Targeted CQA analytical control strategy for commercial antibody products: Replacing ion-exchange chromatography methods for charge heterogeneity with multi-attribute monitoring

Figures & data

Figure 1. Representative IEC chromatogram for the mAb DS. The red dotted lines indicate the collected fractions across the three acidic peaks, the main peak, and the basic peak. The symbols represent the product specific charge variants (CDR deamidation (square), glycation (star), LMWS (oval), N-terminal cyclization (pentagon), and HMWS). The symbols for CQAs and non-CQAs are outlined in red and black font, respectively.

UV chromatographic trace of the mAb DS ion exchange chromatogram showing three acidic peaks, a main peak, and a single basic peak. The main peak is the largest. CDR deamidation, glycation, and LMWS are labeled above the acidic peaks. HMWS and N-terminal cyclization are labeled above the basic peak.

Table 1. Summary of mAb charge variant characterization.

Method	Attribute	mAb DS	Acidic Fraction 1	Acidic Fraction 2	Acidic Fraction 3	Main Peak Fraction	Basic Fraction
Bioassay	Potency (%)^a	108	<25	33	36	98	78
LC-MS/MS	HC N-Terminal Cyclization (%)	1.4	0.6	0.5	0.8	0.2	22.6
	CDR Deamidation (%)	11.7	49.1	78.5	85.5	4.7	9.3
	Fc Deamidation (%)	3.3	3.3	3.5	3.8	3.7	3.9
LC-MS	Total Glycation (%)	5.4	46.4	11.4	6.4	4.3	6.6
IEC	IEC main peak (%)	79.7	1.4	<1.0	1.3	91.9	44.2
	IEC acidic peak (1%)	1.1	91.1	12.8	7.3	<1.0	2.0
	IEC acidic peak (2%)	6.7	5.9	80.7	6.6	4.3	2.3
	IEC acidic peak (3%)	9.7	1.7	5.4	84.6	1.4	8.0
	IEC basic peak (%)	2.8	0	<1.0	<1.0	<1.0	43.4
SE-HPLC	HMWS (%)	1.3	1.2	1.2	1.6	1.0	10.2
CE-SDS	LMWS (%)^b (non-reduced)	1.2	6.8	2.8	1.7	1.2	1.5
	LMWS (%)^c (reduced)	0.3	17.2	9.2	2.0	1.1	1.8

^aResults are reported relative to the reference standard.

^bIncludes sum of peaks smaller than intact IgG.

^cIncludes sum of peaks smaller than HC or LC.

Table 2. Summary of mAb forced degradation results.

Assay	Attribute	Control Sample Range (min-max)^a	Heat (40°C)^b	High pH (pH 8.6)^b	Low pH (pH 4.0)^b
Bioassay	Potency (%)^c	101–109	40	32	Lack of Similarity^d
LC-MS/MS	CDR Deamidation (%)	11.7–13.3	58.0	76.5	12.1
	Fc Deamidation (1%)	3.6–8.0	10.8	47.3	3.8
	Fc Deamidation (2%)	0.5–0.6	30.3	1.4	0.5
IEC	Main Peak (%)	77.3–79.8	27.3	16.7	49.4
	Sum of Acidic Peaks (%)	16.2–16.8	63.0	79.7	10.2
	Sum of Basic Peaks (%)	3.9–6.6	9.7	3.6	40.4
CE-SDS (NR)^e	LMWS (%)	1.2–1.8	9.3	7.8	1.6
CE-SDS (R)^f	LMWS (%)	0.5–0.5	12.0	2.9	0.6
SE-HPLC	Monomer (%)	98.9–99.0	94.2	98.5	67.3
	HMWS (%)	1.0–1.1	5.1	1.4	32.7

^aRepresents the range of results across all control samples in each stress condition.

^bFinal stress point shown. Intermediate data points were collected.

^cResults are reported relative to the reference standard.

^dIndicates the binding curve did meet similarity criteria to the reference standard.

^eIncludes sum of peaks smaller than intact IgG.

^fIncludes sum of peaks smaller than HC or LC.

Figure 2. (a) Linear regression analysis for CDR deamidation measured by the MAM method and the sum of acidic peaks measured by IEC for a) DP stability samples, (b) heat stress DS at 40°C, and (c) high pH stress DS (pH 8.6).

Three correlation graphs are shown plotting the CDR deamidation (%) against the IEC sum of acidic peaks (%) for (a) mAb DP stability (5°C, 25°C, 40ºC) samples, (b) heat stress mAb, and (c) high pH stress mAb. The results show a strong linear correlation over a wide range of deamidation values in all plots.

Figure 3. a) Linear regression analysis for the unmodified (non-CDR deamidation) peptide measured by the targeted MAM method and the main peak measured by IEC for a) DP stability samples, b) heat stress DS at 40°C, and c) mAb high pH stress (pH 8.6).

Three correlation graphs are shown plotting the unmodified peptide (%) against the IEC main peak (%) for (a) mAb DP stability (5°C, 25°C, 40ºC) samples, (b) heat stress mAb, and (c) high pH stress mAb. The results show a strong linear correlation over a wide range of unmodified peptide values in all three plots.

Figure 4. The correlation between HMWS measured by SE-HPLC and the sum of basic peaks measured by IEC is shown for low pH stress mAb.

Correlation plot of the HMWS (%) against the IEC sum of basic peaks (%) for low pH stress mAb. The results show a strong linear correlation.