Revolutionizing Protein Analysis with Microfluidic Modulation Spectroscopy (MMS)

RedShiftBio’s microfluidic modulation spectroscopy (MMS) technology combines laser spectroscopy, signal processing, and microfluidics. It is the main force behind the Aurora protein analyzer. This dynamic tool utilizes mid-infrared to directly probe the protein backbone and generate data on protein stability, concentration, aggregate formation, and higher-order structure.

Unlike other approaches, proteins can be readily assessed over a broad spectrum of concentrations in multiple settings without difficulties related to protein size, light shattering, or background fluorescence.

Aurora, like its predecessors AQS3pro and Apollo, uses a tunable mid-infrared quantum cascade laser to generate an optical beam that focuses through a microfluidic transmission cell along an optical path around 25 μm in length.

The laser runs in continuous wave mode. This allows for a high-resolution (< 0.001 cm^-1 linewidth), low-noise beam with little straying light, which decreases assessment error and enlarges the spectrum of directly measurable concentrations.

The sample (protein) solution and a corresponding water/buffer reference stream are introduced continuously into the transmission cell. The two streams are rapidly modulated over the laser beam path (1–10 Hz) to take turns measuring the reference and sample streams to produce a differential measurement. This approach generates a precise, drift-free, and extremely sensitive absorption assessment.

Following transit through the microfluidic cell, the beam is concentrated onto a thermo-electrically cooled MCT detector. The entire optical system is closed and purged with dry air to decrease interference from the powerful absorption lines of atmospheric water vapor.

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. Image Credit: RedShiftBio

RedShiftBio’s data acquisition system and software were utilized here to gain and process data. All measurements were captured at an ambient temperature of around 21 °C without temperature control of the fluids or measurement cell. Figure 1 displays a straightforward block diagram of the tool.

To produce the sample absorption spectrum, the absorption spectra of the protein were assessed via optical scanning of the amide I band in 4 cm^-1 increments from roughly 1700 to 1600 cm^-1. The amide I band is a direct assessment of the strength of the carbonyl band along the protein backbone. It is extremely sensitive to the surrounding environment and is reflective of the protein sub-structure type and state of aggregation.¹

Figure 2 displays the absorption spectra of four commercially available proteins. Automatic spectral assessment can generate data, including protein concentration and secondary structure. Fluctuations in the adsorption band can also be used to track aggregation and protein stability.

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. Image Credit: RedShiftBio

Higher-Order Structure

Vibrational spectroscopy is well-known as an efficient instrument for studying protein and peptide substructures.² The amide I band (1700–1600 cm^-1) analyzes the C=O stretch vibration of the peptide linkages that make up the protein’s backbone structure.³

The fluctuating patterns of dipole-dipole interactions, hydrogen bonding, and the geometric orientations in the α-helices, β-sheets, random coil structures, and turns lead to varying absorption properties in the amide I band, which correspond to these second-order structures.⁴

An assessment of the absorption spectrum can be utilized to quantify the relative amounts of these substructures. It can thereafter be used as a dynamic probe for characterizing and aggregating proteins as well as thermal and chemical stability.^5-7

While this approach is extremely powerful, assessment is usually confined to concentrations exceeding 10 mg/mL for Fourier transform infrared spectroscopy (FTIR) and 30 mg/mL for Raman.⁸

Ultraviolet circular dichroism (UV CD), among the most widely-used instruments for secondary structure analysis, is quite insensitive to β-sheet formation and has difficulty in detecting the essential intermolecular β-sheet structures that form in the aggregation process.⁸

While UV CD functions at a lower concentration range than FTIR (usually, 0.2–2 mg/mL relative to 10-200 mg/mL),⁹ it cannot directly assess higher concentration ranges typically experienced in formulation.

RedShiftBio’s Aurora can assess protein structure over a wide dynamic range, from 0.1 mg/mL to above 200 mg/mL. This removes the need for steps in sample preparation, including pre-concentration or dilution, which can lead to variability across samples—therefore necessitating several assessments and sample replicates.

The analyzer can additionally assess the protein at the concentration of interest in discovery and formulation. These capabilities are not present in contemporary instruments that measure proteins.

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). Image Credit: RedShiftBio

The quantification of protein secondary structure for α-chymotrypsin was carried out on proteins with good reproducibility via an automated fitting analysis method at a concentration range from around 0.1 to 10 mg/mL (Figure 3). These outcomes exemplify good accuracy, supporting FTIR approaches alongside published values from X-ray and UV CD.

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 %. Image Credit: RedShiftBio

Figure 4 showcases the repeatability of RedShiftBio’s analyzer. Several lysozyme (HEWL) assessments were captured over a month, with secondary structure results displaying a standard deviation of around 1 %.

Most protein analysis technologies are constrained by concentration ranges of around one order of magnitude. RedShiftBio’s MMS analyzer can nevertheless perform assessments at concentration ranges exceeding three orders of magnitude.

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. Image Credit: RedShiftBio

Figure 5 demonstrates an example analysis from bovine serum albumin (BSA) assessments at concentrations ranging from 0.1 to 200 mg/mL, outputting five secondary structure components beneficial for protein fingerprinting.

Stability and Aggregation

RedShiftBio’s analyzer’s capacity to directly assess protein secondary structure makes it a dynamic instrument for exploring the underlying mechanisms of protein aggregation and stability.

The process’s complexities are easy to follow as the proteins are exposed to stress and start to change from their original states. IR assessments are especially sensitive to β-sheet structures, which dominate protein antibody-based therapeutics.

MMS is among the only approaches that can directly monitor how aggregates form owing to its ability to assess intermolecular β-sheet structures.

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. Image Credit: RedShiftBio

Thermal Stability

A high β-sheet content protein at 1 mg/mL was incubated at a higher temperature for different periods. The protein series was assessed using the MMS analyzer. The second derivative spectra were overlaid and plotted to enhance the changes in spectra.

Figure 6 demonstrates the loss of intramolecular β-sheet content as a function of incubation time. The quantity of intermolecular β-sheet structure simultaneously increases, linked to the formation of aggregates. Changes in other areas reflect the protein substructure’s state, providing further information on the denaturation process.

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. Image Credit: RedShiftBio

As the protein was incubated, an insoluble aggregate formed and settled at the bottom of the sample tubes. The researchers decanted and measured only the supernatant fraction. Longer incubation times corresponded with lower overall concentrations of soluble protein (as displayed in Figure 7).

The amide I absorbance lessened by over half of its starting value, equating to under 0.5 mg/mL of protein in solution. The absorption data clearly demonstrates spectral shape changes of the amide I band.

Chemical Stability

Another approach for studying protein stability is chemical stress research. Alcohols are well-known to denature the proteins’ native states, frequently stabilizing the α-helical conformation in unfolded proteins and peptides.¹⁰

A rather high concentration of β-lactoglobulin was prepared in a phosphate buffer at pH 7.4 at concentrations of 0, 20, 40, and 60 % isopropyl alcohol (IPA). The IPA/protein series were assessed via Far-UV CD and MMS to monitor changes in structure.

Figure 8. Far UV-CD studies of the chemical denaturation of beta lactoglobulin in IPA show increasing alpha helix and decreasing beta sheet. Image Credit: RedShiftBio

Figure 8 displays the UV CD data, where an explicit increase in the α-helix structure happens together with an overall reduction in β-sheet.

The RedShiftBio data (displayed in Figure 9) demonstrates a growth in the α-helix form at elevated IPA concentration and a clear and significant shift in the β-sheet type from around 1630 cm^-1 to 1620 cm^-1. This further validates the formation of an intermolecular β-sheet, which is not readily shown by the CD approach otherwise.

The RedShiftBio’s MMS analyzer provides quality insight into the denaturation process and functions over a wide spectrum of concentrations.

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. Image Credit: RedShiftBio

Protein Similarity

Protein similarity is a quantitative approach that assesses the amide I band spectra among proteins to determine small changes in protein secondary structure.

Assessing small spectral variations can be beneficial for monitoring protein biosimilarity, due to how sensitive the amide I band is to changes in protein secondary structure.^11,12 Numerous algorithms have been suggested for this comparison, such as the area of overlap (AO) and the correlation coefficient.^11,12

The AO approach was utilized here to quantify protein similarity. These outcomes can be compared to published results that used other techniques, including FTIR and UV CD, to measure the sensitivity of the MMS approach relative to more traditional approaches.

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. Image Credit: RedShiftBio 

Figure 10 demonstrates the overlaid spectra of BSA at four concentrations acquired by the MMS analyzer (0.1, 1.0, 10, and 200 mg/mL). The similarity is higher than 97 % at the center of the concentration range. This decreases at both the higher and lower extremities.

FTIR values in the literature demonstrate a mean similarity of 86.37 % ± 7.98 % at a single concentration of 10 mg/mL for HEWL for comparison.¹² Utilizing this approach, protein similarity values of 97 % were exclusively obtained at a concentration of 50 mg/mL.

The RedShiftBio MMS analyzer attains superior similarity with less deviation over a concentration range that greatly surpasses the assessment capability of FTIR. The approach also furthers UV CD’s constraints at elevated concentrations, which can necessitate more workflow and dilutions.

Quantitation

Protein quantitation is important for biochemistry research and development, with applications spanning from data provision for biopharmaceutical lot release to enzymatic research. Direct assay approaches include UV and visible absorption assessments corresponding to standard extinction coefficients and indirect assessments via dye-based assays, including Lowry, BCA, and Bradford assays.

Given the specific constraints of each method (such as chemical interferences in dye-based assays, aromatic residue dependency, and the limited dynamic range of the spectroscopic instrument), no approach is universal.¹³

A primary limitation of assessments stems from the spectroscopic instruments themselves. Traditional spectrometers have constrained linearity, principally because of the instrument slit width (resolution), stray light, and detector linearity. Sample absorbance is therefore targeted over an extremely narrow dynamic range, usually between 0.1 and 1.5 au.¹⁴

This restricted range means researchers should adjust either the sample concentration or the cell path length to attain accurate protein quantitation. Either alternative can have a problematic and time-consuming effect on the assessment.

Infrared absorption spectroscopy can be a productive instrument for direct, label-free protein quantitation. It offers an advantage over UV-visible approaches as sample absorption bands in the infrared do not rely on aromatic residues and are far narrower. This makes the approach more selective and less vulnerable to interference.

The IR approach does not rely on a UV chromophore. It probes the carbonyl backbone of the protein, so the variation extinction coefficient is considerably smaller, which can be beneficial when assessing unknown proteins.

IR spectroscopy is not regularly utilized in situ owing to elevated costs, lower sensitivity, and operational difficulties such as water vapor interference, background subtraction, and narrow pathlength cells.

RedShiftBio’s MMS platform can overpower such drawbacks by enhancing sensitivity and significantly reducing errors that are commonly found in traditional spectroscopy. Its high resolution (< 0.001 cm^-1) and low susceptibility to stray light broaden the concentration range for assessment by over two orders of magnitude.

The differential assessment of MMS and its direct control over laser power additionally enhances linearity, preserving high detector linearity over the assessment range and decreasing signal dynamic range. With MMS, only a few wavelengths require measurement.

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. Image Credit: RedShiftBio

Figure 11 demonstrates a plot at around 1656 cm^-1 for BSA between 0.1 and 200 mg/mL, providing a substantial improvement over conventional absorbance-based assays with a minimum measurable concentration of under 10 µg/mL (3 sigma, HEWL) and an upper limit of more than 200 mg/mL.

Conclusion

Powered by MMS, Aurora offers a rapid yet simple system for characterizing proteins over a wide concentration range, using just 50 µL of sample. It simultaneously produces data on secondary structure, aggregation, stability, quantitation, and similarity. Microfluidic referencing improves the reproducibility of the approach, streamlining the workflow by removing the need for independent buffer assessments.

Aurora is an effective and flexible instrument for characterizing proteins in a direct, label-free manner through all phases of biologic drug development, from discovery to formulation and manufacturing.

References and Further Reading

1. Kong J. (2007) Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures. Acta Biochimica et Biophysisx Sinica, 39(8), pp.549–559.
2. Elliott, A. and Ambrose, E. (1950) Structure of synthetic polypeptides. Nature, 165, pp.921−922.
3. Fabian, H. and Mantele, W. (2002) Handbook of Vibrational Spectroscopy. Chalmers, J. M., Griffiths, P. R. (Eds.), John Wiley & Sons, Ltd, Chichester. pp.3399–3425.
4. Koenig, J. K. and Tabb, D. L. (1980) Analytical Applications of FTOIR to Molecular and Biological Systems Durig, J. R. (Ed.) pp.241–255,
5. Aichun, D. Ping, H. and Winslow S (1990) Protein Secondary Structures in Water from Second-Derivative Amide I Infrared Spectra. Biochemistry, 29, pp.3303–3308.
6. Pots, A. et al. (1998) Heat-induced conformational changes of patatin, the major potato tuber protein. Eur J Biochem, 252, pp.66–72.
7. Shivu, B. et al. (2013) Distinct β-sheet structure in protein aggregates determined by ATR–FTIR spectroscopy. Biochemistry, 52(31).
8. Wei Wang, Christopher J. Roberts Aggregation of Therapeutic Proteins Wiley, Aug 30, 2010.
9. Kelly, S. and Price, N. (2017) The Use of Circular Dichroism in the Investigation of Protein Structure and Function Current Protein and Peptide Science, 1, 349–384.
10. Hirota, N., Mizuno, K. and Goto, Y. (1997) Cooperative alpha helix formation of β lactoglobulin and melittin induce by hexafluoroisopropanol. Protein Science, 6, pp.416–421.
11. Kendrick, B. et al. (1996) Quantitation of the area of overlap between second-derivative amide 1 infrared spectra to determine the structural similarity of a protein in different states. J Pharm Sci, 85(2): pp.155–158.
12. D’Antonio, J. et al. (2012) Comparability of Protein Therapeutics: Quantitative Comparison of Second-Derivative Amide I Infrared Spectra. J Pharm Sci, 101(6) pp.2025–2033.
13. Noble, J. and Bailey, MJ. (2009) Quantitation of protein, Methods Enzymol. 463, pp.73-95.
14. Skoog, D., Holler, J. and Crouch, S. (2006) Principles of Instrumental Analysis, 6th Edition, Cengage Learning.

About RedShiftBio

RedShiftBio is redefining the possibilities for analyzing protein structure and concentration.

RedShiftBio has developed a proprietary life sciences platform combining our Microfluidic Modulation Spectroscopy (MMS) and expertise in high-powered quantum cascade lasers that provide ultra-sensitive and ultra-precise measurements of molecular structure. These structural changes affect critical quality attributes governing the safety, efficacy, and stability of biomolecules and their raw materials. This combination of technologies is available to researchers in our fully-automated Aurora and Apollo systems and is backed by a global network of sales, applications, service, and support teams to address all market needs.

Alongside our commitment to further innovation in the formulations and development space, RedShiftBio also supports biopharmaceutical manufacturing with HaLCon, our bioprocess analytics platform, purpose-built to measure protein titer at time of need.

Led by an experienced management team with a proven track record of success in both large instrumentation companies and commercializing disruptive technologies, RedShiftBio is here to support your research, development, and manufacturing goals. Our instruments can be found in the majority of the leading biopharmaceutical companies and CDMOs in the world. We also run product demonstrations and process samples in the StructIR Lab, located in our Boxborough, MA headquarters, as well as at partner sites including the Wood Centre in Oxford, UK, Spectralys/UCB in Brussels, Belgium, and at Sciex laboratories in Redwood Shores, CA.

RedShiftBio is backed by Waters Corporation, Illumina Ventures, Technology Venture Partners, and one undisclosed leading life science company.

Sponsored Content Policy: AZoLifeSciences publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of AZoLifeSciences which is to educate and inform site visitors interested in life science news and information.