A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins

Figures & data

Figure 1. Overlay of UV spectrum with SIR channels. (A) Disulfide bond modifications were monitored between 3 to 7 min. (B) CDR isomerization, PENNY peptide deamidation and Met-252 oxidation were monitored between 16 to 27 min.

Figure 2. Correlation between total afucosylation (sum of all afucosylated glycans) and relative afucosylation to G0f [(G0 + G0-GN + M5) ÷ (G0 + G0-GN + M5 + G0f)] using HILIC oligosaccharide profiling data.

Figure 3. (A) Glycopeptides with G0, G0-GN, M5, and G0f were monitored by SIR between 37 to 41 min. (B) Linear correlation was confirmed between the total afucosylation measured by HILIC and the relative afucosylation to G0f measured by QDa-based MAM.

Figure 4. (A) Modification levels measured by QDa-based MAM in MAB1 stability samples at initial time point (blue), 3 months at 4°C (green), 3 months at 25°C (orange), and 3 months at 40°C (purple). (B) CDR isomerization quantified by LC-UV-based method (blue) and by QDa-MAM (red). (C) CDR deamidation quantified by tryptic peptide mapping (blue) and QDa-MAM (red). (D) PENNY peptide deamidation quantified by tryptic peptide mapping (blue) and QDa-MAM (red). (E) HL thioether bond quantified by reducing gel electrophoresis (blue) and QDa-MAM (red).

Figure 5. (A) Modification levels measured by QDa-based MAM in MAB1 photo-stressed samples at initial time point (blue), dark control (green), light irradiated at the dosage per ICH guideline (1X ICH, orange), and light irradiated at 3 times the strength per ICH guideline (3X ICH, purple). (B) Met-252 oxidation quantified by tryptic peptide mapping (blue) and QDa-MAM (red).

Table 1. Summary of qualification results for repeatability and intermediate precision.

			Intermediate precision
Post-Translational Modifications (PTMs)		Precision-Repeatability (6 preps × 1 injection)	Day-to-Day (3 preps × 1 injection / day, 2 days)	Analyst-to-Analyst (3 preps × 1 injection / analyst, 2 analysts)	Column-to-Column (3 preps × 1 injection / column, 2 columns)	Instrument-to-Instrument (3 preps × 1 injection / instrument, 2 instruments)
CDR isomerization (%)	Mean	3.8	3.9	3.6	3.9	3.9
	% CV	1.9	1.2	7.4	1.7	1.4
CDR deamidation (%)	Mean	3.1	3.2	3.2	3.2	3.2
	% CV	1.4	2.3	2.4	2.4	5.0
PENNY deamidation (%)	Mean	2.7	2.7	2.7	2.6	2.7
	% CV	3.1	2.8	1.9	7.9	2.4
Met-252 oxidation (%)	Mean	1.8	1.8	1.8	1.9	2.0
	% CV	0.9	3.3	7.0	5.0	8.0
HL trisulfide bond (%)	Mean	2.0	2.0	2.0	2.0	1.9
	% CV	3.3	1.4	1.6	2.6	2.0
HL thioether bond (%)	Mean	0.5	0.5	0.5	0.5	0.5
	% CV	9.3	7.1	7.6	7.5	6.4
Afucosylation (%)	Mean	17.0	17.4	17.4	17.2	16.9
	% CV	0.3	2.2	2.8	1.6	0.9

Table 2. Summary of qualification results for linearity, accuracy, Limit of Quantitation (LOQ), and Limit of Detection (LOD).

	Linearity and Range				Accuracy
	R^2	Range	LOQ, from calibration curve, 10 (SD/S)	LOD, from calibration curve, 3.3 (SD/S)	Level	% Recovery	% CV
CDR isomerization (%)	0.9995	4.1 – 65.7	3.3	1.1	Low	107.0	1.7
					Middle	99.0	0.4
					High	100.0	0.8
CDR deamidation (%)	0.9981	3.2 – 10.7	1.2	0.4	Low	106.0	0.3
					Middle	102.0	0.9
					High	103.0	0.5
PENNY deamidation (%)	0.9996	2.5 – 50.1	2.6	0.8	Low	103.0	1.7
					Middle	101.0	2.9
					High	99.0	0.9
Met-252 oxidation (%)	0.9977	3.1 – 46.2	4.8	1.6	Low	104.0	3.9
					Middle	96.0	2.4
					High	100.0	0.0
HL trisulfide bond (%)	0.9970	2.0 – 11.1	1.6	0.5	Low	96.0	0.6
					Middle	105.0	4.5
					High	103.0	8.8
HL thioether bond (%)	0.9979	0.5 – 13.6	1.4	0.5	Low	96.0	1.1
					Middle	106.0	0.1
					High	102.0	2.6
Cys-214 free thiol (%)	0.9936	0 – 3.1	0.6	0.2	Low	160.0	3.6
					Middle	99.0	1.3
					High	104.0	6.0
Afucosylation (%)	0.9917	8.3 – 31.4	6.7	2.2	Low	99.0	0.9
					Middle	98.0	0.3
					High	101.0	0.4

Figure 6. (A) Modification levels measured by QDa-based MAM in serum incubated samples at initial time point (blue), after 0.5 week at 37°C (green), after 1 week at 37°C (orange), and after 2 weeks at 37°C (purple). MAB1 incubated in PBS after 2 weeks at 37°C was tested side by side as control (gray). (B) Slight afucosylation increase in serum incubated samples was mainly contributed by the increase in Man-5.

Figure 7. (A) Modification levels measured by QDa-based MAM in cell culture daily samples. (B) Total afucosylation by HILIC (blue) and predicated afucosylation by QDa-MAM (red, Predicted afucosylation = 0.8118 × Measured relative afucosylation by MAM – 0.1586) in cell culture daily samples.

Figure 8. Modification levels measured by QDa-based MAM in in downstream purification process.