Dedicated Analytical Solutions

Near-Infrared Spectroscopy as a Process Analytical Technology

in the Biotech IndustryPresented to the

Advisory Committee for Pharmaceutical Science

Food and Drug AdministrationApril 13, 2004

ByRobert A. Mattes

Foss

Dedicated Analytical Solutions

Near-infrared Spectroscopy Spectral Region

UV VisibleNear-Infrared

Mid-Infrared

25004,000

Functional Group Analysis

Constituent AssayAppearance

Elemental

70014,285

40025,000

200 50,000

25,000 nm400cm-1

Dedicated Analytical Solutions

Pharmaceutical ManufacturingNIR Applications

Incoming Raw

Materials

Material Dispensary

In-Process: Fermentation

and Cell Culture Monitoring Warehouse

Shipping

Lyophilization

Dedicated Analytical Solutions

BioPharm Process Reactor

Control feedback

On-line monitoring of critical process

parameters

Process control

Raw MaterialsProcess output

Closed loop control (Real-Time)

TemperatureSparging

NIR monitoringNutrient levels

Control Capabilities with NIR/PAT

Moisture level

Melt detection

In Lyo

Process Improvement

Dedicated Analytical Solutions

Organism Supplier Product Fluid Behavior

Analyte (s)

Escherichia coli (unicellular)

Eli Lilly BST (bovine somatotropin)

Non-viscous Newtonian

biomass, substrates, acetate (by-product), product

Streptomyces fradiae

(filamentous)

Eli Lilly Tylosin Viscous non-Newtonian

biomass, substrates, product

Pichia pastoris (unicellular)

British Biotechnology,

PLC

Human Protein

Viscous Newtonian

biomass, substrates, product

CHO-K1 (animal cell-line)

British Biotechnology,

PLC.

Human Protein

Non-viscous Newtonian

Biomass, substrates, product

Industrial Organisms Studied NIR Bioprocess Method Development

Dedicated Analytical Solutions

NIR Spectral Acquisition and Analysis for Bioprocesses

0

1

2

3

400 700 1000 1300 1600 1900 2200 2500Wavelength (nm)

Log(

1/T)

Dedicated Analytical Solutions

NIR Spectral Analysis Analytical Region for Key analytes

0

1 6 00

0

1600 1625 1650 1675 1700 1725 1750Wavelength (nm)

Seco

nd-d

eriv

ativ

e of

Log

(1/T

)

Glycerol, acetate, biomass

t=0, t=27 hr

Dedicated Analytical Solutions

NIR Bioprocess Trends Chart

0

1

2

3

4

0 2 4 6 8 10Time (hours)

Gly

cero

l (g/

L)

0

1

2

3

4

Bio

mas

s (g

/L)

Dedicated Analytical Solutions

NIR Bioprocess Analytical ResultsAnalyte Strategy Ref Error Model SEC/SEP

Biomass (g/L) At-Line 0.7 g/L PLS 1.2/1.4

Acetate (g/L) At-Line 0.7 g/L MLR 1.2/0.7

Glycerol (g/L) At-Line 0.7 g/L MLR 1.1/0.7

Ammonium (mmol/L) At-Line 8 mmol/L PLS 13/7

Carbon Nutrient (%) In-Line 0.05% MLR 0.06/0.06

Glycerol (g/L) Lab 0.3 g/L MLR 0.3/0.3

Biomass Lab 0.2-0.4 g/L MLR 0.2/0.2

Dedicated Analytical Solutions

Near IR applied to Mammalian Cell Cultivation

Industrial CHO-K1 cell line In-situ NIR using transflectance measurements

(1.2 mm pathlength) 2 Liter bioreactor run for 168 –240 hours,

including a temperature change Developed NIR calibrations for:

Glucose Glutamine Lactate Ammonia

Ref: SA Arnold, J Crowley, N Woods, LM Harvey, and B McNeil, Biotech and Bioeng 84(1), 13-19 (2003).

Dedicated Analytical Solutions

Direct In-Line Analysis in Mammalian Cell Cultivation

Direct fermenter interface Standard Ingold ports Optimized for specific

analytes/matrices Dedicated analysis on defined

media Simultaneous results <1

minute Monitoring and closed-loop

control Pilot plant/scale-up operations

and manufacturing

Direct Sample Interface

Dedicated Analytical Solutions

Summary of NIR Results for CHO cell work

Analyte Range SEC R2 SEP

Ammonia 0.5-2.0mM

0.032 0.99 0.036

Glucose 0.7-3.5g/L

0.07 0.99 0.07

Lactate 0.3-1.2g/L

0.008 0.99 0.014

Glutamine 2-6mM 0.12 0.99 0.31

Multifactor PLS calibrations developed on second derivative spectra with 217 samples

Dedicated Analytical Solutions

Enlarged View of Second Derivative Spectra

Used for Analysis of Lyophilized Product

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

1800 1850 1900 1950 2000 2050 2100 2150 2200

Wavelength (nm)

2nd D

eriv

ativ

e Sp

ectra

   

Dedicated Analytical Solutions

NIR offers the advantage of real-time analysis in biotech sterile environments and the possibility of closed-loop feed back process control Benefits in process …

Non-invasive analysis Process optimization Waste reduction Better understanding of processes and

Safer, improved, more consistent product

NIR Benefits for the PAT Biotech Initiative