dedicated analytical solutions near-infrared spectroscopy as a process analytical technology in the...
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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
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