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