QUALITY CONTROL IN BIOMANUFACTURING

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QUALITY CONTROL IN BIOMANUFACTURING

• Kevin Lampe, Sheila Byrne, Laura Roselli, Melanie
  Lenahan and Linda Rehfuss
• BIOMAN 2007, Portsmouth, NH

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Quality The degree to which a set of inherent
properties of a product, system, or process
fulfillsrequirements.
FDA Guidance for Industry Q9
Quality Risk Management
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The quality of drug substances and
products is determined by their

• design
• development
• in-process controls
• GMP controls
• process validation
• specifications applied to them throughout
  development and manufacture.
• (ICH Q6A Guideline)

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A specification is defined as a list of tests,
references to analytical procedures
and appropriate acceptance criteria which are
numerical limits, ranges or other criteria for
the tests described. Specifications are binding
quality standards that are agreed to between the
appropriate governmental regulatory agency and
the applicant. Specifications are chosen to
confirm the quality of the drug substance and
drug product and should focus on those
characteristics found to be useful in ensuring
the safety and efficacy of the drug substance
and drug product.
(ICH Q6A Guideline)
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US CFR United States Code of Federal
Regulations- defines the quality units job
Quality Control Function

• The QC Function may be assigned between a quality
  control and quality assurance group and may be
  integrated into a more comprehensive quality
  system.
• Every manufacturer should establish a written
  plan that describes the role of and
  responsibilities for QC functions.

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QC Functions

• Responsibility for examining the various
  materials used in the manufacture of a drug
  (e.g., raw materials, in-process materials,
  containers, closures, packaging materials, and
  labeling) to ensure that they are appropriate and
  meet defined, relevant quality standards of
  identity, strength, and purity
•   Responsibility for review and approval of
  manufacturing procedures, testing procedures, and
  acceptance criteria
•   Responsibility for releasing or rejecting each
  batch of drug based on a cumulative review of
  completed manufacturing records and other
  relevant information (e.g., procedures were
  followed, product tests performed appropriately,
  acceptance criteria met)
• Responsibility for investigating unexpected
  results or errors that occur during manufacturing
  or complaints received and initiation of
  corrective action, if appropriate

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A significant portion of the CGMP regulations (21
CFR 211) pertain to the quality control
laboratory and product testing.

• SOPs should be complete and adequate and the
  operations of the laboratories should conform to
  the written procedures.
• Laboratory equipment usage, maintenance,
  calibration logs, repair records, and maintenance
  SOPs must be kept.

• If a compendial test method exists, but the firm
  chooses to use an alternate method instead, they
  must compare the two and demonstrate that the
  in-house method is equivalent or superior to the
  compendial method.

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211.194 Laboratory records. shall include...

• (1) A description of the sample with
  identification of source
• (2) A statement of method used in the testing
• (3) A statement of the weight or measure of
  sample
• (4) A complete record of all data secured in the
  course of each test, including all graphs,
  charts, and spectra
• (5) A record of all calculations performed
• (6) A statement of the results of tests and how
  the results compare with established standards
• (7) The initials or signature of the person who
  performs each test and the date(s) the tests were
  performed.
• (8) The initials or signature of a second person
  showing that the original records have been
  reviewed

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QC Microbiology

• Endotoxin Testing
• Air Monitoring
• Microbial Identification
• Mycoplasma Detection

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Endotoxin Testing
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Code of Federal Regulations Title 21, Volume
4 Revised as of April 1, 2008 CITE
21CFR211.167

• For each batch of drug product purporting to be
  sterile and/or pyrogen-free, there shall be
  appropriate laboratory testing to determine
  conformance to such requirements.

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Pyrogen

• a fever producing agent of bacterial origin
  endotoxin.
• Dorland's Illustrated Medical Dictionary. 25th
  E.W.B. Saunders, Philadelphia.

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Endotoxin What is it? A
lipopolysaccharide Where does it come from?
The outer membrane of Gram negative bacteria.
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The term "endotoxin" is usually interchangeable
with the term "pyrogen," although not all
pyrogens are endotoxins and pyrogen testing alone
cannot be used entirely for detection and
characterization of microbial endotoxins. The
release of lipopolysaccaride from bacteria takes
place after death and lysis of the cell.
Bactericidal procedures such as heating,
filtration, or adsorption techniques do not
eliminate pyrogens from parenteral solutions.
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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

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The USP now recognizes two tests

• The Pyrogen Test conducted with rabbits
• Bacterial Endotoxins Test, also termed the
  Limulus Amebocyte Lysate (LAL) Test.

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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..

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LAL Test

• Limulus amebocyte lysate test - based on clotting
  reaction of horseshoe crab (Limulus polyphemus)
  blood cell (amebocyte) lysate to endotoxin
• Developed in 1960s by Drs. Bang and Levin
• Faster, more economical, more sensitive than
  rabbit pyrogen test

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Types of LAL Test

• Gel Clot
• Turbidimetric
• Colorimetric

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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.

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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.

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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.

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Comparison of Methods
Gel Clot Chromogenic Endpoint Chromogenic Kinetic Turbidimetric
Semi- quantitative Quantitative Quantitative Quantitative
Simple, Least expensive, Requires 37C bath Requires spectrophotometer or plate reader Requires incubating plate or tube reader Requires incubating plate or tube reader
Manually read and recorded Manual or can be automated, allows electronic data storage Is automated, allows electronic data storage Is automated, allows electronic data storage
Sensitive down to 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) (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions) (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions) (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)
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Air Monitoring

• Particle Counting
• S. R. Byrne
• Montgomery
  County Community College

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Clean Roomsare separate environments designed
to keep particle contamination at known
controlled levels.

• Pharmaceutical Manufacture
• Microchip Manufacture

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Clean Rooms

• Types of possible particles
• Inert
• Viable
• Controlled by
• Filters and laminar flow
• Gowning - People are prolific
  particle generators.

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Guidelines on Clean Rooms
Federal Standard 209 1963 First comprehensive guideline to clean room classification. English units.
FS 209 E 1992 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
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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
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Classification of Clean Rooms ISO 14644-1
CLASS Number of Particles per Cubic Meter by Micrometer Size Number of Particles per Cubic Meter by Micrometer Size Number of Particles per Cubic Meter by Micrometer Size Number of Particles per Cubic Meter by Micrometer Size Number of Particles per Cubic Meter by Micrometer Size 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
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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.
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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.

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Types of Particle Counters

• Portable Particle Counter

• Facilities Maintenance System

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Air Monitoring

• Microbiological Air Testing
• 
  S. R. Byrne
• 
  Montgomery County Community College

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Pharmaceutical Applications

• Trend analysis of aseptic filling areas
• Determine microbiological quality of laminar flow
  hoods
• Assess decontamination procedures

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Sample Collection Methods

• Passive - Settle plates are exposed for gt 1 hour.
• Active - Electric pump draws preset sample volume
  of air onto nutrient media plate.

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• 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.

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FDA Guidance For Aseptic Processing
FS 209 CLASS ISO CLASS gt0.5 PARTICLES/m3 ACTION LEVELS cfu/m3
100 5 3520 1
1000 6 35200 7
10,000 7 352000 10
100,000 8 3520000 100
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Microbial Identification

• What Do We Identify?
• - Bacteria
• - Yeast
• - Mold

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What Is An Identification?

• Determination of the species, e.g. Escherichia
  coli

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Where Do They Come From?

• Products
• Raw Materials / Water
• Manufacturing Environment
• Manufacturing Personnel

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When Do We Identify?

• When the of microorganisms exceeds an
  acceptable level
• When a microorganism is recovered from a
  presence/absence test

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Identification Methods / Systems

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

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Conventional Method

• Colony morphology and Gram stain
• Series of biochemical tests
• Read reactions
• Refer to Bergeys Manual

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Colony Morphology

• Size, shape, texture, and color

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Gram Stain

• Gram stain reaction
• Size and shape of the cells

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Biochemical Tests

• Fermentation of carbohydrates
• Production of catalase
• Production of indole
• Production of hydrogen sulfide gas

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Limitations of Conventional Method

• Time consuming / labor intensive
• Dependent on the bacterias ability to use the
  biochemicals
• Requires a high level of technical knowledge

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Standardized Identification Systems

• API Strips
• Enterotube

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API Strips

• Miniaturized biochemical tests

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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

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API Strips

• Limitations
• Small database
• Subjective
• Dependent on the bacterias ability to use the
  biochemicals

• Benefits
• Convenient
• Easy to use
• Low cost per ID (6)

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Automated Identification Systems

• Vitek
• Biolog

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• Molecular Microbiology is the wave of the future.
• No single method or system is ideal for all
  identifications

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Mycoplasma Detection

• Laura Anne Roselli
• Burlington County College
• Mt Laurel, NJ

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Mycoplasma Detection

• Laura Anne Roselli
• Burlington County College
• Mt Laurel, NJ

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Cell Culture Contaminants

• Bacteria
• Mycoplasma
• Fungal spores
• Yeast
• Viruses
• Other Cells (Hela)
• Anything not wanted

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Mycoplasma

• Genus of bacteria (Not the same as mycobacterium
  )
• smallest free-living organisms
• 0.2-2 µm in diameter
• restricted in metabolic potential
• do not overgrow the cell lines
• lack a cell wall
• attached to the external surface of host

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Effects of Mycoplasma Contamination

• diminished cell growth
• loss of cultures
• slow growth rate
• lowers saturation density
• agglutination of cells
• alters metabolism

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Prevalance

• 5 - 35 of cell cultures in current use are
  infected with mycoplasma
• We are experts on mycoplasma, not because we
  like them so much, but because about 25-30 of
  the cultures submitted to our cell lines bank
  were infected.
• Hans Drexler, head of the Human and Animal Cell
  Line Department at the German Collection of
  Microorganisms and Cell Cultures

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Of Myco Infections in Cell Cultures...

• 95 are
• Mycoplasma hyorhinis (porcine)
• Mycoplasma arginini (bovine)
• Mycoplasma fermentans (human)
• Mycoplasma orale (human)
• Acholeplasma laidlawii (bovine)

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Where does it come from?

• other contaminated cell cultures
• lab personnel
• primary cultures
• medium components

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How to control?

• Quarantine Lines
• Test
• One Line - One Media Bottle
• One Cell Line in Hood at Time
• Handwashing
• Contaminated? Toss It
• No Cross Use of Pipettes

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Monitoring for Contamination

• Macroscopically
• Microscopically
• Culturing
• DNA Staining
• ELISA/Enzymes
• Electron Microscopy
• PCR

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Microbial Culturing
3-4 weeks
Limit of detection 100 CFU in 10ml ltgt 10
CFU/ml
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Hoechst stain
Fluorescent Stain of DNA
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MycoAlert Mycoplasma Detection Assay

• Mycoplasmal enzymes to react with a substrate
  catalyzing the conversion of ADP to ATP.
• Measurement of ATP before and after the reaction
  indicates whether mycoplasma is present.
• lt 20 minutes

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RD Systems MycoProbe Mycoplasma Detection Kit.

• Oligonucleotide hybridization techniques
• Labeled oligonucleotide probes that hybridize to
  the 16S ribosomal RNA.
• A probe labeled with alkaline phosphatase
• Color change
• 4.5 hours

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MP Biomedicals ImmuMark MycoTest

• Staining with fluorescent antibodies
• The monoclonal antibody used in the test, CCM-2,
  is specific for the mycoplasma species that
  account for over 96 of culture contamination.

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Pros and Cons
http//www.texcell.fr/Zurich_May_06.pps646,15,
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Takara Bio Mycoplasma Detection Kit Principle

• Detection procedure utilizing PCR process to
  amplify this spacer region using two primers (F1
  and R1) on the DNA encoding rRNA of 16S and 23S

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PCR

• Polymerase chain reaction
• Kary Mullis/Cetus
• 1985
• Cetus sold technology
• to Hoffman-LaRoche
• for 300 million dollars
• Perkin Elmer
• first Thermal Cycler

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Polymerase Chain ReactionWhat Do We Need?

• original piece of DNA (template)
• primer (start second strand)
• 4 nucleotides
• DNA polymerase
• Taq (Thermus aquaticus)
• isolated from bacterium
• thermostable

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DENATURATION 93C - 95C
ANNEALING 37C - 65C
25-35 CYCLES
EXTENSION 72C
DENATURATION 93C - 95C
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PCR step 1 Denaturation
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PCR step 2 - Annealing
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PCR step 3 - Extension
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Gel Electrophoresis
Method of physically separating DNA protein
RNA based on electric charge and size AND
physically visualizing it.
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Gel Electrophoresis
1. DNA is negatively charged 2. Agarose
gelatinous (polysaccharide from seaweed) 3.
Sample/Loading buffer glycerol Tris Acetate
EDTA Dyes Bromophenol Blue Xylene Cyanol 4.
Running buffers TAE (faster) TBE
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Gel Agarose Combs Wells Lanes Electrophoresis
(buffer) Chamber Gel Casting Gates (Dams) Gel
Caster Bands Ladder (MW markers)
www.bio-rad.com
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Lane MpHY Marker 1M. hyopneumoniae 2M.
neurolyticum 3M. fermentans 4M. pulmonis 5M.
hyorhinis 6M. orale 7M. capricolum 8M.
arthritidis 9M. salivarium 10M. hominis 11M.
arginini 12U. urealyticum 13human DNA 14mouse
DNA
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• www.biomanufacturing.org
• QUESTIONS ??

Minuteman Regional High School