Purpose: Biotherapeutics are the fastest growing class of drugs among the new molecular entities that are approved by regulatory authorities in the 21st century. These are large complex protein molecules that include monoclonal antibodies, enzymes, hormones, blood-related proteins and toxins among others. These are produced by recombinant technology using eukaryotic biological cells or bacteria. They have increased the treatment options for fatal diseases like cancers via many monoclonal antibodies and conjugated drugs and for congenital disorders via replacement therapies. As proteins are large and complex biomolecules they are susceptible to structural perturbations leading to changes in conformation or oligomerization and aggregation resulting in loss of efficacy. Several studies indicate that aggregation in protein therapeutics lead to unwanted immunogenicity due to proliferation of antidrug antibodies, which in turn diminish efficacy. Therefore, robust formulation resulting in stable structure is important for the success of biologics. In this regard, analytical methods used in the assessment of structural perturbations of proteins need to be sensitive and should facilitate the assessment of the extent of perturbation. Such methods are useful not only in the development of a therapeutic protein and assessment of its quality but will also be useful in the comparative assessment of biosimilars. Here we have examined the utility of fluorescence spectroscopy and light scatter analysis for assessing perturbations to protein structure.
Methods: Intrinsic fluorescence, ANS (8-anilino-1-naphthalene sulfonate) fluorescence with proteins, and static light scatter (SLS) were simultaneously measured in an integrated spectrophotometer, UNit (Unchained Labs, Pleasanton, CA). This spectrophotometer can analyze multiple samples from three sample holders, each with 16 capillary slots (~8 µL of each). Protein samples were analyzed in triplicate and averaged. A 266 nm laser for fluorescence excitation and 473 nm laser for static light scatter were used with sequential illumination. Intrinsic and ANS fluorescence intensities and light scatter intensities were recorded in the range of 250-720 nm with the built-in spectrometer. Temperature was increased in 5 °C steps from 20 to 90 °C with 3 min hold interval at each step.
Results: Protein samples prepared in or pre-reconstituted samples were diluted in 1X PBS buffer for all measurements. Commercially obtained pure beta lactoglobulin (BLG) and alpha amylase were used at a concentration of 2.5, and 10 mg/mL, respectively, and aqueous protein samples were pre-incubated with ANS at room temperature before measurements. Figure 1A shows the intrinsic fluorescence spectra and ANS fluorescence spectra of BLG observed over 20-90 °C. Intrinsic fluorescence intensity of BLG and the fluorescence intensity of ANS both show a progressive decrease with temperature. Fluorescence intensities when plotted as ratios show clear points of inflexion corresponding to significant structural perturbations (Fig. 1B and C; 55 °C and 60 °C). Both intrinsic and ANS fluorescence intensities progressively decreased with increasing temperature for amylase also (Fig. 2A). Interestingly, ratio plots show distinct temperature points of significant structural perturbations for amylase. While the point of inflexion for intrinsic fluorescence is at 55 °C (Fig. 2B), ANS fluorescence shows two distinct points, one at 55 °C and another at 70 °C (Fig. 2C). Static light scatter on the other hand shows the onset of significant perturbation at 60 °C (Fig. 2D). These data indicate that proteins suffer different degrees of perturbation at different temperatures and such analyses may be applied to any other stress variable.
Conclusion: Simultaneous measurement of fluorescence and light scatter intensities of proteins provide information on various degrees of perturbation induced at different points of temperature. We have shown here that temperature induced fluorescence and light scatter intensity changes can be used as a surrogate to assess the protein structure susceptibility. This approach can be useful in the development of a therapeutic protein drug and assessment of its quality as well as in the comparative assessment of biosimilars.
Disclaimer: The conclusions reflect the views of the authors and should not be construed to represent FDA’s views or policies.