Page 43 - Pharmaceutical- Guide to Biopharmaceutical

Characterization Quality Control

Evaluating the Thermal Stability of Proteins DSC-60 Plus

Using a Differential Scanning Calorimeter to benefits

Measure the Thermal Stability of Proteins Cell Line Optimization
click here • The thermal stability of proteins can be easily evaluated.
• The stability due to pH or solvent differences can be evaluated.
Operating Principle and Features DSC
mW • With the built-in liquid nitrogen cooling chamber, the system can be used to evaluate
A differential scanning calorimeter (DSC) can measure the enthalpy the protein effects of freezing.
changes in heat energy generated (endothermic or exothermic) as a
sample is heated or cooled. Sample and reference solutions are placed 0.20 Culture
in approx. 6 mm diameter cells, with the cells placed in thermally
symmetric positions within the furnace, and then the furnace is heated
or cooled at a constant rate. For example, when proteins are heated at
a constant rate, denaturation can cause the three-dimensional structure 0.00
to begin unfolding. DSC systems can measure the thermal changes that
occur during that process as endothermic peaks (thermal denaturation 74.9 ﾟC
temperatures). Due to its superior baseline stability, the DSC-60 Plus
can easily measure the thermal changes of samples in solution. -0.20

Operating Procedure and Measurement 40.00 60.00 80.00 100.00 Purification
Conditions Fig. 1 Endothermic Peak of 2.5 % Lysozyme Solution
Temp [ﾟC ]

Samples were prepared by diluting lysozyme from chicken egg white
with a phosphate buffer solution (pH 7.05) to the concentrations DSC
indicated in Table 1. Then, 20 µL of the sample was sealed with an mW
aluminum hermetic cell. Using 20 μL of the phosphate buffer solution as 0.10
a reference sample, the samples were heated from 35 to 105 °C at a rate
of 5 °C per minute to measure the thermal denaturation temperatures.
To investigate the effect of protein pH, three 0.2 mol/L phosphate
buffer solutions with pH 4.20, pH 7.05, and pH 9.10, were used as Characterization
solvents for preparing and measuring the 10 % lysozyme solutions. 0.00
The temperature was increased from 40 to 100 °C at a rate of 5 °C per 75.1 ﾟ C
minute.
Table 1 Samples Used
Sample Concentration
Lysozyme in Fig. 1 2.5 % of protein -0.10
Lysozyme in Fig. 2 0.2 % of protein Specifications
40.00 60.00 80.00 100.00
Temp [ﾟC ]
Results Fig. 2 Endothermic Peak of 0.2 % Lysozyme Solution Instrument DSC-60 Plus Quality Control
Method Heat flow
With 0.2 % or 2.5 % lysozyme, endothermic peaks from thermal DSC
mW Measurement temperature range -140 to 600 °C (when using liquid nitrogen with cooling chamber included standard)
denaturation appear near 75 °C, which confirms that protein thermal
2.00
denaturation temperatures can be measured in dilute 0.2 % solutions Heat measurement range ±150 mW
(Fig. 1 and 2). ー pH9.10 Baseline noise 0.5 μW max. (RMS value for a blank held at 150 °C)
It also shows that stability is highest for lysozyme with pH 4.20, which ー pH7.05
1.00 Atmosphere Nitrogen, inert gas, or dry air gas flow
had the highest thermal denaturation temperature (Fig. 3). ー pH4.20
External dimensions W 320 mm × D 500 mm × H 290 mm
Conclusion 0.00 Weight 28 kg
Pharmacokinetics
DSC systems can easily measure the thermal denaturation temperatures 72.01ﾟC Power requirement 100 / 120 / 220 / 230 / 240 V AC ±10 %, 50/60 Hz, 800 VA
of proteins and can be used for evaluating the thermal stability -1.00 72.72ﾟC 77.43ﾟC Optional SSCP-1 sample sealer and crimp press
to provide an index for a variety of other evaluations, such as for Cell compatible with crimp attachment *1
evaluating the stability of modified proteins or considering different Cell compatible with sealing attachment *2
storage solvents. -2.00 Aluminum crimped cell *1
Aluminum sealed cell *2
40.00 60.00 80.00 100.00
Application Examples (Shimadzu Application News No.) Fig. 3 Changes in Lysozyme Stability depending on pH *1 Used in Application News T152 Others
Temp [ﾟC ]
*2 Used in Fig. 1 to 3 (p. 42)
• Evaluating the stability of proteins
• Evaluating crystal polymorphism in pharmaceuticals (T152)

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