Nuwiq® is a recombinant Factor VIII (FVIII) product used to treat Hemophilia A, a recessive X-linked disorder characterized by severe FVIII deficiency, primarily affecting men. The product is sold as a lyophilized powder and undergoes extensive quality control before release, including determination of water content. Currently, this is done using the coulometric Karl-Fischer (KF) titration method, a highly accurate and well-established method. However, KF titration requires hazardous chemicals, is time-consuming, labor-intensive, and is sample-destructive.
An alternative method for water content determination could be Near Infrared (NIR) spectroscopy, a non-destructive, simple and faster method. The goal of this project was to evaluate whether NIR spectroscopy could replace KF titration for water content determination in Nuwiq®. To do this, 30 different batches of Nuwiq® were spiked with varying amounts of moisture in a humidity chamber. Spectra of the spiked samples were collected using a FT-NIR Antaris II in diffuse reflectance mode and the Result Operation software. KF titration was then performed on the sample samples to obtain reference values.
The collected spectra were used to build a calibration model in TQ Analyst, using Partial Least Squares (PLS) regression and various chemometric techniques to reduce noise, smooth the data and optimize results. The KF reference values were paired with their corresponding NIR spectra to create the calibration model. The model’s performance was evaluated by analyzing 12 different Nuwiq® batches not included in the calibration set.
The final calibration model revealed strong predictive ability, with a correlation coefficient of 0.99899, a Root Mean Square Error of Calibration (RMSEC) of 0.0362, and a Standard Error of Prediction (SEP) of 0.0346 – suggesting that the model was not overfitted predicted the reference values with high precision. However, in some cases, the model did not predict the water content accurately, indicating a need for further evaluation.
In conclusion, the NIR calibration model shows potential as replacement for KF titration in routine analysis of water content determination in Nuwiq®. However, the calibration sample set should be expanded to 50-100 to improve robustness, accuracy and precision.