Ultracentrifuge Rotor Temperature Measurements and Control

Fabricant, S. J.; Windsor, Alfred A.; Lindgren, Frank T.

doi:10.1063/1.1720224

Cited by 7 publications

(7 citation statements)

References 4 publications

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“…Instead, we have pursued a more direct temperature measurement, continuing the strategy of thermistors located in the rotor [53,56], by using modern inexpensive computer chips that can be gently centrifuged while autonomously logging the rotor temperature. We have supported the temperature corrections to the instrument calibration by independent hydrodynamic measurements of the ensuing effect on the solvent viscosity with simultaneous radial magnification corrections (Figure 6).…”

Section: Discussionmentioning

confidence: 99%

“…Historically, in the Model E, thermistors were placed at the base of the rotor and electrical contact was established through a needle on the spinning rotor inserted into a stationary mercury pool inside the rotor chamber [51]. Alternative designs for the use of thermistors for suspended rotors without electrical contact used radio-telemetry [53,56]. Along this line, another form of wireless device has more recently become available in the form of the Thermochron iButton® (Maxim Integrated Products, Inc.), which is a small steel-enclosed computer chip that can log NIST-traceable calibrated temperature readings at a resolution of up to 0.0625 °C.…”

Section: Introductionmentioning

confidence: 99%

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Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references

Ghirlando

Balbo

Piszczek

et al. 2013

Analytical Biochemistry

106

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Sedimentation velocity (SV) is a method based on first-principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures. We implemented the use of a Thermochron iButton® temperature logger to directly measure the temperature of a spinning rotor, and detected deviations that can translate into an error of as much as 10% in the sedimentation coefficient. We further designed a precision mask with equidistant markers to correct for instrumental errors in the radial calibration, which were observed to span a range of 8.6%. The need for an independent time calibration emerged with use of the current data acquisition software (Zhao et al., doi 10.1016/j.ab.2013.02.011) and we now show that smaller but significant time errors of up to 2% also occur with earlier versions. After application of these calibration corrections, the sedimentation coefficients obtained from eleven instruments displayed a significantly reduced standard deviation of ∼ 0.7 %. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references

Ghirlando

Balbo

Piszczek

et al. 2013

Analytical Biochemistry

106

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“…Systems have been developed that were based on electrical conductance [6,19], optical scanning [8], thermal infrared radiation [9,10], and radio telemetry [20]. Taking advantage of recent developments in integrated circuits, it has become possible to omit any information transfer from the spinning rotor, and instead store the measurements for later retrieval.…”

Section: Discussionmentioning

confidence: 99%

Improved measurement of the rotor temperature in analytical ultracentrifugation

Zhao

Balbo

Metger

et al. 2014

Analytical Biochemistry

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Sedimentation velocity is a classical method for measuring the hydrodynamic, translational friction coefficient of biological macromolecules. In a recent study, comparing various analytical ultracentrifuges, we have shown that external calibration of the scan time, radial magnification, and temperature are critically important for accurate measurements (Anal. Biochem., 2013, doi: 10.1016/j.ab.2013.05.011). To achieve accurate temperature calibration, we have introduced the use of an autonomous miniature temperature logging integrated circuit (Maxim Thermochron iButton ™) that can be inserted in an ultracentrifugation cell assembly and spun at low rotor speeds. In the present work, we developed an improved holder for the temperature sensor located in the rotor handle. This has the advantage of not reducing the rotor capacity and allows for a direct temperature measurement of the spinning rotor during high-speed sedimentation velocity experiments up to 60,000 rpm. We demonstrate the sensitivity of this approach by monitoring the adiabatic cooling due to rotor stretching during rotor acceleration, and the reverse process upon rotor deceleration. Based on this, we developed a procedure to approximate isothermal rotor acceleration for better temperature control.

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“…The result of a density inversion is turbulent flow with concomitant mixing of the two adjacent solutions -convection. Historically, proper temperature control has been a challenging part of AUC's development, where even the presences of a very small positive temperature gradient can lead to density inversion and sample convection due to the amplification effect of the gravitational field [32][33][34][35][36][37][38][39].…”

Section: Figurementioning

confidence: 99%

“…Except for the actual cell alignment in the rotor, AUC users depend on the instrument manufacturer's ability to make sure these critical elements (and their constituent components) deliver the required accuracy and precision. Temperature accuracy and control is of critical importance for the proper interpretation of SV-AUC data due to its impact on solvent density and viscosity (at 20 o C, a 1 degree temperature change results in a 4% sedimentation coefficient change) and to avoid adverse density gradient situations that can lead to convection [27][28][29][32][33][34][35][36][37][38][39].…”

Section: Introductionmentioning

confidence: 99%

Boundary convection during velocity sedimentation in the Optima analytical ultracentrifuge

Berkowitz¹,

Laue

2021

Preprint

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Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate means for characterizing solution hydrodynamic and thermodynamic properties. In recent times AUC has found broad application in the biopharmaceutical industry as a first-principle means for quantitatively characterizing biopharmaceuticals. Boundary sedimentation velocity AUC (SV-AUC) analysis is widely used to assess protein aggregation, fragmentation and conformational variants in the same solvents used during drug development and production. SV-AUC is especially useful for the analysis of drug substance, drug product and dosing solution, where other techniques may exhibit solvent matrix issues or concentration limitations. Recently, the only manufacturer of the analytical ultracentrifuge, released its newest (third generation) analytical ultracentrifuge, the Optima, in early 2017 to replace its aging 2nd generation XL series ultracentrifuges. However, SV-AUC data from four Optima units used in conducting characterization work on adeno-associated virus (AAV) has shown evidence of sample convection. Further investigation reveals that this problem arises from the temperature control system design, which is prone to producing destabilizing temperature induced density gradients that can lead to density inversions. The observed convection impacts both the qualitative and quantitative data generated by the Optima. The problem is intermittent and variable in severity within a given Optima unit and between Optima units. This convection appears to be mainly associated with low rotor speeds and dilute samples in dilute solvents, such as AAV samples in formulation buffers containing relatively low concentrations of salts, sugars, etc. Under these conditions it is found that a sufficiently robust stabilizing density gradient is not always present during sedimentation, making the sample susceptible to convection by localized density inversions. Because SV-AUC is used as an analytical tool in making critical decisions in the development and quality control of biotherapeutics, it is imperative to alert users about this potential problem. In general special attention to data quality needs to be made by those researchers working with very large biopharmaceutical particles (e.g. gene therapy products that involve viral vectors or nanoparticles), where the conditions leading to convection are most likely to occur. It is important to note that the XL series analytical ultracentrifuges do not suffer from this problem, indicating that this problem is unique to the Optima. Attributes that reveal the presence of this problem and strategies for its elimination or minimization are provided.

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Ultracentrifuge Rotor Temperature Measurements and Control

Abstract: This is a library Circulating C~py which may be borrowed for two weeks.

Cited by 7 publications

References 4 publications

Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references

Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references

Improved measurement of the rotor temperature in analytical ultracentrifugation

Boundary convection during velocity sedimentation in the Optima analytical ultracentrifuge

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