Identification of Commercially Available Oligonucleotide Starting Materials Directly Through Containers

Significance of the Topic

Oligonucleotide building blocks known as phosphoramidites are critical reagents in PCR, diagnostics, and therapeutic development. Ensuring their correct identity at receipt is a key step in current Good Manufacturing Practice (cGMP) to prevent supply chain errors, substandard materials, and costly production delays. Handheld Raman spectroscopy, especially Spatially Offset Raman Spectroscopy (SORS), offers a noninvasive, rapid approach to verify raw materials directly through containers, reducing sampling risks and accelerating release into production.

Study Objectives and Overview

This application note evaluates the Agilent Vaya handheld Raman system for qualitative identification of commercially available oligonucleotide starting materials through amber glass bottles. The goals were to demonstrate SORS-based ID verification in a cGMP environment, validate method robustness according to pharmacopeial guidelines, and assess selectivity among structurally similar phosphoramidites.

Used Instrumentation

• Agilent Vaya handheld Raman spectrometer with SORS capability
• Manufacturer-supplied amber glass bottles containing phosphoramidites
• Complementary sample standards and challenge analogs

Methodology

Identification methods were developed by collecting ten Raman scans of each phosphoramidite through its amber container. Closely related analogs were included as negative challenge samples to enhance selectivity. Validation tests comprised expected pass scans on target materials and fail scans on analogs. Post-deployment performance qualification employed the Vaya System Check module to verify photometric accuracy, wavelength precision, and laser power per USP <858> and EP 2.2.48.

Main Results and Discussion

Overlay spectra revealed clear differences among most phosphoramidites in characteristic vibrational regions (~1 000 cm⁻¹ and ~1 500 cm⁻¹). However, three analogs exhibited closely overlapping signatures, leading to potential false positives in an initial challenge matrix. By incorporating additional spectra of analogous compounds into each model, the two-score decision engine (correlation coefficient and linear model coefficient) thresholds were automatically adjusted, yielding perfect pass/fail discrimination across all five phosphoramidites.

Benefits and Practical Applications

• Noninvasive ID through transparent and opaque containers without opening or sampling
• Rapid pass/fail results (< seconds) with color-coded output for nontechnical warehouse staff
• Regulatory compliance via built-in method development, validation workflows, audit trails, and 21 CFR Part 11 features
• Elimination of days-long Mid-IR or NIR testing workflows, enabling same-day material release

Future Trends and Potential Applications

Expanding handheld SORS applications to other sensitive pharmaceutical raw materials, biologics intermediates, and high-value reagents can further streamline quality control. Integration with digital lab management systems and cloud-based spectral libraries may enhance remote monitoring, traceability, and automated release processes. Advances in chemometric algorithms could improve selectivity for ever more similar analogs and complex mixtures.

Conclusion

The Agilent Vaya handheld Raman spectrometer enables reliable, noninvasive identification of oligonucleotide phosphoramidites through amber containers under cGMP conditions. Its SORS capability, coupled with robust method development and validation features, delivers rapid, accurate raw material verification. This approach minimizes sampling risk, shortens release timelines, and supports stringent quality control requirements in pharmaceutical and biopharma manufacturing.

References

1. United States Pharmacopeia <1858> Raman Spectroscopy – Theory and Practice; Qualitative Raman Measurements.
2. European Pharmacopeia Chapter EP 2.2.48 Raman Spectroscopy.
3. Chinese Pharmacopeia General Rule Section 0421 Raman Spectroscopy.
4. Japanese Pharmacopeia (Supplement II, JP XVII) Section 2.26/5.1.
5. ICH Q2(R1) Validation of Analytical Procedures.