Comparability of a Three-Dimensional Structure in Biopharmaceuticals Using Spectroscopic Methods

Table 8

Troubleshooting for DTNB and DyLight 488 Maleimide methods.

Problem	Cause	Solution
Native conditions
High variability on measurements.	Low protein quantity.	Increase protein concentration to 20–30 mg·mL^−1.
	Samples homogeneity.	Always vortex protein solutions.
High background.	Particles in buffer solution.	Filter buffer solutions through 0.2 μm hydrophilic membrane.
High sulfhydryl measurement.	Sample stress.	Avoid high temperatures and analyze samples within 12 hours once prepared. Dialyze samples at 4°C.
	Sample excipients interference.	Dialyze samples against water.
Denaturing conditions
Low sulfhydryl measurement	pH lower than 6.5.	Verify buffer solution pH after GdnHCl addition.
High sulfhydryl measurement using DyLight 488 Maleimide.	pH higher than 7.5.	Verify buffer solution pH after GdnHCl addition.
	Overincubation.	2 hours of incubation must be enough for derivatization.
	Incomplete dialysis.	Increase dialysis time and buffer exchange.
Denaturing-reducing conditions
Low sulfhydryl measurement.	Oxidized thiols.	Degasify all buffer solutions by sonication during 30 min and nitrogen bubbling during 2–15 min prior its use.
High background at 280 nm using DTNB.	TNB interference.	Estimate protein concentration prior derivatization.