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Spectrometers are enjoying increasing popularity in bioprocess monitoring due to their non-invasiveness and in-situ sterilizability. Their on-line applicability and high measurement frequency create an interesting opportunity for process control and optimization tasks. However, building and maintaining a robust calibration model for the on-line estimation of key variables of interest (e.g., concentrations of selected metabolites) is time-consuming and costly. One of the main drawbacks of using IR spectrometers on-line is that IR spectra are compromised by both long-term drifts and short-term sudden shifts due to instrumental effects or process shifts which might be unseen during calibration. The effect of instrumental drifts can normally be reduced by referencing the measurements against a background solution, but this option is difficult to implement for single-beam instruments due to sterility issues. In this work, in order to maintain the robustness of calibration models for single-beam IR and to increase resistance to process and instrumental drifts/offsets, planned spikes of small amounts of analytes were injected periodically into the monitored medium. The corresponding measured difference spectra were scaled-up and used as reference measurements for updating the calibration model in real-time based on Dynamic Orthogonal Projection (DOP). Applying this technique lead to a noticeable decrease in the standard error of prediction of metabolite concentrations monitored during an anaerobic fermentation of the yeast Saccharomyces cerevisiae.