to a specific wavelength (LEDs exposure) in Milliflex Quantum reader and emit fluorescence

38 ATP and Fluorescent Detection of Micro-colonies Number of Organisms Micro Colonies = Results Up to 50-75% faster than conventional methods Typical Growth curve 10 6 Micro colonies Visible Colonies 10 2 T1 T5 Time (days)

39 ATP & Fluorescence Performance Examples 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science

40 Sterile water contaminated with ATCC or environmental germs Escherichia coli ATCC hours Pseudomonas aeruginosa ATCC 9027 TSA 12 hours Bacillus subtilis ATCC ± 2.5 C 8 hours Staphylococcus aureus ATCC hours Candida albicans ATCC SDA 24 hours 22.5 ± 2.5 C Aspergillus brasiliensis ATCC hours Caulobacter sp. 30 hours Micrococcus sp. Environmental strains R2A 32.5 ± 2.5 C 18 hours Ralstonia sp. 24 hours

41 Examples of slow growing microorganisms detected within 30 hours Acidovorax temperans Env. strain R2A / 32.5 C Ralstonia sp. KN1 Env. strain R2A / 32.5 C Staphylococcus aureus subsp. aureus MSSA476 Env. strain R2A / 32.5 C Caulobacter sp. Env. strain R2A / 32.5 C Ralstonia sp. C1 Env. strain R2A / 32.5 C Bacillus cereus Env. strain R2A / 32.5 C Micrococcus spp. Env. strain R2A / 32.5 C Candida catenulata SDA / 22.5 C Candida albicans SDA / 22.5 C Candida pelliculosa Customer strain SDA / 22.5 C Saccharomyces cerevisiae 7754 SDA / 22.5 C Aureobasidium pullulans Customer strain SDA / 22.5 C Aspergillus brasiliensis (fd) SDA / 22.5 C

42 Detection of Contaminants in Mammalian Cell Culture Incubation time on TSA at 37 C required for the detection of different bacteria with a rapid system B. cereus 8 hours S. epidermidis 9 hours P. acnes 48 hours

43 2013 Laboratory Conference: Unraveling the Mystery of Pharmaceutical Laboratory Science THANK YOU!