High pressure cell for Bio-SANS studies under sub-zero temperatures or heat denaturing conditions

Teixeira, Susana; Leão, Juscelino B.; Gagnon, Cedric; McHugh, Mark A.

doi:10.3233/jnr-180057

Cited by 10 publications

(7 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The HP-SANS cell unit used in this project was built at the National Institute of Standards and Technology (NIST) Center for Neutron Research (NCNR), in Gaithersburg, MD. This system is designed for studies of biological macromolecule solutions (BioSANS) over a broad temperature range, as described by Teixeira et al 55 The sample volume requirement is ∼3 mL. Data were collected at a neutron wavelength of 6 Å and a wavelength spread of Δλ/λ = 0.138, covering a scattering vector amplitude range of 0.003 < q (Å −1 ) < 0.25.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

View full text Add to dashboard Cite

show abstract

“…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 .…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

View full text Add to dashboard Cite

The effects of high pressure and low temperature on the stability of two different monoclonal antibodies (MAbs) were examined in this work. Fluorescence and small-angle neutron scattering were used to monitor the in situ effects of pressure to infer shifts in tertiary structure and characterize aggregation prone intermediates. Partial unfolding was observed for both MAbs, to different extents, under a range of pressure/temperature conditions. Fourier transform infrared spectroscopy was also used to monitor ex situ changes in secondary structure. Preservation of native secondary structure after incubation at elevated pressures and subzero ° C temperatures was independent of the extent of tertiary unfolding and reversibility. Several combinations of pressure and temperature were also used to discern the respective contributions of the isolated Ab fragments (Fab and Fc) to unfolding and aggregation. The fragments for each antibody showed significantly different partial unfolding profiles and reversibility. There was not a simple correlation between stability of the full MAb and either the Fc or Fab fragment stabilities across all cases, demonstrating a complex relationship to full MAb unfolding and aggregation behavior. That notwithstanding, the combined use of spectroscopic and scattering techniques provides insights into MAb conformational stability and hysteresis in high-pressure, low-temperature environments.

show abstract

“…The pressure cell used was capable of a maxim and a maximum temperature of 80 °C. A detailed design for a temperature-controlled high-pressur logical applications, specifically protein denaturation studies, w et al [33]. The sample cell was capable of a low temperature of − 2 kbar.…”

Section: Pressurementioning

confidence: 99%

“…The pressure jumps were on the order of esting variation here is that the data acquisition time per frame w ing with a time frame length of 0.05 s that was elongated by a fact thus anticipating that events evolved fastest immediately after th ously, this has some consequences regarding the statistical qua experiment only had to be repeated five times. This is another a A detailed design for a temperature-controlled high-pressure cell developed for biological applications, specifically protein denaturation studies, was described by Teixeira et al [33]. The sample cell was capable of a low temperature of −18 • C with a pressure of 2 kbar.…”

Section: Pressurementioning

confidence: 99%

Soft Matter Sample Environments for Time-Resolved Small Angle Neutron Scattering Experiments: A Review

et al. 2021

View full text Add to dashboard Cite

With the promise of new, more powerful neutron sources in the future, the possibilities for time-resolved neutron scattering experiments will improve and are bound to gain in interest. While there is already a large body of work on the accurate control of temperature, pressure, and magnetic fields for static experiments, this field is less well developed for time-resolved experiments on soft condensed matter and biomaterials. We present here an overview of different sample environments and technique combinations that have been developed so far and which might inspire further developments so that one can take full advantage of both the existing facilities as well as the possibilities that future high intensity neutron sources will offer.

show abstract

High pressure cell for Bio-SANS studies under sub-zero temperatures or heat denaturing conditions

Cited by 10 publications

References 57 publications

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

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

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

Soft Matter Sample Environments for Time-Resolved Small Angle Neutron Scattering Experiments: A Review

Contact Info

Product

Resources

About