Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and Thioflavin T binding

Figures & data

Figure 1 Key structures of the mAb probed by fluorescent dyes. N and U are native and unfolded monomers, respectively. “n” reactive monomers form aggregates.

Figure 2 Binding of ANS to the mAb. (A) ANS binding equilibrium to native mAb with increasing dye concentration. Inset: Double reciprocal representation.20 (B) Temperature-dependence of mAb unfolding studied with ANS binding. Dye binding rates were determined from these data as described in the text and shown in Table 1.

Figure 3 Arrhenius plots for unfolding and aggregate formation of the mAb as a function of temperature (63–70°C). Rate constants were calculated with the first-order reaction for both of the proteins' unfolding and aggregation with the empirical sigmoid fit function. SEC-HPLC results (55–68°C) were calculated using the second-order reaction rate. Data fit was performed using Arrhenius equation (ln k_obs = ln A − E_a/RT). The activation energies for the mAb unfolding and aggregation from the ANS and ThT binding and SEC-HPLC experiments are 610, 544 and 549 kJ/mol, respectively.

Table 1 Observed unfolding and aggregation rate constants of the mAb by the dye-binding studies

	Unfoldinga	Aggregationb
T (°C)	kb (min^−1)	kb (min^−1)
63	0.007	0.038
65	0.031	0.091
67	NDc	0.163
68	0.115	0.667
70	0.758	2.000

a Unfolding rates were determined by the ANS binding. The mAb was incubated at 0.2-mg/ml at shown temperatures. At this concentration mAb only unfolds.

b The aggregation rate constants were determined by the ThT binding. The mAb was incubated at 150-mg/ml at 63–70°C, and then diluted to 0.2-mg/ml.

c ND, not available.

Van Nuland NAJ, Meijberg W, Warner J, Forge V, Scheek RM, Robillard GT, et al. Slow cooperative folding of a small globular protein HPr. Biochemistry 1998; 37:622 - 637; PMID: 9425085; http://dx.doi.org/10.1021/bi9717946

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