Protein Similarity

Determining protein similarity for biologics is much more challenging than with small molecules due to their larger size and structural complexity. Along with functional comparisons, measurement and analysis of the structural similarity between proteins is an effective method of demonstrating bioequivalence. Microfluidic Modulation Spectroscopy (MMS) measures protein secondary structure, reveals very small conformation differences between different proteins, and provides information as to where those differences occur. These capabilities make MMS a powerful tool in the analysis and development of biosimilars .

Quantitating Protein Similarity by Comparing the Amide I band

Protein similarity is a quantitative approach for detecting small changes in protein secondary structure by analyzing and comparing the amide I band spectra between proteins. As the amide I band is very sensitive to changes in the secondary structure of proteins, the ability to measure small differences in the spectra can be a powerful tool in monitoring the biosimilarity of a protein. A number of algorithms have been proposed for this comparison, including the correlation coefficient and the area of overlap. These results can be compared to published results using other methods to assess the sensitivity of the MMS method relative to more traditional methods such as FTIR or UV-CD.

The following figure shows the overlaid second derivative spectra of a monoclonal antibody (mAb ) run at a concentration of 0.5 mg/ mL on the AQS³pro. The spectra overlay with superior precision and the similarity of the measurements for samples run at both 0.5 mg/mL and 10 mg/mL are all above 98%.

Triplicate data (above) for a mAb run at 0.5 mg/mL, second derivative and calculated similarity scores (below) for these samples and samples run at 10 mg/mL..*
Triplicate data (above) for a mAb run at 0.5 mg/mL, second derivative and calculated similarity scores (below) for these samples and samples run at 10 mg/mL..*

For comparison, FTIR values found in the literature show a mean similarity of 86.37% +/- 7.98% at a single concentration of 10 mg/mL for HEWL (Hen Egg White Lysozyme). Using FTIR, protein similarity values at the 97% level could only be obtained at a concentration of 50 mg/mL. For all tested applications, the AQS³pro achieves better similarity with less deviation over a concentration range that far exceeds the measurement capability of FTIR, while also addressing the limitations of UV-CD (circular dichroism) which, limited dynamic range which requires dilutions to a concentration range of detection technology and interference with many formulation excipients causing issue with analysis of samples in relevant conditions.

*Data Source: Poster: Microfluidic Modulation Spectroscopy (MMS) - a novel automated infrared (IR) spectroscopic tool for secondary structure analysis of biopharmaceuticals with high sensitivity and repeatability; Dipanwita Batabyal et al.

Related resources:

Analyzing Structural Similarity of Biotherapeutics with Microfluidic Modulation Spectroscopy

  • Microfluidic Modulation Spectroscopy for Protein Characterization: A Case Study with EcoCRM®, CRM197 Carrier Protein

Microfluidic Modulation Spectroscopy for Measuring Protein Secondary Structure and Similarity

  • Webinar: “Using Microfluidic Modulation Spectroscopy to Monitor Protein Misfolds and Structural Similarity”
  • Application note: Microfluidic Modulation Spectroscopy (MMS) Fills an Analytical Gap with a Lower LOQ for Measuring Protein Misfolds and Structural Similarity

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