HOS Study for IgG Samples Spiked with Different Amount of BSA by Microfluidic Modulation Spectroscopy

An innovative and powerful system from RedShiftBio (RedShift Bioanalytics), based on Microfluidic Modulation Spectroscopy (MMS), is now available for the structural analysis of proteins. This device uses infrared spectroscopy, but is much more sensitive, operates under a larger range of measurement conditions and has significantly improved accuracy.

It is extremely useful in determining the secondary structure of a protein. It is based on a tunable laser which emits light in the mid-infrared range, with quantum cascade characteristics which enable the generation of a light beam about one hundred times as bright as that produced by FTIR spectroscopy. The source brightness means that detector design can be simplified, avoiding the need for cooling with liquid nitrogen.

Both the sample solution of protein, as well as a matching stream of buffer for reference, enter the microfluidic flow cell automatically, and the two liquids undergo rapid modulation at 5 Hz or so, passing across the path of the laser beam, generating measurements which are background-subtracted for almost zero drift. The figure below shows a simplified diagram of the working of the system, as well as an MMS Beta Unit.

Methods

In this experiment MMS measurements were taken on two sets of spike samples. The spike samples were as follows:

  • At 20 mg/mL prepared by combining different ratios of stock BSA in 20 mg/mL IgG to obtain varying BSA percentages: 0, 2%, 4%, 6%, 8%, 10% and 100%.
  • At 1 mg/mL prepared in the same way but using 1 mg/mL IgG to obtain 0, 2%, 4%, 6%, 8%, 10% and 100%.

Differential absorbance was utilized to obtain spectra at 1 Hz 5 Psi, and the data was then processed by the proprietary software built into the device.

Results

The results obtained from the experiment are summarized as follows:

Using MMS to measure IgG spike samples with 0-10% BSA at 20 mg/mL
When the quantity of BSA spiked into IgG was increased, there was an increase in the alpha-helix peak at 1656cm-1 and a reduction in the beta-sheet peak at 1637cm-1 as well.

When the peak changes at 1656cm-1 and 1637cm-1 were tracked, the difference in structure could be traced in a spike sample at 2% BSA.

When the data was compared for similarity, results show the accurate and reproducible nature of MMS measurements using 20mg/mL spike samples, and its ability to detect structure change at 2% BSA spike sample.

The result of the HOS analysis confirms that the detection of structure change is possible at 2% BSA spike sample.

Using MMS to measure IgG spike samples with 0-10% BSA at 20 mg/mL

When BSA was spiked into IgG at increasing levels, there was an increased in the alpha-helix peak at 1656cm-1 with a corresponding decrease at 1637cm-1 in the beta sheet peak.

When the changes in peaks are tracked at 1656cm-1 and 1637cm-1 the difference in structure can be seen in a spike sample at 4% BSA.

Comparing the data for similarity shows the precise and repeatable nature of MMS measurements at 1mg/mL, with structural changes being detected at 6% BSA spike sample.

The result of the HOS analysis confirms that detection of change in structure is possible in spike samples at 4% BSA.

Using FTIR to measure IgG spike samples with 0-10% BSA at 20 mg/m

When FTIR was used on 8 – 10% BSA spike samples, differences in secondary structure were detected at 20 mg/ml. It should be noted that the operator time required with this method on ten samples is about five hours. On the other hand, the use of the MMS autosampler requires only one to two hours under the same conditions.
 

Conclusions

The MMS system from RedShiftBio was used to test IgG spike samples containing 0-10% BSA at levels of 20 mg/mL and 1 mg/mL, showing that as the amount of spike BSA in IgG went up, the signal at 1656 cm-1 due to alpha-helix structure increased but the signal at 1637 cm-1 due to beta-sheet structure decreased.

At a level of 20 mg/ml, the use of MMS enabled changes in secondary structure to be detected in spike samples at 2% BSA or even less, and at 4-6% BSA spike samples at 1 mg/ml.

At 20 mg/ml the secondary structure change was detected at 8-10% BSA spike sample.

The actual operating time required to perform the procedure on ten samples was about two hours and five hours using MMS and FTIR respectively. In addition the system is fully automated with walk away operation.

Thus compared to traditional FTIR methods, MMS yields quicker results with higher sensitivity in HOS analysis, as well as providing more high quality data and more information through analytical software.

MMS is a highly adaptable and powerful platform to perform direct characterization of proteins without the use of labels, at all stages of drug development.

About RedShiftBio

See change. Change in how you take measurements. Change in your ability to detect and monitor protein conformation. Change in how you collect, analyze and present results. Change for the better. RedshiftBio help researchers see change better using the innovative tools and technologies.

RedshiftBio's origins are outside of the Life Sciences, but they are fast learners and listen closely to the needs of customers. Being outsiders allowed them to see how new technologies from other fields could be combined to better solve problems within the Life Sciences.

RedshiftBio developed a Microfluidic Modulation Spectroscopy (MMS) platform in response to a customer’s request for a better solution, and rather than repurpose outdated tools and methods designed originally for other applications, they started anew, and developed MMS from the ground up specifically with the protein scientist in mind.

In 2015, in response to the unique capabilities and potential of MMS, they pivoted their spectroscopy business to focus entirely on bringing innovative analytical solutions to the protein scientist. And so RedshiftBio are changing, too.

RedShiftBio develop tools to help protein scientists see change more clearly. And that’s definitely change for the better.


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Last updated: Aug 10, 2018 at 4:40 AM

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