# Presentation: 796206

## Near-Infrared Spectroscopy as a Process Analytical Technologyin the Biotech Industry

- Presented to the
- Advisory Committee for Pharmaceutical Science
- Food and Drug Administration
- April 13, 2004
- By
- Robert A. Mattes
- Foss

## Near-infrared Spectroscopy Spectral Region

**UV**

**Visible**

***Near-Infrared***

**Mid-Infrared**

**2500**

**4,000**

**Functional Group Analysis**

**Constituent Assay**

**Appearance**

**Elemental**

**700**

**14,285**

**400**

**25,000**

**200 **

**50,000**

**25,000 nm**

**400cm-1**

## Pharmaceutical ManufacturingNIR Applications

- Incoming Raw Materials

- Material Dispensary

- In-Process: Fermentation and Cell Culture Monitoring

- Warehouse Shipping

- Lyophilization

## Control Capabilities with NIR/PAT

- BioPharm Process Reactor

- _Control feedback_
- On-line monitoring of ***critical process*** parameters

- Process control

- Raw Materials

- Process output

- Closed loop control (Real-Time)

**Temperature**
**Sparging**
**NIR monitoring**
**Nutrient levels **

**Control Capabilities with NIR/PAT**

- Moisture level
- Melt detection
- In Lyo

- Process Improvement

## Industrial Organisms Studied NIR Bioprocess Method Development

| 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

## NIR Spectral Acquisition and Analysis for Bioprocesses

## NIR Spectral Analysis Analytical Region for Key analytes

**Second-derivative of Log(1/T)**

- Glycerol, acetate, biomass

- t=0, t=27 hr

##  NIR Bioprocess Trends Chart 

## NIR Bioprocess Analytical Results

## 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).

## 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**

## 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

## Enlarged View of Second Derivative Spectra Used for Analysis of Lyophilized Product

## 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

- 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**