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

Table 1

Validation results for DTNB and DLM methods.

Validation test	Acceptance criteria	Method result
		DTNB	DyLight 488
Precision (repeatability)	<20%	RSD: 2.52%; (denaturing-reducing conditions).; RSD: 17.3% (native conditions).	RSD: NT%; (denaturing-reducing conditions).; RSD: 13.3% (native conditions).
Accuracy	Total recovery between 95 and 105% in respect to theoretical value.	99.5% recovery using Somatropin (Cys/Protein ratio 3.98)	101.0% recovery using HSA (Cys/Protein ratio 1.01)
Intermediate precision	RSD < 10%	RSD: 3.8%	RSD: 0.66%
Linearity	𝑟^2 ≥ 0.980; Ordinate = 0	𝑟^2 = 0.999 native and denaturing-reducing conditions.; Slope = 0.01285 𝜇M^−1; 𝑟^2 = 0.983 denaturing conditions.; Slope = 0.0093 𝜇M^−1; Forced through zero.	𝑟^2 = 0.990 native and denaturing conditions.; Slope = 465,834 𝜇M^−1; Forced through zero.
Quantification limit	Report value	5 𝜇M	0.1 𝜇M
Selectivity	Cysteine-free protein has the same signal of placebo.	GA: 0.0010; Placebo: 0.0005	PM: 0.0005; Placebo: 0.0005

NT%: not tested.