Application Notes

Importantly, since the single intramolecular interactions are weak, differences in the expression or purification process can introduce structural alterations of the biosimilar when compared to the originator. Controlled heating of the biosimilar gradually dissipates intramolecular interactions required for protein folding, resulting in multiple unfolding transition events. nanoDSF offers highly precise and reproducible detection of thermal unfolding patterns and therefore precisely recapitulates conformational stability and similarity between distinct unfolding events. Here we show that the similarity in the unfolding profiles of biosimilar candidates serves as a direct measure for their conformational similarity to the originator. The nanoDSF thermal unfolding profiling approach allows for rapid screening of a large number of biosimilar variants in different stages of development in order to narrow down the number of candidates and to optimize the biosimilar development process. Results A CHO cell line expressing candidates biosimilar to a marketed Fc-fusion therapeutic protein was generated at UGA Biopharma. During early-stage biosimilar cell line development, sixteen different combinations of basal and feed media were screened in fed-batch mode. The aim of media screening was to identify a baseline cell culture fed-batch process resulting in maximum comparability of biosimilar candidates to the reference molecule. Candidate biosimilars were purified from clarified cell culture fluid by capture chromatography using modified protein A. Biosimilar candidates, as well as the reference molecule, were then diluted to approximately equal concentrations in a sample buffer comparable to the formulation buffer of the reference product. We measured the thermal unfolding of 16 candidates and the reference by nanoDSF. Measurements were performed in triplicates in 2 runs. The variance in unfolding transition temperatures (T m values) and unfolding patterns between the different molecules was rather large: While the reference molecule showed a single T m value at 57.5 °C in the F350/F330 fluorescence ratio, almost all candidates, except for sample #11, displayed two unfolding transitions (Figure 1A). The T m 1-values differed by as much as 12 °C, ranging from 55.7 °C (sample #11) to 67.9 °C (sample #1) (Figure 1B). Interestingly, unfolding in the single-wavelength fluorescence showed two clear unfolding transitions for all samples, including the reference molecule and sample #11 (Figure 2). These differences in the unfolding signals are likely caused by differences in the secondary structures of the candidates. The fact that the reference molecule and sample #11 show a second transition only in the single wavelength and not in the F350/F330 ratio suggests that the responsible tryptophan(s) is/are already surface-exposed and thus do not undergo a red-shift. Figure 2: Comparison of triplicate thermal unfolding signals from the F350/F330 ratio and the single wavelength at 350 nm. While sample #13 displays two unfolding transitions in both signals, the reference and sample #11 display only one transition in the F350/F330 ratio. Since the candidates not only differed in T m , but even more in the entirety of their unfolding profiles, including unfolding amplitudes, unfolding transition slopes, and number of unfolding events, we quantified the overall similarity of their thermal unfolding patterns. For this, the first derivative data of the F350/F330 signal were analyzed by directly comparing the profile of the reference molecule to the unfolding profile of each candidate. The result is an "overlap index" which allows for ranking of

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