The traditional method
Protein concentration monitoring is the standard method to determine the step yield of a particular protein production run, as well as its capacity and efficiency. Accurate protein quantitation for downstream processes, such as protein purification and buffer exchange for final formulations, is important for quality control. Traditionally, batch testing has been performed using standard cuvette methodology. This technique required volumes of product to be pulled from the manufacturing line and sent to another area for quality control analysis. Results were then sent back to the manufacturing floor to calculate the step yield, determine the amount of product needed for subsequent steps and make any adjustments to ensure the system reaches a desired target amount.
Microvolume UV analysis
In contrast to the traditional cuvette methodology, microvolume UV analysis is conducted with a microvolume spectrophotometer that measures multiple protein samples using a sample-retention system that requires small amounts (~2μL) of protein sample. Using the inherent physical properties, namely surface tension, to hold samples in place during measurement, a microvolume spectrophotometer eliminates the need for cuvettes, capillaries, or other containment devices.
Microvolume UV testing performed using the Thermo Scientific NanoDrop 8000 spectrophotometer incorporates optical pedestals that can hold and measure as many as eight microvolume protein samples at a time. The removal of classic containment devices allows the path length between the optical pedestals to change during the measurement cycle. The ability of optimising path length results in an extensive dynamic range of possible protein concentrations that can be measured (e.g., 0.1 mg/mL to 120mg/mL for human serum albumin), essentially eliminating the need to perform time-consuming dilutions and the errors associated with preparing such dilutions.
Industry benefits
The bioprocessing industry benefits from microvolume UV analysis in several ways. Firstly, the molecular integrity of the sample remains intact during testing, which is highly preferred from a regulatory standpoint. Microvolume UV analysis also reduces waste and allows real-time adjustments to be made immediately at the processing line. Additionally, the multi-sample capability of an eight-pedestal spectrophotometer allows for convenient replicate sampling, providing a broader data set for quality assurance. Finally, having a common platform for sample testing facilitates training and integration of this technology throughout a facility.
Microvolume testing enables facilities to provide real-time results on the protein manufacturing floor, which enables quick adjustments if necessary. The microvolume analysis system also generates a large set of data, which, in turn, provides greater confidence in the condition of the product batch. This technology minimises the amount of material lost during quality control assessment. During an entire process run, traditional UV testing typically requires pulling several cuvette-filled samples for analysis. Using microvolume analysis, 1–2 microlitre samples can be continually pulled off the product stream with minimal loss of product.
Industry challenges
As biomanufacturing methodologies continually push the threshold of product production, detection systems need to co-evolve with the process. The dynamic range of concentration measurement capabilities of a microvolume spectrophotometer such as the NanoDrop 8000 allows measurements of the protein product to be performed without dilution. The integrity of the batch biomolecule is thus maintained, providing a robust and accurate assessment of the protein product in its natural state.
With protein drugs, the pharmaceutical industry has moved to high concentration therapeutics, and anytime dilutions are performed, the protein product may change its molecular configuration. UV microvolume technology allows measurement of the product in its neat configuration, giving companies greater confidence in the final drug being delivered to the patient. The ability to analyse what is truly in a sample vial gives greater assurance to the regulatory authorities that the biomolecule has not changed during testing.
Conclusion
Biomanufacturers have found at-line microvolume UV testing can provide significant savings of time and at least a five-fold improvement in efficiency. Microvolume UV testing using the NanoDrop 8000 can improve confidence in the processing results and fidelity of the end product. Testing can be performed on the manufacturing floor in real time with minimal loss of product, providing greater overall confidence in protein products. This type of production methodology offers significant benefits for the future of bioprocessing optimisation.
The authors
Dr Philippe Desjardins, Scientific marketing & Manju Sethi, Product Manager
Thermo Fisher Scientific
Wilmington, DE, USA
www.thermoscientific.com/nanodrop
