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Chet French
Total Protein Determination by A280
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Spectrophotometry: Use of light absorbance to measure concentration. Many biological substances (including proteins) absorb light. The spectrophometer measures absorbance of light at specific wavelengths
Absorbing substance concentration can be calculated based on absorbance at 280 nm:
A = × c × l (Beer-Lambert Law) A = absorbance = extinction coefficient*c = concentrationl = path length
*Variable for Substance of Interest
Total Protein Determination by A280
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Tryptophan TyrosineCysteine
Ultraviolet light Absorption by Proteins at 280nm wavelength is due to the presence of the ring structures on the side chains of three amino acids
UV Light Source
DetectorA280
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Bubble Point TestIntegrity Test
Conducted as a confirmation of filter specification
Formula for Bubble Point Test
Where:P = bubble point pressured = pore diameterk = shape correction factorcos θ = liquid-solid contact angle
ó = surface tension
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Cleaning Process Parameters: Action
Lower = Better
Higher = Better
Shear force (impingement) acting on the surface of equipment under the cleaning process
Lowest level of action (corresponding to worst case locations) are identified
Reynolds Number
Re = (Dvρ)/µ Where, D: pipe id
v: fluid velocity ρ: fluid density µ: fluid viscosity
Spraywand
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Column Geometry
Column geometry influences resolution
Equal SampleVolumes
Equal ResinVolumes
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Resin Particle Size
Resin Particle Size determines maximum flow rate and chromatographic resolution
Smaller Particles (Fine)Lower Flow Rates Higher Resolution
Larger Particles (Medium)Higher Flow Rates Lower Resolution
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Bioseparation Methods
UltracentrifugationAdsorptionAffinity Separation
Ultrafiltration/DiafiltrationChromatographyMembrane Chromatography
Cell disruptionPrecipitationCentrifugationLiquid-liquid extractionFiltrationDialysis
Res
olut
ion
Throughput Low High
Lo
wH
igh
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Targeting Capacity
Inadequate Capacity
Cost of Lost Sales
Estimated loss of operating profit (50% shortage): >>$10 M/monthly
Does not include other costs (reputation, competition, etc.)
Excess Capacity
Carrying Cost of Facility and Organization:
Estimated carrying cost of a facility operating at 50% capacity: <<$10 M/monthly
What’s the cost of being wrong?
Estimating the range of probable outcomes is important
Source: Mallik, A. et al, The McKinsey Quarterly, 2002 Special Edition: Risk & Resilience
Monoclonal antibodies represent the fastest growing segment of the pharmaceutical industry
Approximately 65% of all biopharmaceutical products in development are produced in mammalian cell culture
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Civil Servants - Strong belief in purpose
Specialized training (frequent and recurring):
Human Psychology
Interview Techniques
Inspection Team
1-5 Investigators with Lead – CDER Office of Compliance– Office of Biotechnology Products– CDER District Office
Mix and Match
1- 3 weeks duration
No vested interest in organizations’ success…
FDAThe Investigators & Inspection Team
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Inspection LogisticsInside the Inspection Room
Runners
Scribe
HostSMEs
Front RoomManager
Alert: Never hand anything directly to an Investigator; instead give it to the host
Scribe
Host SME
SME
FDA Investigators
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DNA & RNA RemovalBenzonase Treatment
Benzonase: A non-specific Restriction Nuclease
High Specific Activity
Converts DNA & RNA to 2-5 bp fragments
Plasmid DNA
Chromosomal DNA
• Cheap• Little Impact to Product Yield
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SDS-PAGESlab Gel
Sample Lanes
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The Rushton Turbine
The standard Rushton turbine is most commonly used in microbial fermentations
Also used in some mammalian cell culture bioreactors
Its diameter d is 1/2 - 1/3 of the inner tank diameter
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Adherent vs. Suspension Cells
Adherent cells: Cells must grow while attached to a
substrate/matrix
Cell growth is limited by available surface area
Can use microcarriers in suspension
Suspension cells Cells are free-floating in culture
Cell growth is limited by nutrient availability and waste product accumulation
Microbial and mammalian cells are maintained in suspension
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Validation Cycle Development – Types of Biological Indicators
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Spore Suspensions
Pure spore suspension of the desired challenge organism which can be inoculated onto the surface of a material
Spore Dots
Circular pieces of fibrous paper impregnated with the spore suspension
Spore Strips
Narrow strips of fibrous paper impregnated with a bacterial spore suspension contained in a glassine envelope
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Compact Plan Production Floor Layout
Personnel Flow
Access Corridor
Materials Flow
Production Module
Gray Space
Production Module
Gray Space Access
Gray Space Access
In Out
InOut
Schering-PloughProventil® Asthma Inhaler Timeline
1998 1999 2000 20011998 1999 2000 2001
… … Q3 Q4Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 … Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 …
PuertoRicoIRE
KenilworthNJ
PuertoRico Kenilworth
NJ
FDA
FDA
AACFDA
190KUnits
60MMUnits
Biotech Industry Overview
Source: PhRMA – 324 Biotechnology Medicines in Testing Promise to Bolster the Arsenal Against Disease – Oct 2004
In Development: 324 New Medicines & Vaccines 150 Disease Indications
Currently: 18 products over $1 Billion annually 10 products over $2 Billion!
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Human B-CellAntibody Producing
Human Macrophage
Human T-Cell
Understanding Nature: Finding a TargetExample 2: Humoral Immune Response
Antibodies
Bacteria
Variable Region
Constant Region
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Special Challenges Size Considerations for Processing – Virus
Removal
Proteins
HSA
MAbFVIII
Hb
FIX
Monoclonal Antibodies
Interleukins
EPO
tPA
Viruses
ParvoPolio, EMC
SV40BVDV
HIVMuLV
Nanometers1 10 1000100
MW (KD)10 100 1,000 10,000
Biopharmaceutical macromolecules are not much smaller than viral particles!
Core Technology
Hybridoma (Cloning)
Proteomics
Recombinant DNA
Biosimilars
Genomics
Target MarketHuman Therapeutics
Human Diagnostics
Vaccines
Veterinary
Agriculture
Industrial
Most biotechnology companies focus on pharmaceutical discovery; not commercialization: Low volume, high value Relatively low plant, property and equipment requirements
Target Indication(s)
Cancer
Neurological Disorders
Cardiovascular Disorders
Autoimmune Disorders
Diabetes
Infectious Disease
Constraints:MoneyTechnologyMarket Factors
Where to Start?