RSB AQS3 Product
Measurements

Structure

The overall structural make-up of a protein biomolecule is complex and governed by folding, intramolecular, and intermolecular interactions. Repetitive patterns allow the identification of structural populations and monitoring the presence and changes in these structural populations reveals important information relating to the stability and ultimately the safety and efficacy of a biotherapeutic. The most common structures that are important in the characterization of folded proteins include parallel and anti-parallel beta sheets, alpha-helices, turns, and unordered structures. By tracking relative amounts of these components and comparing them to known reference materials, it is possible to gain confidence in the integrity of a biotherapeutic as well as identify undesired changes in functionality that will be critical to control and remedy at all stages in the development and formulation workflows.

Analysis of secondary structure in proteins using Infrared spectroscopy has a long tradition and is a well-accepted industry practice. At first glance, a typical IR spectrum focusing on the Amide I region appears broad, so extraction of useful information requires spectral enhancement. One process of extracting secondary structure information from a spectrum typically involves a band narrowing and curve fitting approach. For instance, Fourier self-deconvolution curve fitting, and partial least squares analysis are two well-known approaches. However, second derivative analysis is the most prominent method used and is the method employed in the delta software package.  

Since the various structural components of the Amide I spectrum absorb at a range of wavelengths (Table 1), the Amide I band tends to be broad. The goal of a band fitting approach is to deconvolute the Amide I band into the various component bands which can then be assigned to the different types of secondary structure. A band narrowing step is first necessary to assign the relevant bands and can be performed by either a second derivative analysis or by Fourier deconvolution.  

Table 1: Deconvoluted Amide I band frequencies and assignments to secondary structure.

An example of Gaussian curve fitting can be seen in Figure 1 where the red line represents the inverted and baselined second derivative spectrum of an antibody, and the colored gaussian curves represent the deconvoluted contributions of each spectral feature as outlined in Table 1.

Figure 1:  Example curve fitting process of an Amide 1 spectrum obtained from MMS using the AQS3pro.

The advantage of using Microfluidic Modulation Spectroscopy (MMS) powered by the AQS³ delta software to elucidate the structural composition of a protein therapeutic is that it is intuitive and easy to specify the populations that should be monitored. Post-analysis processing gives clear and meaningful results to show which secondary structural motifs have been maintained and which may have been compromised due to changes in experimental or formulation conditions.  

Figure 2: HOS analysis of four insulin samples tested at different pHs demonstrating an increase in alpha-helical structures relative to an increase in pH. Application Note Using Microfluidic Modulation Spectroscopy to determine the Structural Effect of pH on the Peptide Hormone Insulin.
HOS analysis of four insulin samples tested at different pH and demonstrating an increase in alpha-helical structures with an increase in pH. Application Note Using Microfluidic Modulation Spectroscopy to determine the Structural Effect of pH on the Peptide Hormone Insulin.

The overall structural make-up of a protein biomolecule is complex and governed by folding, intramolecular, and intermolecular interactions. Repetitive patterns allow the identification of structural populations and monitoring the presence and changes in these structural populations reveals important information relating to the stability and ultimately the safety and efficacy of a biotherapeutic. The most common structures that are important in the characterization of folded proteins include parallel and anti-parallel beta sheets, alpha-helices, turns, and unordered structures. By tracking relative amounts of these components and comparing them to known reference materials, it is possible to gain confidence in the integrity of a biotherapeutic as well as identify undesired changes in functionality that will be critical to control and remedy at all stages in the development and formulation workflows.

Analysis of secondary structure in proteins using Infrared spectroscopy has a long tradition and is a well-accepted industry practice. At first glance, a typical IR spectrum focusing on the Amide I region appears broad, so extraction of useful information requires spectral enhancement. One process of extracting secondary structure information from a spectrum typically involves a band narrowing and curve fitting approach. For instance, Fourier self-deconvolution curve fitting, and partial least squares analysis are two well-known approaches. However, second derivative analysis is the most prominent method used and is the method employed in the delta software package.  

Since the various structural components of the Amide I spectrum absorb at a range of wavelengths (Table 1), the Amide I band tends to be broad. The goal of a band fitting approach is to deconvolute the Amide I band into the various component bands which can then be assigned to the different types of secondary structure. A band narrowing step is first necessary to assign the relevant bands and can be performed by either a second derivative analysis or by Fourier deconvolution.  

Table 1: Deconvoluted Amide I band frequencies and assignments to secondary structure.

An example of Gaussian curve fitting can be seen in Figure 1 where the red line represents the inverted and baselined second derivative spectrum of an antibody, and the colored gaussian curves represent the deconvoluted contributions of each spectral feature as outlined in Table 1.

Figure 1:  Example curve fitting process of an Amide 1 spectrum obtained from MMS using the AQS3pro.

The advantage of using Microfluidic Modulation Spectroscopy (MMS) powered by the AQS³ delta software to elucidate the structural composition of a protein therapeutic is that it is intuitive and easy to specify the populations that should be monitored. Post-analysis processing gives clear and meaningful results to show which secondary structural motifs have been maintained and which may have been compromised due to changes in experimental or formulation conditions.  

Figure 2: HOS analysis of four insulin samples tested at different pHs demonstrating an increase in alpha-helical structures relative to an increase in pH. Application Note Using Microfluidic Modulation Spectroscopy to determine the Structural Effect of pH on the Peptide Hormone Insulin.
HOS analysis of four insulin samples tested at different pH and demonstrating an increase in alpha-helical structures with an increase in pH. Application Note Using Microfluidic Modulation Spectroscopy to determine the Structural Effect of pH on the Peptide Hormone Insulin.

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.

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RedShiftBio AQS3 Product Detail

Request a Demo Today! 

RedShiftBio AQS3 Product Detail