QUALITY CONTROL IN BIOMANUFACTURING

Kevin Lampe, Sheila Byrne, Laura Roselli, Melanie Lenahan and Linda

RehfussBIOMAN 2007, Portsmouth, NH

Quality Control Toolbox• Endotoxin Testing

• Air Monitoring

• Microbial Identification

• Pipette Calibration Check

Quality

Ensure purity- # 1 goalAlso identity and strength

Quality Assurance

• A unit that is part of the whole

• US CFR – United States Code of Federal Regulations- defines the quality unit’s job

• Approve or reject all materials associated with production of any product – includes containers, components, labels, other packaging materials

Quality Control (QC) within QA

• QC is a testing function

• Defined as fitness for use

• Need to be integrated throughout process

Endotoxin Testing

EndotoxinWhat is it? a lipopolysaccharide Where does it come from? the cell membrane of Gram negative bacteria

Endotoxin Testing

Which products are tested?

• injectable drugs and medical devices

which will contact blood or spinal fluid

• includes raw materials, water and

in process monitoring

LAL Test

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

• Based on clotting reaction of horseshoe crab blood to endotoxin

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

Types of LAL Test

• 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.

Comparison of MethodsGel Clot Chromogenic

EndpointChromogenicKinetic

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

Can be automated,allows electronicdata storage

Can be automated,allows electronicdata storage

Can be 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)

Air Monitoring

Particle Counting

Clean Roomsare separate environments designed to keep particle

contamination at known controlled levels.

• Pharmaceutical Manufacture

• Microchip Manufacture

Clean rooms

Types of possible particles

Inert

Viable

Controlled by

Filters and laminar flow

Gowning - “People are prolific particle

generators.”

Guidelines on Clean RoomsFederal Standard 209 First comprehensive

guideline to clean room classification. English units. 1963

FS 209 E Fifth revision added metric or SI units

FS 209 Class 1 to 6

1992

ISO 14644-1

ISO 14644-2

International Society for Standardization

ISO Class 1 to 9

2001

Classification of Clean RoomsFederal 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

CLASS Number of Particles per Cubic Meter by Micrometer Size

  0.1 µm 0.2 µm 0.3 µm 0.5 µm 1 µm 5 µm

ISO 1 10 2        

ISO 2 100 24 10 4    

ISO 3 1,000 237 102 35 8  

ISO 4 10,000 2,370 1,020 352 83  

ISO 5 100,000 23,700 10,200 3,520 832 29

ISO 6 1,000,000 237,000 102,000 35,200 8,320 293

ISO 7       352,000 83,200 2,930

ISO 8       3,520,000 832,000 29,300

ISO 9       35,200,000 8,320,000 293,000

Classification of Clean RoomsISO 14644-1

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.

Portable Particle Counter

Air Monitoring

Microbiological Air Testing

Pharmaceutical Applications

Trend analysis of aseptic filling areas

Determine microbiological quality of laminar flow hoods

Assess decontamination procedures

Sample Collection Methods

Passive - Settle plates are exposed for > 1 hour.

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

After samples are collected on nutrient media, the plates are incubated at 30-35 degrees C. for 48 hrs. to promote growth of bacteria, yeast and mold.

The plate colonies are counted and reported as colony forming units 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

Microbial Identification

What Do We Identify?

Bacteria

Yeast

Mold

What Is An Identification?

Determination of the species - Escherichia coli

Where Do They Come From?

• Products• Raw Materials /

Water• Manufacturing

Environment• Manufacturing

Personnel

When Do We Identify?

• When the # of microorganisms exceeds an acceptable level

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

Identification Methods / Systems

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

Gram Stain

• Gram stain reaction

• Size and shape of the cells

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®

API Strips®

Miniaturized biochemical tests

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®

Pipette Calibration

Why check pipettes?_________________________________________Every scientist and laboratorian understandsthe impact of unnecessary inaccuracy andimprecision on scientific data yet pipettes and pipetting technique tend to be the least controlled process in the laboratory.

Pipettes and GMP Compliance

Regulatory Guidelines 21 Code of Federal Regulations Parts 210 and 211

The calibration of instruments... at suitable intervals in accordance with an established written program containing specific directions, schedules, limits for accuracy/precision, and provisions for remedial action in the event accuracy and/or precision limits are not met. Instruments... not meeting established specifications shall not be used.2

The ARTEL PCS

In the case of the PCS Pipette Calibration System, a photometer with extraordinarily low noise is coupled with NIST-traceable reagents to measure liquid delivery colorimetrically.

• Molecular Microbiology is the wave of the future.

• No single method or system is ideal for all identifications.

www.biomanufacturing.org

QUESTIONS ??Minuteman Regional High School