The microfluidic modulation spectroscopic system (AQS3pro) from RedShiftBio uses a powerful combination of laser spectroscopy, with microfluidic technology and sophisticated signal processing, to give a direct characterization of the protein secondary structure using mid-infrared laser, as well as data on protein concentration, stability and aggregation.
The advantage of this platform is that it lends itself to measurement of proteins in several types of conditions and with a broad range of concentration without much difficulty, avoiding artifacts due to background fluorescence and light scattering or issues caused by protein molecule size.
The heart of the optical apparatus is a tunable quantum cascade mid-infrared laser. The resulting beam is brought to focus by a microfluidic transmission cell which has an optical path length of about 25 um. The measurement achieves extremely high resolution with line width of less than 0.001 cm-1 by the use of laser in continuous wave mode. This also results in very low levels of stray light and so low noise in the measurements, increasing the accuracy and the directly measurable concentration range.
The protein sample solution is passed continuously into the transmission cell along with a reference stream of water or buffer to match, and the two streams of solution are alternately fed into the cell, or modulated, at a high speed of 1-5 Hz across the path of the laser beam. This results in alternate measurement of the sample and the reference stream, generating a differential measurement. This is called Microfluidic Modulation Spectroscopy, or MMS.
The outcome is a very accurate absorption measurement with low drift values, and high sensitivity. The laser beam next falls on an MCT detector which undergoes thermoelectric cooling. This whole system is protected against interference by atmospheric water vapor (which produces strong absorption lines in the spectra) by sealing the system and purging it with dry air.
The data is acquired and processing is carried out with the data acquisition system from RedShiftBio, which is equipped with its own software. The fluids and measurement cell were at room temperature, about 21 °C, throughout the measurement cycle. Figure 1 shows a simple block diagram of MMS.
The absorption spectra generated by the protein were measured by scanning across the amide I band, from about 1700 to 1600 cm-1 in increments of 4 cm-1 to obtain the spectrum of absorption for the sample. The amide I band is in direct proportion to the carbonyl band strength, situated along the backbone of the protein. The amide I band responds quickly to changes in the local environment, which in turn varies with the structure and aggregation of the protein.
Figure 2 represents the absorption spectra of four commercially obtained proteins. Each of these spectra can be analyzed automatically to yield information about the secondary structure and concentration of the protein, and changes in adsorption bands are also useful in helping to detect protein instability or aggregation.
Figure 1. Simplified block diagram of the protein analyzer shows the tunable laser which probes the protein solution through a microfluidic cell. The microfluidic cell rapidly alternates between sample and reference (buffer) streams to continuously refresh the instrument referencing to dramatically improve measurement precision, accuracy, and signal-to-noise.
Figure 2. Representative measurements of commercially available proteins made with RedShiftBio’s MMS analyzer at 10 mg/mL. The laser scans the amide I band and directly probes the protein backbone. The shape of the band reveals the protein sub-structure making it a powerful tool for protein characterization.
Higher Order Structure
The use of vibrational spectroscopy has for many years revolutionized the unearthing of protein and peptide structures. The amide I band from 1700 – 1600 cm-1 produces spectra based on the stretch vibration in the C=O bond within the peptides which form the protein backbone. Various other geometries within the secondary structures such as random coils, beta-sheets, turns and alpha-helices, as well as varying types of hydrogen bonding patterns, and interactions between molecular dipoles are all correlated with differing absorption spectral features, which can thus help render a quantitative visualization of each of these substructural elements.
With MMS being an IR technique as opposed to vibrational, MMS is therefore capable of throwing much light upon the various characteristics of the protein under study, its stability in various chemical and thermal conditions, and protein aggregation. Current Analytical techniques are very powerful but they are not able to detect proteins at concentrations over 10 mg/ml with methods such as Fourier Transform Infrared Spectroscopy (FTIR), and 30 mg/mL for Raman.
Ultraviolet Circular Dichroism (UV-CD) is widely used at present to analyze the secondary structure of proteins, but has the disadvantage of not showing beta-sheet formation between molecules that form during protein aggregation. It functions at a concentration range below that of FTIR, at about 0.2 – 2 mg/mL (for the latter, it is about 10 – 200 mg/mL), but cannot effectively measure proteins in higher ranges as are found in formulation stages.
The MMS analyzer has a wide dynamic range from 0.1 mg/mL to over 200 mg/mL, and so the sample does not need to be diluted or pre-concentrated. This avoids an important source of variability between samples which then makes replication of samples and multiple measurements necessary. Another advantage is that the protein can be measured at the stage where it is actually found, from discovery to manufacture, unlike current protein measurement systems.
The secondary structure of the protein alpha-chymotrypsin was carried out using automated fitting analysis. The analysis was shown to be reproducible over a range of concentrations from about 0.1 to 10 mg/mL, as seen in Figure 3. This shows high accuracy for this method, comparable to FTIR, X-ray and UV-CD values.
Figure 4 shows that the values obtained on measurements of lysozyme (HEWL) with the RedShiftBio analysis are repeatable. These were taken over one month and the results had a standard deviation of about 1%.
Most protein measurement technologies are able to function within concentration ranges that vary within an order of magnitude; RedShiftBio’s MMS system can cover ranges of concentrations over more than three orders of magnitude. This is illustrated in Figure 5 showing bovine serum albumin measurements at a concentration range of 0.1 to 200 mg/mL, which yielded five components of the secondary structure which helped to develop a protein fingerprint.
Figure 3. Secondary structure for alpha-chymotrypsin measured over 2 orders of magnitude of concentration (0.1 to 10 mg/mL). Results agree with conventional FTIR results (likely Dong paper, 20 or 30 mg/mL).
Figure 4. Secondary structure for hen egg white lysozyme (HEWL) from 7 separate measurements of 10 mg/mL samples, taken over one month, showing standard deviation of about 1%.
Figure 5. RedShiftBio system measurements of protein secondary structure of Bovine Serum Albumin over a range of concentration from below 0.1 mg/mL to 200 mg/mL showing good analysis results over three orders of magnitude in concentration.
Stability and Aggregation
The ability of the RedShiftBio analyzer to monitor protein stability and aggregation as well as understand the underlying processes is related to its power to directly measure the secondary structure of the protein. When a protein is stressed it starts to show changes in its structure, and these can be visualized easily using this device.
In particular, infrared measurements respond to the presence of beta-sheets which are prominent within drugs based upon protein antibody molecules. MMS is one of the very few which is able to monitor aggregation of proteins because it is able to measure the occurrence of beta-sheets between molecules. The rest of the article shows how this system can be used to monitor stability in two common settings, namely, thermal and chemical stability.
For this experiment, a protein with a lot of beta-sheet content at a concentration of 1 mg/mL was used. After incubating it at high temperature for varying intervals, measurements were taken using the MMS. The type of protein used and the details of the experiments are withheld because the samples are proprietary, provided by a customer.
Following MMS measurements, the second derivative spectra were overlaid and a plot was generated to improve the level of spectral change. Figure 6 shows data which reveal that beta-sheet configuration is lost within molecules as incubation time increases, but at the same time the beta-sheet formation between molecules increases, leading to protein aggregation. Other secondary structure characteristics also change, reflected in other regions, and mirroring other components of protein denaturation.
As incubation proceeded, the development of an insoluble aggregate was noted, which settled at the bottom of the tubes containing the sample. The supernatant fraction was decanted off and measured. This led to a lower overall concentration of soluble protein as the incubation period increased.
Figure 7 shows this relationship, with a decrease in amide I band absorption values to less than 50% of the original, or below a concentration of 0.5 mg/mL of protein in solution, and changes in the shape of the amide I band spectrum as well as the data shows.
Protein stability is also studied by chemical stress experiments. Alcohols cause denaturation of proteins and enhanced alpha-helix stability in proteins and peptides which have unfolded in this process. In the current study beta-lactoglobulin at a high concentration was formulated with Isopropyl Alcohol (IPA) at concentrations of 0, 20, 40 and 60% in phosphate buffer at a pH of 7.4 and the solutions were then measured using Far UV-CD and MMS to monitor the changes in structure.
Figure 8 shows the UV-CD results revealing increased alpha-helical content and overall reduction in beta-sheet content.
In Figure 9, which shows the results of measurement with the RedShiftBio analyzer, the alpha-helix content is increased at higher IPA concentration as well as intermolecular beta-sheet formation, shown by the band shift from 1630 cm-1 to 1620 cm-1 which was not shown clearly by UV-CD. Thus this analyzer helps understand the denaturation of proteins more clearly and over a wider concentration range as well.
Figure 6. Incubation of a 1 mg/mL high beta sheet containing protein incubated at elevated temperature from 0 to 24 hours. As the incubation time increases the (intramolecular) beta sheet content decreases and the intermolecular beta sheet increases, indicative of aggregate formation.
Figure 7. As the protein was denatured insoluble aggregate formed and precipitated out of solution. As only the supernatant of the sample was measured the intensity of the amide I band decreases, which is a direct indicator of soluble protein concentration.
Figure 8. Far UV-CD studies of the chemical denaturation of beta lactoglobulin in IPA show increasing alpha helix and decreasing beta sheet.
The detection of similarity in protein structure is another way in which changes in protein secondary structure are identified. This is carried out by amide I band spectrum analysis and comparison. This band responds with high sensitivity to any change in secondary protein structure and therefore this helps immensely to monitor the extent of biosimilarity of a protein.
Several algorithms have been developed to compare protein similarity, such as correlation, coefficient and overlap area. In this experiment, the area of overlap was the method of choice. The results obtained by this method are compared to the results obtained from other methods and published previously, to establish the level of sensitivity of MMS in comparison with conventional methods including UV-CD and FTIR.
Figure 10 shows the results of overlaying the BSA spectrum at four levels of concentration, namely, 0.1, 1.0, 10 and 200 mg/mL, using spectra acquired with MMS. The similarity was found to be above 97% in the middle range of concentration, falling off to lower levels at both ends.
Published values in the literature show that FTIR values show a mean similarity of 86.37% +/- 7.98% when the concentration was 10 mg/mL for HEWL. A similarity value of 97% or more was possible only with a concentration of 50 mg/ml.
This shows that the RedShiftBio MMS analyzer not only performs much better at similarity measurements, while causing less deviation, but can be used over a range of measurements that is greatly above the levels possible with FTIR. This analyzer also overrides the inability of UV-CD to take accurate measurements at higher concentrations, which need more input to achieve comparable values.
The need to quantify protein is essential in many biochemical research applications and drug development laboratories, covering all types of studies from enzyme studies to data gathering in order to release a biopharmaceutical lot. Some methods of protein quantitation include direct assays, which use UV and visible absorption to measure protein concentrations relative to a standard established by extinction coefficients.
Another method is to use indirect measurements which depend on dye-based assays, such as Bradford, BCA, and Lowry assays. No single approach can be applied to every situation, because each has its limitations. These range from chemical interferences in the case of dye-based arrays, to aromatic residue dependency, or the smaller dynamic range of concentrations covered by spectroscopy.
Spectroscopic measurements are limited by the tool design itself, in most cases. The traditional design of a spectrometer allows only limited linearity because of the phenomenon of stray light and resulting noise, as well as the slit width of the instrument, which determines its resolution, and the linearity of the detector.
This limits the dynamic range of the sample absorbance to a low level, typically 0.1 to 1.5 au. To overcome this and achieve accurate quantitation of protein, the cell path length or the sample concentration must be adapted. However, these are both cumbersome and can adversely affect the measurements.
Infrared absorption spectroscopy is thus a useful tool in ensuring that protein content can be measured directly and in a label-free manner. It is superior to UV/VIS techniques in that the infrared absorption spectra are independent of aromatic residue and they are narrow in comparison to those acquired by the former.
Figure 9. Protein characterization results obtained using RedShiftBio’s MMS analyzer not only show the expected increase in alpha helix with higher alcohol concentrations, but also shows a shift in beta sheet to the aggregate form of intermolecular beta sheet.
Figure 10. Protein similarity of BSA is shown over the range from 0.1 mg/ml to 200 mg/ml, again demonstrating the viability of the measurement technique to compare protein characteristics across multiple steps of protein development.
This makes it selective and less likely to suffer from interference. The infrared absorption affects the carbonyl bonds in the protein backbone, which means it is not necessary to have a UV chromophore. This means that the variation extinction coefficient is significantly lower and this helps greatly when unknown proteins are being measured.
The problem with infrared spectroscopy studies in situ for routine use lies in the higher expense, lower sensitivity and inconvenient operation in the form of avoiding interference by water vapor, background subtraction and cells which use a narrow pathlength.
RedShiftBio’s MMS analyzer succeeds in offering streamlined performance by using a design which enhances the sensitivity and reducing the measurement errors frequently seen with traditional spectroscopy. It offers a resolution below 0.001 cm-1 and avoids noise due to stray light, which means the range of linear concentrations is boosted by over two orders of magnitude.
The MMS method also provides differential measurement and enables the laser power to be directly controlled, which further enhances the linearity, because it decreases the dynamic range of the signal and keeps the detector linearity high throughout the range of measurement.
When the MMS analyzer is used, it is only necessary to measure a few, or in most cases just one, wavelength. As Figure 11 shows, when BSA is plotted at about 1656 cm-1 between 0.1 and 200 mg/mL, the minimum measurable concentration is below 10 μg/mL (3 sigma, HEWL) and the maximum above 200 mg/mL, which represents a clear and marked superiority to traditional assay methods which use absorbance.
RedShiftBio’s MMS analyzer is a system which enables the secondary structure of proteins to be rapidly determined in a simple way, over a broad range of concentrations, while also providing data on protein stability, concentration, similarity and aggregation. The use of microfluidic cells allows good reproducibility while simplifying the process, by avoiding the steps required for buffer measurement in a separate phase. Thus the MMS platform represents a powerful but flexible solution to perform direct rapid label-free protein characterization in all stages of development of a biologic drug, whether discovery, formulation or manufacturing.
Figure 11. Differential absorbance at ~1656 cm-1 (BSA peak) plotted as a function of concentration at 0.1, 1, 10, and 200 mg/mL.
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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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