Demonstration of a compact deep UV Raman spatial heterodyne spectrometer
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Demonstration of a compact deep UV Raman spatial heterodyne spectrometer Michael Foster,1 William Brooks1, Philipp Jahn2 1 IS-Instruments Ltd, Pipers Business Centre, 220 Vale Road, Tonbridge, Kent, TN9 1SP TOPTICA Photonics AG, Lochhamer Schlag 19, 82166 Graefelfing (Munich), Germany

ABSTRACT As Raman technology has advanced more challenging applications have been explored, where high fluorescence and low concentration of samples are present. Monoclonal antibodies and antibody fragments are increasingly important classes of biotherapeutics. However, these products are both challenging and expensive to manufacture. New process analytical tools (PAT) used to monitor these products during their manufacture are of significant interest. Deep UV Raman spectroscopy promises to provide the required specificity and accuracy, however instruments, have historically been large and...

and Sotrovimab treatment for Covid-19. Despite these successes, antibody production is still burdened with challenges that impact both time and cost of development. Monoclonal antibodies and antibody fragments are expensive and any system that can provide data to improve their production is of interest. Antibody fragments (e.g., Fab, scFv, Dab, etc.) are an increasingly important class of protein-based biotherapeutics. Due to their structure and smaller size, antibody fragments possess advantageous properties (e.g., easier tissue penetration) that suit a range of diagnostic and therapeutic...

to maintain a compact form, whilst yielding excellent stability and performance characteristics. The SHS allows for a large optical throughput resulting in a comparably large laser spot at the target thereby mitigating against power density-induced sample damage. The instrument has been demonstrated to successfully capture the Raman spectra of a range of biochemical samples, including immunoglobulin (IgG) at varying concentrations, tryptophan, and a series of domain antibody (dAb) samples. The dAb samples were extracted at different stages of a pilot bioreactor manufacturing process and at...

achieved by combining the SHG process with an optical enhancement cavity, which allows for a conversion efficiency of more than 13 % and a maximum UV power of 20 mW. Optical components used in the UV stage of the laser, especially the non-linear crystal, are prone to UV induced degradation, therefore the full UV compartment is hermetically sealed to ensure stable operation in target deployment environments. To further extend the lifetime, the UV compartment is equipped with an optics shifter, which can move the nonlinear crystal (without manual alignment) inside the cavity and provides...

A dynamic positioning stage on which the sample is sited is also shown in Fig2 (below sample stage). This facilitates a scanning routine throughout the duration of the Raman measurement ensuring the samples were not damaged by prolonged laser exposure, a function presented and discussed in detail in the Results and Discussion section. 2.3 Spectrometer The spectrometer has a spatial heterodyne configuration as shown in Fig3. Fig 3. Spatial heterodyne spectrometer design as used by the UVRRS instrument The design lends itself well to operation in the deep UV, where resolution, power density...

The spectrometer was calibrated using cyclohexane which exhibits a series of well-known discreet peaks from 801 cm-1 to 1444 cm-1. 3. Results and Discussion The spectrometer was calibrated using cyclohexane which exhibits a series of discreet peaks from 801 cm-1 to 1444 cm-1 as shown in Fig 4. Cyclohexane spectra were routinely acquired to confirm instrument stability was maintained. Fig 4. Raman spectrum of cyclohexane captured in 1s 3.1 IgG and Tryptophan Whilst operating in the deep UV, target samples can experience very high radiant energies. This can result in degraded spectra being...

Fig 5. Raman spectra of IgG (30 second integration time, average of 10 frames). Solid black line: IgG is measured in static configuration; Blue line: IgG measured with sample rotating during the observation; Red line: IgG measured with a complex motion applied using a rotation and linear stage In the static configuration, the spectra indicate a considerable amount of damage has occurred to the sample, with no clear peaks being present between 700 cm-1 and 1500 cm-1. Using a rotation stage improves the quality of the data obtained with peaks now becoming prominent in this region however they...

Fig 6. Raman spectra of IgG at a range of concentrations as labelled (all spectra presented are the averaged output of 10x 30 second frames); Solid black line = 0.1 mg/ml, dotted black line = 0.4 mg/ ml, blue line = 0.8117 mg/ml, dotted red line = 2.0173 mg/ml All spectra are the product of 10 averaged frames, each captured in a 30s integration time. The spectra illustrate that as the IgG concentration reduces towards 0.1 mg/ml the water spectrum becomes more prominent. Extrapolation indicates a detection limit of IgG in water of 0.08 mg/ml. To determine the potential of the instrument for...

Fig 7. IgG PCR-calculated vs. actual sample concentration The error bars are computed from the standard deviation of the individual frames with accuracy levels varying from 0.03 mg/ml for the low concentration sample to 0.1 mg/ml for the higher concentration samples. The data shows an excellent linear relationship with a regression coefficient of 0.99. To further assess instrument utility, the common amino acid, tryptophan was examined. The Raman spectrum of this sample was acquired by averaging 10 frames, each captured in a 30 second integration time and is shown in Fig 8.

Fig 8. Raman spectrum of tryptophan The sample concentration was 1.038 mg/ml and the sample was rotated and linearly translated to avoid sample degradation as detailed previously. The interferogram produced by the spatial heterodyne spectrometer undergoes minimal processing prior to the FFT algorithm. This comprised of flat field correction to remove instrument aberrations, and a spline fit to remove the fibre intensity profile. No spectral smoothing has been applied. Kumamoto et al. [8] has previously presented a well-resolved tryptophan Raman spectrum using a 2 cm-1 resolution...