Contamination and Beyond - cbinet.com IVT 05Jun16_FINAL...growth/gross contamination Visual turbidity, microbial cells visible on slides, odor Direct Steps with open processing Recurring

Contamination and Beyond

Hilary Chan

QC Sr. Scientist

Shire

Lynn Johnson

QC Scientist

Shire

IVT Microbiology Week

June 7-9, 2016

Detection, Remediation, Control, and the

Integral Role of the Microbiologist

To be as brave as the people we help.

2

Session Outline

Objective and Introduction

Building Blocks of an Effective Contamination Investigation

Interactive Exercise Part I – Investigation Case Study

Contamination Control – Microbial Risk and CAPA

Interactive Exercise Part II – CAPAs

Contamination Control - Preparedness


3

OBJECTIVE AND INTRODUCTION


4

• How to detect, investigate, and respond effectively to a

contamination

• Tools for improved lab support for investigation

• Tactics for continual improvement of contamination

response and investigation

Objective


5

Introduction

• Determining the root cause of a microbial contamination

can be time consuming and difficult

• Potential product loss

• Production down time

• Unique circumstances/“uncharted territory”

Patient safety is #1 priority

5


6

Scrambling around to figure out what to do

No time for lengthy studies

Difficult to pinpoint root cause

Shotgun approach to mitigation

Rely on contamination response “formula”

Scope and risk may be unclear

Microbiologist may be left out of early stages of investigation

Common Pitfalls

Goal:

A systematic approach for investigating

microbial contaminations

Ideal Scenario

Introduction


7

• Aseptic vs. non-sterile/low bioburden

• aseptic facility – a finished product free of any microorganisms

• non-sterile or low bioburden facility - a microorganism presence may

be expected/allowed, but the type and number are tightly controlled

• Intrinsic vs. extrinsic sources of microbial contamination

Introduction

• Examples - bacterial contamination from raw material, biofilm in water system

Intrinsic

• Example - bacterial contamination from poor gowning practices, HEPA failure

Extrinsic


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• What constitutes a “contamination” at a non-sterile/low

bioburden facility?

• “Gross” contamination/out of specification or action-level result

• A more subtle loss of microbiological control

• Trend (product, EM, utility)

• Recovery of microorganism that is considered “of concern” or

high risk due to characteristics and sample source

Can be more difficult to detect and investigate

Can progress and severely impact your process/product

Introduction:

Signs and Symptoms of Microbial Contaminations


9

Introduction:


Endospores: Bacterial (typically Gram positive) spores form in stressful environments and are prime for dispersal in the environment

Ultramicrobacteria: The ability of microorganisms to reduce in size as a means of starvation survival, and at the same time, allow them to pass through traditional 0.45um and 0.2um membrane filters

Mold: Spores are ubiquitous in nature and can readily grow in diverse environments; concern surrounds pathogenicity, mycotoxin production, allergic reactions, and invasiveness in the facility

Endotoxin: Lipopolysaccharide complex associated with the outer membrane of Gram negative bacteria. Can indicate the presence of Gram negative bacteria and elicit inflammatory responses in humans

Biofilm: Microorganism creates an enclosed environment and adheres to surfaces under nutrient limited conditions


10

Susceptible Systems Signs of Contamination Detection Mode Contamination

Type

Nutrient-rich

environments (e.g., cell

culture, media)

System parameter shifts (e.g.,

pH, DO), loss of host cell viability

Indirect High-level

growth/gross

contamination

Visual turbidity, microbial cells

visible on slides, odor

Direct

Steps with open

processing

Recurring low level bioburden

recovery

Direct bioburden

detection, indirect

trend detection

Low level

bioburden trend

Water systems, nutrient-

poor environments

Sporadic bioburden recovery,

similar/reoccurring isolates,

endotoxin

Indirect Biofilm

Areas with lower

environmental

classification/ transition

points (e.g., gowning

rooms, airlocks)

Similar/reoccurring isolates

(especially spore-forming bacilli

and fungi), frequent excursions

Direct EM trend

Production operations

with heavy personnel

reliance

Similar/reoccurring isolates

(especially G+c), frequent

excursions specific to operator(s)

Direct Personnel trend

Introduction:



11

BUILDING BLOCKS OF AN EFFECTIVE

CONTAMINATION INVESTIGATION


12

Building Blocks of an Effective Contamination Investigation:

Introduction

The role of the microbiologist is not limited to testing and

reporting of data; the impact a microbiologist has is best

summarized in the statement below:

“The mission of a microbiologist is to develop in the pharmaceutical

organization a foundation for understanding of microbial origin, and

parameters for proliferation and survival; to continuously improve/

embed the concepts for protection, exclusion, reduction, removal or

destruction of contaminating microbiological entities.”

Singer, D.C. (2012) “A Strategy for Developing Robust Pharmaceutical

Microbiological Control.” American Pharmaceutical Review 15(4)


13


Collaborative Contamination Response

Anatomy of a Contamination

Response Team

Key Representatives

Visual Management

Problem Solving

Tools

Participation


14


An Overview of a Contamination Investigation - Examples

Immediate actions/containment

Investigation plan

Problem statement

Root cause analysis

CAPA

or

Define

Measure

Analyze

Improve

Control


15


Root Cause Analysis

Root Cause Analysis Tools

Checklists, tables,

flowcharts Five Whys

Fishbone/ 6M/Ishikawa

diagram

Contradiction matrix/TRIZ

Is/Is not


16


Root Cause Analysis

RCA Tool Benefits Limitations

Checklists, tables,

flowcharts

Customizable, wide range of usage, scope

setting

Unstructured, less efficient

Five Whys Easy to use, no hypothesis testing,

determine relationship between root causes

Higher risk of failure to find root

cause (less comprehensive)

Fishbone Diagram Brainstorming, cause-and-effect,

categories, identifies multiple possible

causes/thorough

Time-consuming, can create

divergence, difficult to

represent interrelatedness

Contradiction Matrix Precedent solutions used to resolve

contradictory elements of a problem,

established matrix reduces analysis time.

Examine facts and causes to see if they are

relevant

Lack of formalization, difficult

to implement as official tool

Is/Is Not Comparative analysis, structured thinking,

can be used when partial knowledge of

situation (determine scope/boundaries),

process of elimination

Focus on differences/changes


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Root Cause Analysis - Example


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• After framing the contamination through initial discussions, a

deeper dive can be taken using one or a combination of the

data collection plan approaches outlined below to help focus the

investigation:


Investigation Tools and Methods

Walk the Process

• GEMBA walk

• Process map

• Material flow diagrams, equipment drawings

• Timeline of sequence of events

• Standard knowledge baseline for all team members on relevant areas of focus

Containment/Scope Setting Sampling

• “Survey” samples to help define scope or problem statement

• Additional process or environmental monitoring locations and/or increased sampling frequency

Hypothesis Testing

• Hypothesis-specific sampling

• Challenge each hypothesis using facts, observations, and data

• Rule in or rule out

• Work cross functionally between functional areas based on topic

• Review data

Laboratory Studies

• Increase knowledge base

• Hypothesis-specific experiments

• Data gathering/ scenario testing


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The Role of the Microbiologist

Recurring contamination? Increasing vs. variable recovery

level? Same microorganisms/strains?

Is endotoxin OOS?

Matrix Evaluation:

What are the microbial growth-

effecting attributes?

Challenge Assumptions:

Is a 0.45µm filter 100% retentive

of a 0.5µm microorganism?

Asking the right questions in order to determine the

appropriate tools and test methods to use


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Gram positive spore forming or Gram negative bacilli that are ubiquitous in nature

Microorganisms that persist in areas that are inaccessible to cleaning processes

Microorganisms that are capable of surviving in stressful conditions


Assessment of Microbial Risk

Typical characteristics of a pharmaceutical contamination


21

Examples of manufacturing environments associated with contaminating

microorganisms




22


Investigation Tools and Methods

Situation Studies Purpose

Contaminant found in a

tank or “pool” sample

Growth kinetics study

using process isolate,

matrix, and maximum

process hold time

Use growth curve to determine

doubling time in order to back-

calculate point of entry

Same species

recovered in other

steps/batches/WFI/EM

or investigational (e.g.,

swab) sampling

Strain type analysis of

isolates

Strain relatedness to determine

common source

Recurring

contamination with

variable recovery levels

Biofilm formation and

disinfection

Determine if isolate(s) form

biofilm on manufacturing surfaces

and determine effective method

of eradication

High levels of endotoxin

with low bioburden

Combined

endotoxin/growth

kinetics study

Evaluate microbial growth kinetics

and relationship to endotoxin

production

Hypothesis testing Investigational sampling

or scenario testing

Rule in/out


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CONTAMINATION INVESTIGATION

Interactive Exercise Part I


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Objective of Part I: teams will form hypotheses based on clues

provided by instructors and rule in/out for root cause

1. Break into teams

2. Instructors will guide the teams in performing a mock

contamination investigation based on an actual case study

• Instructors are a resource representing all SMEs required for the

investigation, providing any requested information or data to the

team

• Reference session material as needed

Interactive Exercise Part I: Instructions


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• Form investigation team

• List any critical immediate actions

• Determine problem statement

• Perform root cause analysis

• Establish CAPAs (Interactive Exercise Part II)

Interactive Exercise Part I: Investigation Plan


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• Low level (1-2 CFU/mL, below action limit) mold (Acremonium

kiliense) was detected from in-process bioburden samples for

two sequential drug substance purification batches

• In-process data review revealed increased frequency in purification bioburden recovery over prior two months, including:

• Stenotrophomonas maltophilia (Gram negative bacilli)

• Microbacterium lacticum and Microbacterium species (Gram

positive bacilli)

• Ralstonia pickettii (Gram negative bacilli)

Interactive Exercise Part I: Background

http://jcm.asm.org/content/49/6/2342.full


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• Form investigation team

• List the areas/departments that will be represented as part of the

investigation team

• Immediate actions

• Risk assessment for continued production to ensure controls in place

contained issue

• Confirming scope – walkthrough with survey sampling

• Purification production hold

• Full facility sporicidal cleaning and VHP disinfection

• Equipment cleaning

• Problem statement

• “Increased frequency of low level bioburden recovery from drug

substance purification over a two month period of time, including

recovery of mold”

Interactive Exercise Part I: Step 1


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Root cause analysis strategy:

• Identify RCA tools and activities

• Comparative analysis “Is/Is Not” tool

• Fishbone diagram

• Investigational sampling of equipment and environment

• Strain typing

• Generate hypotheses based on clues from RCA



29

An atypically large quantity of equipment was returned to the facility

from offsite warehouse storage

• Storage in uncontrolled/unclassified area at warehouse

• Inadequate cleaning process for moving equipment into the manufacturing facility permitted an increased microbial challenge to the purification suites

Interactive Exercise Part I: RCA Clue #1

Equipment Transfer into Facility


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Cabinet Design

• In preparation for facility sporicidal deep clean, discoloration of floor and wall was discovered behind purification cabinetry

• The edges of the cabinetry where they met the walls and floor were sealed, generating a niche area inaccessible for routine facility cleaning

• No backing on the cabinet, which created an area behind and underneath the cabinets that was routinely exposed to the environment when drawers were opened/closed

• Small parts used on the chromatography skid stored in these drawers

Exposed Floor

and Wall

Section after

Drawer

Removal


31


Condensate on Wall in Purification Room • During the prior year facility shutdown, a freezer insulation project caused a

change in temperature between the purification and adjacent freezer room,

resulting in areas of the wall having a lower temperature

• Condensate formed on the lower part of the wall, confirmed by moisture mapping

• Cabinet from prior slide located on this wall

Temperature Difference

(~20°C)

Highest Level of Moisture (%)


32


Difficult to Clean Parts in Equipment

• Incomplete welds and rouging of internal chromatography skid

surfaces observed

• Check valves on skid difficult to clean due to design


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• Test hypotheses

• Detailed observations (e.g., borescoping)

• Hypothesis-specific investigational sampling

• Strain typing

• Rule in/out hypotheses

• Based on hypothesis testing and supporting data



34

• An atypically large quantity of equipment was returned to the

facility from offsite warehouse storage

• Storage in uncontrolled/unclassified area at warehouse

• Inadequate cleaning process for moving equipment into the manufacturing facility permitted an increased microbial challenge to the purification suites

• Investigative sampling demonstrated one genus level and one

species level match between equipment stored at the warehouse

and the growth behind the cabinet

Interactive Exercise Part I: Hypothesis Testing

Equipment Transfer into Facility


35


Cabinet Design

• In preparation for facility sporicidal deep clean, discoloration of floor and wall was discovered behind purification cabinetry and confirmed as microbial growth

• Acremonium kiliense, Microbacterium species, and Stenotrophomonas maltophilia/Stenotrophomonas maltophilia were recovered from this area

• The edges of the cabinetry where they met the walls and floor were sealed, generating a niche area inaccessible for routine facility cleaning

• No backing on the cabinet, which created an area behind and underneath the cabinets that was routinely exposed to the environment when drawers were opened/closed

• Small parts used on the chromatography skid stored in these drawers

Exposed Floor

and Wall

Section after

Drawer

Removal


36


Condensate on Wall in Purification Room

• During the prior year facility shutdown, a freezer insulation project caused a

change in temperature between the purification and adjacent freezer room,

resulting in areas of the wall having a lower temperature

• Condensate formed on the lower part of the wall, confirmed by moisture mapping

• The moisture provided an environment that was conducive to growth of microorganisms

Temperature Difference

(~20°C)

Highest Level of Moisture (%)


37


Difficult to Clean Parts in Equipment • Incomplete welds and rouging of internal chromatography skid surfaces

observed

• Check valves on skid difficult to clean due to design

• Acremonium kiliense was found in check valves and incomplete welds on skid

• Strain match of Stenotrophomonas maltophilia was found between skid outlet

and the bioburden samples

• Strain match of a different strain of Stenotrophomonas maltophilia was found

between the check valves and from behind the cabinet


38

12 Hypotheses Ruled Out: • Increased movement of material, cages and carts

• Column packing room exposure to uncontrolled spaces

• Mold spores/organisms colonizing under floor scales

• Product-specific hose aging

• Exposure of skids to WFI stored during plant shut down

• WFI float installation in WFI storage tanks during plant shut down

• Purified water system preventive maintenance and cleaning

• WFI flow orifices in purification room

• Installation of HEPA fans

• Floor drain repair during plant down

• Pipe insulation can harbor microorganisms

• Upstream material introducing microorganisms to purification

Interactive Exercise Part I: Case Study Hypotheses

• Visual inspections - facility walkthroughs and equipment borescoping

• Investigational sampling - environmental and equipment, scenario testing

• Microbial characterization/strain typing

Hypotheses Testing and Rule In/Out

3 Hypotheses Ruled In: • Equipment transfer from storage with

ineffective cleaning allowed an increased microbial load to facility

• Microorganisms behind cabinet transferred onto bags of small parts stored in cabinet by turbulent airflow from opening/closing of drawers

• Use of contaminated small parts on purification skid led to equipment contamination (biofilm formation on difficult to clean areas)


39

Interactive Exercise Part I: Case Study Root

Cause Summary

Hard to Clean Pump Heads / Check Valves

Cabinets

with Small Parts

Mobile

Equipment Warehouse PROCESS

Process

Equipment

Stenotrophomonas

Stenotrophomonas Strain Match

Strain Match

Acremonium Acremonium

Species Match

Species Match Species Match

Facility

Environment

( Typical plant flora )

Species Match

Loss of Control Maintaining Control Process “In Control”

Species / Strain Match

Between Sites

Primary Proposed

Microorganism Transfer

Secondary Proposed

Microorganism Transfer

Microorganism Growth behind the Cabinet in Purification Room

Ingress of Microorganisms from Equipment Shipment

Formation of Biofilm in Chromatography Skid


40

CONTAMINATION CONTROL

Microbial Risk and CAPA


41

• Assessment of microbial risk is the foundation for the determination

of impact to product/process/material

• Allows for evaluation of the impact that microorganisms have on the

quality and safety of the product based on data and scientific

knowledge

• Microorganisms should be evaluated on a case by case basis by

assessing various microbial hazards that may include:

• Number of microorganisms present in the sample

• Downstream endotoxin data

• Downstream bioburden data

• Secondary metabolite/toxin production

• Analytical assays that demonstrate product integrity and product purity

• Considerations such as multiple compounding issues, bracketing data,

limitations of particular process monitoring

Contamination Control:



42


Control vs Monitoring

Control

Actively managing contamination

Monitoring

Evaluating the process state of control

Cleaning/Sanitization (facility, equipment) Water monitoring

Sporicidal treatment Air monitoring

Water sanitization Surface monitoring

Physical barriers Personnel

Incoming raw material and in-process

acceptance criteria Raw material and in-process monitoring

Procedures Finished product testing

Sutton, S. (2013) “The Contamination Control Plan in Facility Validation.” Contamination

Control in Healthcare Product Manufacturing, PDA, Bethesda, MD.


43

• Actions must be complete and thorough (both big picture and specific) to be

effective


Key actions following the event

Remediation:

• Actions taken to bring impacted system(s)/area(s) back to normal condition

• E.g., disinfection, passivation

Corrective actions:

• Actions taken to fix specific failure(s) causing a contamination situation

• E.g., fix leak, replace damaged elastomer

Preventative actions:

• Actions taken to reduce risks of repeated failure specific to causes of contamination:

• E.g., implement or improve preventative maintenance program


44

• Effective immediate actions

• Interim controls to enable

production to resume

• Long term improvements for

sustainment

• Implement improvements to

bolster detection and monitoring

capabilities

• Examples of CAPAs:

• Improving disinfection and

cleaning program

• Implementing or improving in-

process bioburden/endotoxin

trending

• Effectiveness checks to ensure

CAPAs are successful


CAPA

Remediation

Preventative Actions

Effectiveness Checks

Corrective Actions


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CORRECTIVE AND PREVENTATIVE ACTIONS

Interactive Exercise Part II


46

• Review:

• Multiple compounding root causes

• Continuation of investigation plan:

• Assessment of microbial risk

• Determine appropriate remediation, corrective, and preventative actions

Interactive Exercise Part II – Corrective and


Equipment transfer

from storage/

ineffective cleaning

Organisms behind cabinet

transferred onto bags of small parts

Use of contaminated small parts on

purification skid led to

biofilm


47

• General:

• Break into same teams

• Each team will develop remediation and corrective and preventative

actions using the actual case study

• Instructors will function as all SMEs required, providing any requested

information or data

• Reference slide material as needed

• Establish CAPAs (Interactive Exercise Part II) Instructions:

1. List areas of microbial risk (equipment, materials, processes, etc.)

2. Determine appropriate remediation

3. Determine corrective and preventative actions

4. Determine appropriate effectiveness checks

Interactive Exercise Part II – Corrective and



48

• Perform remediation activities:

• Remove cabinets in purification room and repair/replace wall

• Repair incomplete welds in the chromatography skid

• Treat purification equipment with caustic and a sporicidal disinfectant

(multiple cycles) followed by passivation

Interactive Exercise Part II – Remediation

http://www.atilimkimya.com/paslanmaz-celik-asindirma-ve-pasivasyon-uygulamalari/?lang=en


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Identify CAPAs:

• Implement sporicidal disinfection of equipment and materials

entering manufacturing facility

• Improvements to equipment movement and offsite storage

• Evaluate all fixed cabinetry, replace cabinets with wheeled

cleanroom-approved cabinets, and approve all fixture purchases

• Implement procedures for facility “return to service” from work that

is potentially environmentally disruptive

• Assess check valve/pump design replacement, cleaning

improvements, and improved equipment maintenance

• Improvements to microbial control strategy

• DE testing for equipment cleaning solutions

• Supplemental bioburden challenge recovery with process isolates

• Strategy for in-process bioburden trending

Interactive Exercise Part II – CAPAs

http://www.directindustry.com/prod/bi

oclean/product-121651-

1376205.html


50

Implement Effectiveness Checks:

• Prior to resuming production:

• Additional non-routine bioburden and EM sampling

• Confirmatory cleaning process qualifications (CPQ)

Interactive Exercise Part II – Effectiveness Checks

http://microqa.com/wp-content/uploads/2014/08/MQA-Environmental-

Monitoring-Services.jpg


51

CONTAMINATION CONTROL

Preparedness


52


Creating a comprehensive preparedness strategy

Goal: Be proactive and prepared in the event another

contamination occurs

The Benefits: Significantly decrease response time during investigation

Avoid or reduce time-consuming studies

Easily accessible data and information for immediate reference

Gain familiarity with contamination support tools

Understand impact of process parameters and matrices on microbial growth kinetics and endotoxin production

Understand susceptibility of manufacturing surfaces for biofilm formation and efficacy of routine cleaning cycles and chemistry on biofilms

Data to support specific, effective, remediating actions


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• Compile data and references in a consolidated resource for

accessibility

• Literature searches, published resources

• Build database of relevant in-house data

• Past contaminations

• Evaluation/development/validation studies

• Disinfectant efficacy studies

• Properties of prior contaminants against disinfection, growth properties,

identified source

• Microbial evaluation of site matrices

• Categorize based on likelihood to promote, sustain, or inhibit microbial growth

using attributes such as pH, water activity, nutrient source

Helps identify if the contamination in question is an isolated event or

recurring event that is an indication of insufficient controls


Building your knowledge base


54

Trending of microorganisms isolated from the process provides

useful detail on:



Seasonal shifts in recovery is there an increase in isolate recovery

certain times of the year?

Effectiveness of disinfectant program is there an increase in

recovery of spore forming organisms, necessitating implementation of

a sporicide?

Comparing the type(s) of microorganism recovered: within a specific

product, between products manufactured at the same facility, the

environment and in process steps

Environmental and bioburden levels for a particular process or

atypical growth

Strain information


55

• Experimentation to collect data prospectively using

facility isolates and production matrices

• Growth kinetics

• Endotoxin production

• Endospore formation

• Spike/recovery

• Hold time studies

• Biofilm formation

• Disinfectant efficacy

• Filter challenge




56



Contamination Investigation Laboratory Studies update

Type Tool Purpose Strengths Weaknesses

Basic Lab consumables &

equipment (e.g., sterile

flasks & tubes,

incubator)

Microbial growth

kinetics, disinfectant

efficacy, biofilm

formation (certain

surfaces)

Simple &

inexpensive

Can be challenging

to replicate exact

process conditions

Special Bench Scale Bioreactor

(e.g., CelliGen®)

Microbial growth

kinetics, disinfectant

efficacy

More accurately

replicate some

process conditions

Requires expertise,

can be challenging

to maintain sterility

Biofilm Reactor Biofilm formation &

disinfection

Simple & relatively

inexpensive, wide

range of surfaces

Studies can be

lengthy

Strain level

identification (e.g.,

Riboprinter®)

Strain relatedness

between isolates

Can help pinpoint

contamination

source

Higher cost,

expertise

necessary

RMMs Rapid endotoxin,

bioburden, and ID

instruments (e.g., Scan

RDI®, MCS™/PTS™,

MALDI Biotyper)

Rapid testing of

water and in-

process samples

and rapid microbial

identification

Significant

reduction in time

to result, helps

advance

investigation

Some are relatively

expensive and

require expertise


57

• Successful contamination control is achieved through

expedient detection, thorough investigation, and effective

actions (mitigate, correct, and prevent)

• A wide range of tools can be utilized for improved

microbiological support for investigations

• Contamination responsiveness/preparedness requires

continuous improvement and a broadening knowledge

base

Summary


58

• Singer, D.C. (2012) “A Strategy for Developing Robust

Pharmaceutical Microbiological Control.” American

Pharmaceutical Review 15(4)

• Prince, R., ed. (2008) Microbiology in Pharmaceutical

Manufacturing, Vol. 1, 2nd edition, PDA

• Madsen, R.E. and Moldenhauer, J., eds. (2013) Contamination

Control in Healthcare Product Manufacturing Vol. 1, PDA

References


59

QUESTIONS?

Documents

Contamination and Beyond - cbinet.com IVT 05Jun16_FINAL...growth/gross contamination Visual turbidity, microbial cells visible on slides, odor Direct Steps with open processing Recurring