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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

4

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

55

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

7

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?