FDA Expectations Regarding Bioburden Control in Biotech Processes

Patricia 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
Column Chromatography

Harvest Tank
Harvest Filters VirusClearance

Concentration One or More Bioreactors

Cell Culture

Purification

Drug Product Active Drug Substance

Aseptic processing into finished dosage

Analytical Testing, Filtration, Filling, Quality Testing

Bulk Material Formulated

Media
Media are sterile filtered or sterilized by moist heat prior to use
Media for cell culture is typically filtered through 0.1m 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 0.2m 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: Inprocess 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 Crude extract UF/DF prefiltration Action Limit 100 1000 Batch 1 < 10 95 Batch 2 < 10 295 Batch 3 < 10 < 10 Batch 4 < 10 420

UF/DF postfiltration, storage

CEX UF/DF-2

500
100 500

475
10 10

TNTC1130
10 5

10
20 < 10

140
50 65

AEC
AEC after adjustment HIC 5

100
100 100

< 10
< 10 < 10

20
< 10 15

15
< 10 < 10

10
< 10 < 10

HIC after storage

UF/DF-3 SEC

100
500 10

30
760-220 314-335

< 10
260 10917.5

< 10
1030-1055 15-40

25
870-680 <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 0.2 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