Physicochemical and biological impact of metal-catalyzed oxidation of IgG1 monoclonal antibodies and antibody-drug conjugates via reactive oxygen species

Figures & data

Figure 1. Effects of metal catalyzed oxidation (MCO) by Cu(II)/Asc and Fe(II)/H₂O₂ on Fc (a) M255 and (b) M431 by LC-MS Tryptic Peptide Mapping.

Figure 2. Effects of MCO on (a) trastuzumab and (b) T-DM1 using Pearson correlation analysis. Pearson’s r-values outlined in yellow have P-value <0.05.

Table 1. Impact of MCO on Fc Met255 and Met431 oxidation, size variants changes, and carbonyl formation for mAbs and ADCs after various time points at 37°C. n = 3.

			Peptide map		SEC		ELISA
Sample	Treatment (mM)	Timepoint	Fc Met255 (% Oxidation)	Fc Met431 (% Oxidation)	Total (% HMWF)	Total (% LMWF)	(nmoles carbonyls/ mg protein)
trastuzumab		T0	3	1.1	0.1	0.2	0.71
T-DM1			3.1	1.3	2.0	0.2	0.46
NaPi2b			3.5	1.6	0.3	0.2	0.61
NaPi2b-vc-MMAE			3.7	1.9	0.4	0.2	0.30
trastuzumab	0.01 mM Cu(II), 1 mM Asc	6 hr, 37°C	7.4	2.5	18.3	24.9	40.13
T-DM1			7.4	2.5	25.8	20.0	41.18
NaPi2b			5.6	2.5	21.1	21.2	40.54
NaPi2b-vc-MMAE			7.0	3.5	32.2	23.3	39.55
trastuzumab	0.01 mM Cu(II), 1 mM Asc	24 hr, 37°C	29.4	13.1	25.6	25.1	40.28
T-DM1			28.4	11.7	38.4	21.6	38.55
NaPi2b			22.0	8.1	23.7	24.2	36.62
NaPi2b-vc-MMAE			27.5	11.9	44.4	23.3	34.69
trastuzumab	0.05 mM Fe(II) 1 mM H2O2	3 hr, 37°C	48.5	25.5	0.5	1.2	4.95
T-DM1			48.1	25.3	2.7	1.0	5.21
NaPi2b			47.3	25.7	0.5	1.2	5.74
NaPi2b-vc-MMAE			48.8	29.0	1.2	4.8	2.99
trastuzumab	0.5 mM Fe(II), 0.1 mM H2O2	3 hr, 37°C	18.3	12.5	7.1	10.7	2.16
T-DM1			19.1	13.0	12.6	4.1	1.79
NaPi2b			17.8	12.5	6.6	9.6	1.97
NaPi2b-vc-MMAE			18.9	16.9	13.2	14.4	1.16

Figure 3. Effects of MCO on carbonylation (nmols carbonyls/mg protein) for (a) trastuzumab, (b) T-DM1, (c) NaPi2b, and (d) NaPi2b-vc-MMAE after various time points at 37°C. The cross (+) indicates results of the untreated control and values that are not significantly different from the untreated control. Error bars represent standard deviation where n = 3. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 4. Impact of MCO on mAb and ADC peptide level (a, c) % oxidation and (b, d) % carbonylation as detected by LC-MS Tryptic Peptide Mapping. Treatment conditions = 0.01 mM Cu(II)/1 mM Asc 24 hr, 37°C and 0.05 Fe(II)/1 mM H₂O₂ up to 3hr, 37°C.

Table 2. Effects of MCO on trastuzumab binding to FcRn, FcγRIIIa, and HER2 antigen after metal catalyzed oxidation as measured by SPR.

	FcRn binding^a			FcγIIIa binding^a			HER2 Antigen Binding^b
SAMPLES	ka (M^−1s^−^Citation1) x 10^5	kd (s^−1) x 10^–2	KD (µM)	ka (M^−1s^−^Citation1) x 10^5	kd (s^−1) x 10^–2	KD (µM)	ka (M^−1 s^−1) x 10^5	kd (s^−1) x 10^–6	KD (pM)
trastuzumab (control)	26.1 ± 2.9	2.9 ± 0.7	0.011 ± 0.001	0.5 ± 0.1	1.7 ± 0.09	0.33 ± 0.08	15.5 ± 8.4	4.6 ± 3.0	4.1 ± 4.2
0.01 mM Cu(II)/1 mM, Asc, 6 hr,37°C	0.077 ± 0.007	2.3 ± 1.5	3.1 ± 2.2	NB	NB	NB	11.8 ± 7.0	39.8 ± 36.0	51.7 ± 6.1
0.01 mM Cu(II)/1 mM, Asc, 24 hr,37°C	0.006 ± 0.003	1.2 ± 0.4	24.8 ± 19.8	NB	NB	NB	5.3 ± 0.28	90.3 ± 6.2	171.0 ± 2.8
0.05 mM Fe(II)/1 mM H2O2, 1.5 hr, 37°C	12.4 ± 0.3	2.6 ± 0.5	0.021 ± 0.003	0.2 ± 0.03	1.6 ± 0.02	0.76 ± 0.11	7.1 ± 0.82	4.9 ± 4.9	6.6 ± 6.1
0.05 mM Fe(II)/1 mM H2O2, 3 hr, 37°C	8.3 ± 0.2	1.6 ± 1	0.019 ± 0.012	0.2 ± 0.04	1.6 ± 0.03	0.67 ± 0.09	7.4 ± 1.2	36.9 ± 50.5	55.7 ± 76.7

NB = no binding

^an = 3

^bn = 2

Figure 5. Effects of MCO on Antibody-Dependent Cellular Cytotoxicity (ADCC) for (a) trastuzumab, (b) T-DM1, (c) NaPi2b, and (d) NaPi2b-vc-MMAE at 37°C after various timepoints. The cross (+) indicates results of the untreated control and values that are not significantly different from the untreated control. Error bars represent standard deviation where n = 3. *p < 0.05, **p < 0.01. Note that signal with anti-NaPi2b-vc-MMAE exceeded the upper limit of quantification and was therefore not included in panel d for statistical analyses.

Table 3. Effects of MCO on BT-474 cell proliferation for trastuzumab.

Treatment Conditions	Relative Specific Activity (Compared to Control) ^a
Control (reference material)	1.0
0.01 mM Cu(II)/1 mM Asc, 6 hrs, 37°C	No Activity^b
0.01 mM Cu(II)/1 mM Asc, 24 hrs, 37°C	No Activity^b
0.05 mM Fe(II)/1 mM H2O2 1.5 hrs, 37°C	0.79
0.05 mM Fe(II)/1 mM H2O2, 3 hrs, 37°C	0.23^c

^aMethod measured in specific activity (x10^4 U/mg)

^b“No Activity” is slope = 0

^cFailed slope ratio, estimated calculation

Figure 6. Impact of MCO on autophaghy for (a) trastuzumab, (b) T-DM1, (c) NaPi2b, and (d) NaPi2b-vc-MMAE.

Figure 7. Effects of MCO on the mAb and ADC CDR, hinge, FcRIII and FcRn binding sites. IgG1 homology structure with mapped oxidation levels impacted by either (a) Fe(II)/H₂O₂ (red) or (b) Cu(II)/Asc (green) to the functional sites. Note: hinge residue “H224” is oxidized specifically by Cu(II), and not Fe(II). Cu(II) Model of IgG1 constructed using 1BJ1 and 1IGY crystal structures

Chinthaginjala H, Kalpana K, Manchikanti SP, Reddy PG, Karamthoy S, Vennapusa NR. Biotherapeutics as drugs, its delivery routes and importance of novel carriers in biotherapeutics. Int J Of Pharma Sci Res. 2021;12:44–19.

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