Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry

Figures & data

Figure 1. SEC-UV chromatograms of mAb1 (a) and mAb2 (b) stability samples at T0 and after 3-month incubation at 25°C (“3M25C”). Inserts in panel (a) and (b): enlarged area to display the HMW peaks.

SEC chromatogram of mAb1 with an insert showing enlarged area of the HMW peaks. SEC chromatogram of mAb2 with an insert showing enlarged area of the HMW peaks.

Table 1. Major species detected by SEC-UV-native MS in mAb1 stability samples at T0 and after 3-month incubation at 25°C (“3M25C”).

Sample name	Retention time (min) and peak naming	Detected mass (Da)	Assigned identity	Theoretical mass^# (Da)	% UV peak area
T0	6.3 (HMW)	290.5 k	mAb1 dimer	290,194	0.6
	7.1 (main peak)	145,094	mAb1	145,097	98.0
	7.7 (LMW1)	98,950	Fab/c*	98,958	1.4
3M25C	6.3 (HMW 1)	290.5 k	mAb1 dimer	290,194	1.8 (total HMWs)
	6.5 (HMW 2)	244.5 k	mAb1-Fab/c	244,054
	6.6 (HMW 3–1)	198.5 k	Fab/c dimer	197,915
	6.7 (HMW 3–2)	191.0 k	mAb1-Fab	191,254
	6.9 (main peak)	145,092	mAb1	145,097	95.6
	7.7 (LMW1)	98,960	Fab/c*	98,958	2.1
	8.6 (LMW2)	46,159	Fab*	46,157	0.4

*Masses of Fab/c and Fab fragments were calculated based on cleavage site KSCDKTH/TCPPC.

^#All the theoretical masses of mAb1 and Fab/c were calculated based on two copies of FA2 glycan were included.

Table 2. Major species detected by SEC-UV-native MS in mAb2 stability samples at T0 and after 3-month incubation at 25°C (“3M25C”).

Sample Name	Retention time (min) and Peak Naming	Detected mass (Da)	Assigned identity	Theoretical mass (Da)	%UV peak area
T0	6.6 (HMW)	289.4 k	mAb2 dimer	288,882	0.8
	7.5 (main peak)	144,441	mAb2	144,441	97.4
	8.0 (LMW1)	97,118	Fab/c*	97,123	1.8
3M25C	6.5 (HMW)	289.4 k	mAb2 dimer	288,882	2.8
	7.4 (main peak)	144,442	mAb2	144,441	94.5
	8.0 (LMW1)	97,124	Fab/c*	97,123	2.1
	9.3 (LMW2)	47,335	Fab*	47,336	0.6

*Fab/c and Fab fragments were identified with cleavage site KSCDKTH/TCPPC.

Figure 2. Generation of Fab/c fragment to study Fab/c associated aggregation. (A) Limited IgdE enzymatic digestion of mAb1 and mAb2 to generate Fab/c fragments; (b) separation of digests by hydrophobic interaction chromatography; (C) stability study on the collected mAbs and Fab/c fractions from HIC and their mixture followed by SEC-UV-native MS characterization.

A schematic describing the limited IgdE digestion of mAb1 and mAb2 where cutting site is KSCDKT/HTCPPCP and the digest contain Fab/c and Fab fragment. HIC chromatograms of mAb1 IgdE digest and mAb2 IgdE digest showing four components, Fab, Fc, Fab/c, and left mAb, were separated. A schematic illustrating the design of the stability study on purified Fab/c fraction, mAb fraction and their 1:1 mixture of two mAbs. Briefly, they were placed on thermal acceleration for three months and monitored by the SEC-UV-native MS method at four time points.

Figure 3. Characterization of three mAb1 stability samples containing Fab/c fragments after a three-month incubation at 25°C by the SEC-UV-native MS method. a) SEC-UV chromatograms of mAb1 fraction, 1:1 mixture and Fab/c fraction at time point of three months (“3M25C”); insert panel: enlarged area to display the HMW peaks; b) mass spectra of HMW peaks, with the deconvoluted masses and the corresponding dimer species shown; the charge states highlighted in blue were used for the construction of XICs and peak area calculation; the charge states highlighted in orange were interference peaks from mAb1 dimer; c) plots of total HMWs in mAb1 fraction, 1:1 mixture and Fab/c fraction based on UV peak area; d) plots of major species in mAb1 fraction, e) 1:1 mixture and f) Fab/c fraction based on UV peak area.

Figure 4. Plots of dimer species formation based on UV peak area in (a) mAb1 fraction, (b) 1:1 mixture and (c) Fab/c fraction. Plots of dimer species formation based on XIC peak area in (d) mAb1 fraction, (e) 1:1 mixture and (f) Fab/c fraction.

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Plots of HMW1, HMW2, and HMW3 based on UV peak area in mAb1 fraction. All three HMWs increased with a comparable level and in a similar pattern. Plots of HMW1, HMW2, and HMW3 based on UV peak area in 1:1 mixture. HMW1 almost remained the same level throughout the incubation. HMW2 and HMW3 increased at a comparable rate. Plots of HMW1, HMW2, and HMW3 based on UV peak area in Fab/c fraction. HMW1 was barely detected and remained unchanged throughout the incubation. HMW3 increased significantly followed by HMW2. Plots of mAb1 dimer, mAb1-Fab/c dimer, Fab/c dimer, and mAb1-Fab based on XIC peak area in mAb1 fraction. mAb1 dimer and mAb1-Fab showed a similar trending. Similar observation was for mAb1-Fab/c and Fab/c dimer. The increase rate of the first two was higher than the second two. Plots of mAb1 dimer, mAb1-Fab/c dimer, Fab/c dimer, and mAb1-Fab based on XIC peak area in 1:1 mixture. The ranking of the increase rate from high to low is Fab/c dimer, mAb1-Fab/c, mAb1-Fab, mAb1 dimer. Plots of mAb1 dimer, mAb1-Fab/c dimer, Fab/c dimer, and mAb1-Fab based on XIC peak area in Fab/c fraction. The ranking of the increase rate from high to low is Fab/c dimer, mAb1-Fab/c, mAb1-Fab, mAb1 dimer. Fab/c shows a much higher increase rate than the other three.

Figure 5. Characterization of three mAb2 stability samples containing Fab/c fragments after three-month incubation at 25°C by the SEC-UV-native MS method. a) SEC chromatograms of mAb2 fraction, 1:1 mixture and Fab/c fraction; insert panel: enlarged area to display the HMW peaks; b) Mass spectra of detected dimer species under the HMW peak; deconvoluted masses and cartoon illustrated identifications are shown on the right side; the charge states labeled and highlighted in blue of each dimer species were used for the construction of XICs and peak area calculation; the charge states highlighted in orange were interference peaks from mAb2 dimer.

Figure 6. Plots of total dimer species change during 25°C stability incubation for (a) mAb2 fraction, (b) 1:1 mixture and (c) Fab/c fraction based on UV peak area. Plots of formation of each dimer species in (d) mAb2 fraction, (e) 1:1 mixture, and (f) Fab/c fraction based on mass spec XIC peak area.

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A plot of total dimer species vs. incubation time based on UV peak area in mAb2 fraction. The increase throughout the incubation was steady with a level change of 0.6%. A plot of total dimer species vs. incubation time based on UV peak area in 1:1 mixture. The increase throughout the incubation was steady with a level change of 0.5%. A plot of total dimer species vs. incubation time based on UV peak area in Fab/c fraction. The increase throughout the incubation was steady with a level change of 0.2%. Plots of mAb2 dimer, mAb2-Fab/c, and Fab/c dimer based on XIC peak area in mAb2 fraction. The ranking of the increase rate from high to low is mAb2 dimer, mAb2-Fab/c, Fab/c dimer. Plots of mAb2 dimer, mAb2-Fab/c and Fab/c dimer based on XIC peak area in 1:1 mixture. The ranking of the increase rate from high to low is Fab/c dimer, mAb2-Fab/c, mAb2 dimer. Plots of mAb2 dimer, mAb2-Fab/c and Fab/c dimer based on XIC peak area in Fab/c fraction. Fab/c dimer increased significantly while mAb2-Fab/c only increased slightly. mAb2 dimer was barely detected and remained at minimal level.

Figure 7. SEC-UV chromatograms of a) deglycosylated mAb1 and b) control sample at T0, one-month (“1 M”), two-months (“2 M”) and three-month (“3 M”) time points of incubation at 25°C. The two post monomer peaks with retention times of approximately 19.5 min and 23.0 min were the fab/c fragment and fab fragment, respectively. C) a plot of %HMWs vs. incubation time (month) based on UV peak areas of deglycosylated mAb1 and control sample.