QUALITY CONTROL MICROBIOLOGY

Maggie Bryans Sheila Byrne John Hasyn

BIOMAN 2011 MiraCosta CollegeOceanview, California

The degree to which a set of inherent properties of a product, system or process fulfills requirements. FDA Guidance for Industry Q9 Quality Risk Management

Quality

Quality Control (QC):• Testing performed during biopharmaceutical

manufacturing to verify that appropriate standards of product quality are attained

• The actual organizational group (QC unit) who execute this work

Biopharm QC Testing Scheme

• Figure 7-2 P-257

“ Quality should be built into the product and testing alone cannot be relied on to ensure product quality.”

Testing Performed by QC Microbiology

• Environmental monitoring• Utility testing• Sterility testing• Microbial content testing

– Bioburden– Microbial Limit

• Bacterial Endotoxin (LAL)• Microbial identification• Antimicrobial Effectiveness Testing• Cleaning validation• Media fills

Environmental monitoring includes…..

1) Water and clean steam monitoring,2) Air monitoring- non-viable3) Air monitoring – viable4) Microbial surface testing using RODAC plates,5) Gown and fingertip RODAC testing.

Air Monitoring

Particulate (Non Viable) Air Monitoring

Cross sectionof a hair

How large is a human hair?How large is a particulate?

Guidelines for Clean Rooms

Federal Standard 2091963

First comprehensive guideline to clean room classification. English units.

FS 209 E1992

Fifth revision added metric or SI units

FS 209 Class 1 to 6

ISO 14644-1

ISO 14644-2

2001

International Society for Standardization

ISO Class 1 to 9

Classification of Clean Rooms - Federal Standard 209

≥ 0.1µm

Particles/ft3≥ 0.2µm

Particles/ft3≥ 0.3µm

Particles/ft3≥ 0.5µm

Particles/ft3≥ 5.0µm

Particles/ft3

Class

1 35 7.5

3 1

Class

10 350 75

30 10

Class

100

750

300 100

Class

1000

1,000 7

Class

10,000

10,000 70

Class

100,000

100,000 700

Selected Equivalent Classes

FS 209 Classes

Class

1

Class

10

Class

100

Class

1,000

Class

10,000

Class

100,000

ISO 14644-1 Classes

Class

3

Class

4

Class

5

Class

6

Class

7

Class

8

ISO 14644-1 Class is equivalent to FS 209 Class above it.

Particle Detection

• The validation of a clean room is ongoing

• The air quality of a clean room must be monitored

• An optical particle counter is used to monitor air quality– “Real-time” test results

Types of Particle Counters

Portable Particle Counter

Facilities Maintenance System

Microbial Air Monitoring

• Passive - Settle plates are exposed for specified time period.

• Active - Electric pump draws preset sample volume of air onto nutrient media plate.

Pharmaceutical Applications

• Trend analysis of aseptic filling areas

• Determine microbiological quality of laminar flow hood air

• Assess decontamination procedures

Inspection of Agar Plate and Count

• Total microbial count– Bacteria– Mold

• The colonies are counted and reported as colony forming units (CFU) per cubic meter of air

FDA Guidance For Aseptic Processing

FS 209CLASS

ISOCLASS

>0.5 PARTICLES/m3

ACTION LEVELS

cfu/m3

100 5 3520 1

1000 6 35200 7

10,000 7 352000 10

100,000 8 3520000 100

RODAC Plate

0

20

40

60

80

100

120

140

Test#1

Test#2

Test#3

Test#4

Test#5

Test#6

Test#7

Action Level: a test result that is ___?__ .

Alert Level: indicates ___?__.

Passing Level: are __ ? __ results.

Acceptable / Action / Alert - LevelsEnvironmental Monitoring Testing Results

Alert Level

Action Level

Knowledge Management

ICH Q10

Microbial Identification

What Do We Identify?

- Bacteria- Yeast- Mold

What Is An Identification?

Determination of the genus and species, e.g. Escherichia coli

When Do We Identify?

• When the # of microorganisms exceeds an acceptable level– Class 100– Class 10,000

• When a microorganism is recovered from a presence/absence test

Identification Methods / Systems (Phenotypic Methods)

• Bacteria• Conventional Method• Standardized Identification Systems• Automated Identification Systems

Conventional Method

• Colony morphology and Gram stain• Series of biochemical tests • Read reactions • Refer to Bergey’s Manual

Colony Morphology

Size, shape, texture, and color

Biochemical Tests

• Fermentation of carbohydrates

• Production of catalase• Production of indole • Production of hydrogen

sulfide gas

Limitations of Conventional Method

• Time consuming / labor intensive• Dependent on the bacteria’s ability to

use the biochemicals• Requires a high level of technical

knowledge

Standardized Identification Systems

• API Strips®• Enterotube®

Miniaturized biochemical tests

API Strips®

API Strips® - Method

• Gram stain• Prepare a suspension of the bacteria• Inoculate with the suspension• Incubate strip• Read the pattern of reactions (color

changes)• Refer to index

API® Strips

Benefits• Convenient• Easy to use• Low cost per ID

($6)

Limitations• Small database• Subjective• Dependent on the

bacteria’s ability to use the biochemicals

Automated Identification Systems

• Vitek®• Biolog®

• Molecular Microbiology (genotypic methods) is the wave of the future.

• No single method or system is ideal for all identifications

Genotypic Methods

Endotoxin Testing

What is it? A lipopolysaccharide

Where does it come from? The outer membrane of Gram negative bacteria.

Endotoxin

Which products are tested?

• injectable drugs and medical devices which will contact blood or spinal fluid

• includes raw materials, water and in process monitoring

Endotoxin Testing

The USP now recognizes two tests –

• The Pyrogen Test conducted with rabbits

• Bacterial Endotoxins Test, also termed the Limulus Amebocyte Lysate (LAL) Test.

Pyrogen Assay

• USP XIX considers a solution to be pyrogenic when 10 ml/kg is injected into a rabbit and there is a rise of temperature of 0.6 C or more for any rabbit, or a total rise of more than 1.4 C for three rabbits in a three rabbit test group.

LAL Test

• Limulus amebocyte lysate test - based on clotting reaction of horseshoe crab (Limulus polyphemus) blood cell (amebocyte) lysate to endotoxin

• Developed in 1960’s by Drs. Bang and Levin

• Faster, more economical, more sensitive than rabbit pyrogen test

Types of LAL Tests

• Gel Clot

• Turbidimetric

• Colorimetric

Gel Clot Method

• Original method

• The official “referee test”

• The specimen is incubated with LAL of a known sensitivity. Formation of a gel clot is positive for endotoxin.

Chromogenic Method

• Endotoxin concentration is measured as a function of color intensity

• LAL contains enzymes that are activated in a series of reactions in the presence of endotoxin. The last enzyme activated in the cascade splits the chromophore, para-nitro aniline (pNA), from the chromogenic substrate, producing a yellow color.

Turbidimetric Method

• In the presence of endotoxin LAL becomes turbid and, under appropriate conditions,

forms a solid gel-clot.

• In the kinetic turbidimetric LAL method, endotoxin concentration is measured as a function of either the rate of increase in turbidity or the time taken to reach a particular level of turbidity.

Comparison of Methods

Gel Clot ChromogenicEndpoint

ChromogenicKinetic

Turbidimetric

Semi-quantitative

Quantitative Quantitative Quantitative

Simple, Least expensive,Requires 37°C bath

Requiresspectrophotometeror plate reader

Requiresincubating plate or tube reader

Requiresincubating plate or tube reader

Manually read and recorded

Manual or can be automated,allows electronicdata storage

Is automated,allows electronicdata storage

Is automated,allows electronicdata storage

Sensitive downto 0.03 EU/ml

Sensitive down to 0.1 EU/ml

Sensitive down to .005 EU/ml

Sensitive down to .001 EU/ml *

* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)