Bioburden FDA Hughes, Patricia
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Transcript of Bioburden FDA Hughes, Patricia
FDA Expectations Regarding
Bioburden Control in Biotech
ProcessesPatricia F. Hughes, Ph.D.
Team Leader
Biotech Manufacturing Team
CDER/Office of Compliance
DMPQ
September 23, 2010
Scope
• Microbiology controls and issues
– Regulatory framework
– Importance of microbial control
– Elements of an overall microbial control
strategy
– Examples
Regulatory Framework
Microbial Control
Current Laws
• Public Health Service Act
– Section 351 (a)(2)(B) -- Licensure of biological establishments and products
• The biological product must be safe, pure and potent
• The facility in which the biological product is manufactured, processed, packed, or held must meet standards designed to assure that the biological product continues to be safe, pure and potent
• Federal Food, Drug, and Cosmetic (FD&C) Act (1938, 1962, 1997, 2007)
– Interprets that “biological products” are also “drugs”
• The FFD&CA applies to a biological product, except no application required under section 505
• Inspection under both the provisions of both the PHS Act and the FD&C Act
Regulatory Requirements
Applicable regulations for Biological Products
• Title 21 Code of Federal Regulations (CFR)
–Parts 210 and 211 – Current Good Manufacturing Practice (CGMP)
–Parts 600-680 – biologics regulations
Manufacturers must comply with their biological license application (BLA) commitments and applicable standards
FDA Guidances for Contamination
Control• General:
– FDA Guidance for Industry 2006, “Quality Systems Approach to Pharmaceutical CGMP Regulations.”
– ICH Q9 Quality Risk Management
• Drug Substance:
– ICH Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin
– ICH Q5D Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products
– ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products
– ICH Q7A Good Manufacturing Practices for APIs
• Drug Product:
– FDA Guidance for Industry 2004, “Sterile Drug Products Produced by Aseptic Processing –Current Good Manufacturing Practice.”
– FDA Guidance for Industry 1994, “Guidance fro Industry for the Submission Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products.”
ICH Q6B: Key Concepts on
Microbial Control
• Manufacturing processes should be designed to limit microbial contamination / proliferation in non sterile process intermediates
• In-process testing should be conducted at critical decision making steps (e.g. end of cell culture process)
• Manufacturing process must be able to produce a sterile product with a high degree of assurance
• Some processes are intended to produce a sterile bulk drug substance
• Finished biotech drug products are sterile
ICH Q7A: Section 18
• This document provides guidance on how to control bioburden, viral contamination, and/or endotoxins during manufacturing
• Provides guidance on what and when to monitor processes
Susceptibility to Microbial
Contamination
• Biotech processes and products are prone to microbial contamination because,
– Products are heat labile and cannot be terminally sterilized
– Raw materials, personnel and the manufacturing environment are a source of bioburden
– Products, process intermediates and raw materials support microbial growth
Consequences of Inadequate Microbial
Control in Biotech Manufacturing
Health Hazard to Consumer:
- Defective product:- Loss of drug efficacy (degraded product)
- Loss of drug safety/quality (impurities, metabolites, endotoxins)
- Drug shortages - medically necessary drugs are unavailable or in short supply
Manufacturer:
• Inconsistent, unpredictable manufacturing outcomes – rejection of lots because of contaminations or failed release or stability
specifications
• Shutdown of facility - disruptions in manufacturing operations
• Recalls– Uncertain quality of products on the market
Elements Of An Overall Microbial
Control Strategy
Microbial Control Strategy
• Use of risk assessment tools to design control and mitigate microbial contamination risks
• Identify, control, and monitor potential microbial entry points
• building and facilities
• equipment
• raw materials
• process
• Minimize hold steps and personnel interactions
• Validate critical process steps to eliminate potential adventitious agents
Facilities and Equipment:
Design for Bioburden Control
General Facility and Equipment
Design• Facility design:
– Area classifications, segregation, pressure differentials appropriate for risk of operations
• Segregation of CIP and AHU between live cell and cell free areas.
– Adequate design, qualification, maintenance, and monitoring of utilities, air, water, process gases, etc.
– Layout designed to allow for adequate process, material, personnel flow and to minimize potential of cross contamination through touch points and cross overs
• Equipment
– Closed pipes & vessels with CIP/SIP capabilities
– Closed sampling on vessels and pipes
– Maximize automated transfer to avoid manual connections; minimize manual open handling
– Use in-line monitoring instrumentation to avoid frequent sampling.
– Dedicated equipment for each unit operation
– Dedicated chromatography resins & filter media (e.g. UF, MF) for each product
– Effective preventive maintenance and calibration program in place
Raw Materials: Microbial
Control
MCB and WCB
• Use of working cell bank (WCB) derived from the master cell bank (MCB)
• 21 CFR 610.18 Cultures
– (c)(1)(iv) Tested for the presence of detectable microbial agents
– (c)(2) Tests. ..necessary to assure the safety, purity, and potency …
– (d) Records. Records for cultures… prepared and maintained as required by
211.188 and 211.194
• ICH Q5A Cell Line Qualification
• ICH Q5D Test of Purity
Raw Materials
• Raw materials should be screened for bioburden and endotoxin, where appropriate
• Certain cell culture raw materials, especially those that are biologically derived (peptones, phytones, soytones), are prone to contamination with bacteria, endotoxin, mycoplasma or viruses
– Low levels of certain adventitious agents in raw materials are often impossible to detect
– However, once introduced into a cell culture process, the agents can replicate. The spread of adventitious agents to other bioreactors and throughout a facility is a great concern
• Mycoplasma and virus contaminations have interrupted manufacturing operations at major firms for significant amounts of time leading to product shortages with public health impact and significant economic consequences
Raw Materials: Issues
• Use of biologically derived complex raw materials (e.g., serum) should be avoided, whenever possible. – If use cannot be avoided, then
• Materials should be handled in segregated areas to prevent contaminations of facilities, equipment and process streams
• Treated prior to use in cell culture:
– inactivation procedures such as sterilization or pasteurization,
– 0.1micron filtration
– irradiation
• Screened and tested for adventitious agents prior to use
Production and Process
Controls: Microbial Control
Biopharmaceutical Manufacturing
Drug Product
PurificationCell Culture
HarvestTank
Concentration
Virus-Clearance
Column Chromatography
HarvestFilters
One or MoreBioreactors
Active Drug
Substance
Analytical Testing, Filtration, Filling,
Quality Testing Bulk MaterialFormulated
Aseptic processinginto finished dosage
Media
• Media are sterile filtered or sterilized by moist heat prior to use
– Media for cell culture is typically filtered through 0.1µm filters and /or heat treated
• Hold conditions for media should be validated at scale using production equipment
• Media held under worst case conditions (worse than during routine manufacturing) for temperature and time
– Media is held in SIP tanks with vent filters or gamma irradiated bags
– Media growth promotion
Cell Culture / Fermentation
• Main goal is to maintain culture purity
– Inoculation, seed expansion: • Involves open aseptic processing operations in
BSC to maintain culture purity– T-flasks, roller bottles, shake flasks, spinner flasks
– ISO 5, ISO 7 or ISO 8 background
• Seed expansion may occur in closed systems– Bioprocessing bags or bioreactors
– ISO 8 or ISO 9 areas
Bioreactors
• Bioreactors are cleaned and sterilized prior to use– Validated for sterilization in steam penetration studies with
biological indicators
– Media hold or media simulation studies to demonstrate maintenance of sterility in the bioreactor are recommended
– Pressure hold tests initially, periodically and between campaign to demonstrate closed system
– Critical liquid filters should be integrity tested
– Critical air filters should be integrity tested
Microbial Control in Microbial
Fermentations• Microbial fermentations are generally of short duration (1-3 days
long) and involve rapidly growing organisms (e.g., E. coli)
– Less chance that microbial contaminants can overtake the
production organisms in the bioreactor
– Medium is often sterilized (SIP) in production the vessel
– Cell banking and small scale inoculum generation will need
aseptic processing
– Measures should be in place to prevent phage contamination of
microbial cultures (“phage – out”)
Microbial Control in Cell Culture
Process• Microbial control in cell culture is critical because contaminants can
grow at a faster rate than mammalian cells in culture.
– Doubling times of common bacteria:
• The doubling time for E. coli in a glucose salt medium is reported to be ~17 minutes ; the doubling time for Bacillus megaterium in a sucrose salt medium is 25 minutes
– Doubling times for mammalian cells in culture can vary from 18 to 48 hours or longer
Consequences of Rapidly Growing
Contaminants in a Mammalian Cell
Culture Process
• Cell culture conditions are very favorable for contaminating microorganisms:
– Microorganisms, if initially present even below detectable levels, will increase exponentially and at a faster rate than the cells in culture
– The presence of contaminating microorganisms in a cell culture process will lead with time to a process failure
– Consider that some mammalian cell culture processes occur over a period of 50 –100 days in the same bioreactor
• Aseptic conditions must be maintained throughout this time frame!
Cell Culture/Fermentation
Bioburden Test Methods
• Purpose to demonstrate culture purity– Use membrane filtration (preferred), pour plate or direct plate
count
– Demonstrate method suitability: recoverability of microorganisms in the presence of the test sample (product interference)
• Dilution, neutralization of interference
– Growth promotion of media
– USP microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus niger (A. brasiliensis), possible environmental and bioburden isolates)
– Sample volume preferably 10 to 100 mL, when possible
– Report results in CFU per sample volume tested
– Reference USP <61>
Harvest, Recovery and
Purification
Recovery Operations
• Centrifuges and microfiltration systems are exposed to cell culture/fermentation broth streams that are rich in organic matter
– Bioburden and endotoxin control is challenging
• Equipment, transfer lines, membranes and recovery vessels should be adequately cleaned and sanitized/sterilized after use
Purification Operations
• Purification column resins and UF/DF membrane systems are typically cleaned and sanitized after each use
– The cleaning and sanitization procedures should be validated for microbial control
– The columns and the membranes should be monitored for bioburden before each use.
– The first columns or membrane systems are more susceptible to bioburden contamination or biofilm problems because of the nature of the process streams from the cell culture / fermentation/ harvest areas (rich in organic matter)
Purification Buffers
• Buffers are typically filtered through 0.2µm filters into sterilized vessels or sterile bioprocessing bags
– Buffers should have bioburden and endotoxin limits
– Hold conditions for buffers should be microbiologically validated at scale using production equipment
• Growth promoting buffers may be used to simulate hold times
• Buffer hold should simulate routine production conditions
Microbial Control Strategy for
Purification• In process purification intermediates (column eluates, pre-UF/DF
intermediates) are typically filtered through 0.2 micron filters.
– Intended to protect the column resins from colonization with bioburden
• In process purification intermediates (column eluates, pre-UF/DF process streams, pre-filtration process streams) should be monitored for bioburden
– Bioburden limits are typically set at 10 – 1000 CFU/ 100mL
• In process purification intermediates (column eluates, UF/DF retentates) held under conditions that support microbial proliferation (e.g., 24 hours, RT) should be monitored microbiologically
• In process purification intermediates hold conditions should be validated microbiologically at scale, not just monitored
Microbial Control Strategy for
Purification (cont.)
• Column resins and membranes from UF/DF
systems should be cleaned, sanitized after each
use
– Procedures should be effective in controlling
bioburden and endotoxin
– Resins should be used only within the validated use
time
– Resins and membrane should be stored under
conditions that do not promote microbial growth
Filtration of the Bulk Drug
Substance
• The final filtration step in a drug substance manufacturing process is often a bioburden reduction step intended to reduce bioburden load in the Bulk Drug Substance
– Filters should be integrity tested
Filling of the Bulk Drug Substance
• After filtration bulks may be filled into stainless steel vessels, bottles or sterile bioprocessing bags
– Liquid Bulks – that is bulk to be stored at 2-8 C
• The fill process may occur using aseptic processing conditions in an ISO 5/6 area
• The environment and personnel should be monitored
• Containers/closures should be cleaned and sterilized using validated cycles
• The aseptic operations should be qualified in media simulation studies
• The suitability of the container closure should be demonstrated
– Frozen bulks – to be stored frozen at -20 - 60 C
• The fill process may occur in an ISO 7/8 area when all operations are closed
• The environment and personnel should be monitored
• Containers/closures should be cleaned and sterilized using validated cycles
• The suitability of the container closure should be demonstrated
Bioburden Test Methods: In-
process and Release• Use <61> Microbiological Examination of Nonsterile Products:
Microbial Enumeration Tests
Membrane filtration or plate-count methods (pour or direct plate)
• Method must allow testing sufficient sample size to judge compliance with specification
• Suitability of the method must be established
– The ability of the test to detect microorganism in the presence of product must be established
• Use of USP test strains
• Negative control
• Growth promotion properties of the media
– Test each batch of medium
• Sample volumes 10-100 mL
Bioburden Test Method: Bulk
Release• Bioburden release specifications:
– Bioburden limits for a BDS that is stored at 2-8 C should be <1 CFU/10 mL or <10 CFU/100 mL
• The sample size should be 10 - 100 mL
• Frozen BDS may not have release bioburden specifications
– Bioburden is monitored and controlled throughout the manufacturing process, including the final pre-filtration step
Purification: Microbial Control
Issues• Current culture based methods for bioburden provide
results after 2 to 3 days after process intermediates have been forward processed – This may allow for the spread of bioburden in the process
• Filtration of contaminated process streams does not assure product quality– Filtration of process intermediates with high bioburden or
endotoxin will result in process or product failures. • altered impurity profiles
• product instability and OOS results
• Purification must be conducted under tight microbial control
Examples
In-process Bioburden –Example 1
• High bioburden levels for the in-process intermediates and bulk drug substance– Discovered during the pre-license inspection
• 483 observation:
– Inadequate control of the production process:
• Multiple lots of drug substance were released which had unacceptably high levels of bioburden during the final purification steps
– In addition, bioburden limits were not been established for each step in the purification process
Lots were ultimately rejected
In-process bioburden - Example 2
• 483 observation during a pre-license inspection
– Inadequate control of the production process:
• Hold times for four column eluates [column steps
x, y, z] have not been adequately validated
microbiologically
– No microbial limits are established
– Results show high bioburden levels (>1000
cfu/mL)
In-process Bioburden:
Clinical and Process Validation Lots
In Process Bioburden (CFU/mL)Step Action
Limit
Batch 1 Batch
2
Batch 3 Batch 4
Crude extract 100 < 10 < 10 < 10 < 10
UF/DF pre-
filtration
1000 95 295 < 10 420
UF/DF post-
filtration, storage
500 475 TNTC-
1130
10 140
CEX 100 10 10 20 50
UF/DF-2 500 10 5 < 10 65
AEC 100 < 10 20 15 10
AEC after
adjustment
100 < 10 < 10 < 10 < 10
HIC 5 100 < 10 15 < 10 < 10
HIC after storage 100 30 < 10 < 10 25
UF/DF-3 500 760-220 260 1030-1055 870-680
SEC 10 314-335 109-
17.5
15-40 < 1
Outcome of Corrective Actions
• Process redesign:
– Improvements in equipment cleaning and sanitization/sterilization (vessels, transfer lines, use of gamma irradiated bioprocessing bags)
– Addition of 0.2µ filters prior to each column or UF/DF step
– Column cleaning/sanitization studies
– Reduction of hold times
– Bioburden levels were in both cases brought under control to < 10 CFU/ml
Conclusions
• Appropriate management of microbial control in a Biotech process will lead to
– More consist process and product
– Fewer failures or deviations both upstream and downstream
– Management of change control and continuous improvement post - approval
– May provide for a more cost effective process
Acknowledgements
• Richard Friedman
• Kalavati Suvarna
• Ingrid Markovic
• Anastasia Lolas
• Brian Hasselbalch