In Situ Monitoring of Protein Unfolding/Structural States under Cold High-Pressure Stress

Abstract

Biopharmaceutical formulations may be compromised by freezing, which has been attributed to protein conformational changes at a low temperature, and adsorption to ice–liquid interfaces. However, direct measurements of unfolding/conformational changes in sub-0 °C environments are limited because at ambient pressure, freezing of water can occur, which limits the applicability of otherwise commonly used analytical techniques without specifically tailored instrumentation. In this report, small-angle neutron scattering (SANS) and intrinsic fluorescence (FL) were used to provide in situ analysis of protein tertiary structure/folding at temperatures as low as −15 °C utilizing a high-pressure (HP) environment (up to 3 kbar) that prevents water from freezing. The results show that the α-chymotrypsinogen A (aCgn) structure is reasonably maintained under acidic pH (and corresponding pD) for all conditions of pressure and temperature tested. On the other hand, reversible structural changes and formation of oligomeric species were detected near −10 °C via HP-SANS for ovalbumin under neutral pD conditions. This was found to be related to the proximity of the temperature of cold denaturation of ovalbumin (T CD ∼ −17 °C; calculated via isothermal chemical denaturation and Gibbs–Helmholtz extrapolation) rather than a pressure effect. Significant structural changes were also observed for a monoclonal antibody, anti-streptavidin IgG1 (AS-IgG1), under acidic conditions near −5 °C and a pressure of ∼2 kbar. The conformational perturbation detected for AS-IgG1 is proposed to be consistent with the formation of unfolding intermediates such as molten globule states. Overall, the in situ approaches described here offer a means to characterize the conformational stability of biopharmaceuticals and proteins more generally under cold-temperature stress by the assessment of structural alteration, self-association, and reversibility of each process. This offers an alternative to current ex situ methods that are based on higher temperatures and subsequent extrapolation of the data and interpretations to the cold-temperature regime.

High-Pressure, Low-Temperature Induced Unfolding and Aggregation of Monoclonal Antibodies: Role of the Fc and Fab Fragments

“…For MAb1, no significant pressure effects up to 300 MPa were observable in the Kratky plots. These results are not consistent with conclusions drawn from fluorescence measurements, although we note solution conditions were altered to lower pH, citrate buffer salt, and 5 mg/mL protein …”

“…The average structure/morphology of partially unfolded species was also characterized in situ with small-angle neutron scattering (SANS), which is a complementary technique to fluorescence with somewhat higher resolution to assess conformational changes and aggregation induced by pressure. Prior work reported SANS measurements for MAb1 and will be compared to those for MAb2 below Figure A shows isothermal SANS profiles measured at ambient temperature for MAb2 up to 200 MPa and after returning to atmospheric conditions.…”

“…Small-angle neutron scattering was measured for MAb2 samples at 10 mg/mL by using the previously described liquid insertion pressure system on the NGB30SANS beamline at the NIST Center for Neutron Research (Gaithersburg, MD) . Similar high-pressure SANS experiments were conducted for MAb1 in prior work . Three sample-to-detector distances of 2, 8, and 13 m were used to collect scattering data in the Q range between 0.001 and 0.30 Å –1 , where Q = 4π­(sin θ)/λ and 2θ is the scattering angle.…”

“…45 Similar high-pressure SANS experiments were conducted for MAb1 in prior work. 18 Three sample-to-detector distances of 2, 8, and 13 m were used to collect scattering data in the Q range between 0.001 and 0.30 Å −1 , where Q = 4π(sin θ)/λ and 2θ is the scattering angle. A neutron wavelength (λ) of 6.0 Å was used for medium and long sample-to-detector distances, and 4.5 Å was used for the 2 m configuration, with a wavelength spread Δλ/λ of 0.14 for all configurations.…”

“…Aggregation rates in solutions below 0 °C have recently been measured with an “isochoric method” (see Figure for context). , Rosa et al used the isochoric method to establish an empirical link between high- and low-temperature aggregation rates for bovine hemoglobin. The isochoric method enables the study of proteins at storage temperaturesincluding below 0 °Cwhile maintaining the liquid phase.…”