Quantitation
The ability to quantitate a sample and compare its measured concentration against a nominal value is a built-in benefit of the Aurora and Apollo, powered by Microfluidic Modulation Spectroscopy (MMS). The Aurora and Apollo are unique IR spectroscopic tools that utilize a quantum cascade laser (QCL) and a microfluidic flow cell to provide secondary structural characterization of biomolecules. As opposed to using a chromophore, fluorophore or a fluorescent tag, IR spectroscopy probes absorption in the range of 780 nm to 1.0 mm resulting from the vibration of bonds present in a chemical structure. For proteins, the vibrations of interest result from bonds present in the peptide backbone, primarily carbonyl bending and stretching vibrations. Quantitation is automatically determined during routine sample analysis from the same differential absorbance spectra as the other spectral measurements. The improved sensitivity and real-time buffer referencing generated by MMS technology produces highly sensitive and linear results across a wide working concentration range of 0.1 to > 200 mg/mL.
The value of concurrent quantitation among the other sample measurements using the Aurora and Apollo lies in the ability to correlate concentration information with structural measurements as well as orthogonal measurements. This allows the scientist to assemble the information into actionable decisions and monitor the status of the biotherapeutic through the development and formulation processes.
Quantitation results for a monoclonal antibody measured across the experimental concentration range of 1 to 150 mg/mL and linear with a correlation coefficient R2= 0.999. Application Note Monoclonal Antibody Analysis by Microfluidic Modulation Spectroscopy in a Complex Formulation Buffer (1 to 150 mg/mL).
Related Biomolecules
The ability to quantitate a sample and compare its measured concentration against a nominal value is a built-in benefit of the Aurora and Apollo, powered by Microfluidic Modulation Spectroscopy (MMS). The Aurora and Apollo are unique IR spectroscopic tools that utilize a quantum cascade laser (QCL) and a microfluidic flow cell to provide secondary structural characterization of biomolecules. As opposed to using a chromophore, fluorophore or a fluorescent tag, IR spectroscopy probes absorption in the range of 780 nm to 1.0 mm resulting from the vibration of bonds present in a chemical structure. For proteins, the vibrations of interest result from bonds present in the peptide backbone, primarily carbonyl bending and stretching vibrations. Quantitation is automatically determined during routine sample analysis from the same differential absorbance spectra as the other spectral measurements. The improved sensitivity and real-time buffer referencing generated by MMS technology produces highly sensitive and linear results across a wide working concentration range of 0.1 to > 200 mg/mL.
The value of concurrent quantitation among the other sample measurements using the Aurora and Apollo lies in the ability to correlate concentration information with structural measurements as well as orthogonal measurements. This allows the scientist to assemble the information into actionable decisions and monitor the status of the biotherapeutic through the development and formulation processes.
Quantitation results for a monoclonal antibody measured across the experimental concentration range of 1 to 150 mg/mL and linear with a correlation coefficient R2= 0.999. Application Note Monoclonal Antibody Analysis by Microfluidic Modulation Spectroscopy in a Complex Formulation Buffer (1 to 150 mg/mL).
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Frequently Asked Questions
What is the HaLCon "trap-and-elute technique" and how does it work?
Trap and elute means the analyte (IgG) is fully captured on the column, everything else passes through, then the analyte is released and the detector sees one peak. In terms of flow it differs from traditional liquid chromatography in that there is no gradient, just 100% reagent A to capture or trap IgG, then 100% reagent B to elute.
How does HaLCon react to other Immunoglobulins and other impurities?
The Tridex uses a Protein A media. Anything not bound to the Protein A passes through the column to waste.
How must the sample be prepared for HaLCon?
The sample needs to be filtered through a 0.2 µm or 0.45 µm filter to ensure it is free of cells and cell debris. Alternatively, the sample can be spun down and the supernatant sampled with a syringe. Users doing this need to be careful not to stick the syringe too deep in the centrifuge vial. For users familiar with HPLC, the sample can be prepared as if it were being run on HPLC because HaLCon uses a similar column and pre-column filter.
How much sample material or sample volume is required to run HaLCon?
We recommend 100 µL per sample. If you have a lower desired sample volume, please contact us and one of our experts can help evaluate the feasibility of the proposed sample volume.
Does HaLCon require any connections, e.g. water or gas, in addition to a power connection?
Nothing else but a power connection is needed.
What type of pump is used (traditional binary HPLC pump, peristaltic pump) for HaLCon?
HaLCon uses displacement/dispense pumps. They work like a syringe pump except they are composed of more durable materials for longer, maintenance-free use. Since HaLCon uses low-pressure Liquid Chromatography, the maximum pressure recommended is 200-250 psi.
What type of maintenance is required for HaLCon? And, will this maintenance be performed during a yearly PM?
No regular maintenance is required beyond changing the reagent pack and analysis module every 3 months or after running 1,000 samples, whichever comes first. It is recommended to run a flush before running samples if the system has been idle for a few days.
How do you ensure that no cells from the fermenter enter the HaLCon measuring device?
Samples should be spun down, and care should be taken to not disturb any pellets or to remove the entire sample from the centrifuge tube. Alternatively, the samples can be filtered through a 0.2 µm or 0.45 µm filter.
Does HaLCon have a built-in filter or a separator?
HalCOn does not have a built-in filter for removing samples, that must be done before loading the sample.
How can the user determine if the sample purification is functioning correctly when using HaLCon?
The best way to validate performance is by running a control sample of known concentration and verifying that the correct concentration value is returned by the software.
Can HaLCon also control the process, such as cooling down or emptying the fermenter when a specific threshold is reached? Or is it solely for monitoring purposes?
HaLCon is purpose-built for measuring protein titer, nothing else. A sample is added either manually or via an autosampler and HaLCon provides a concentration. HaLCon is compatible with multiple autosamplers, including the Flownamics Director, which may be able to automate the sampling from a bioreactor.
