Structural analysis of a therapeutic monoclonal antibody dimer by hydroxyl radical footprinting

Figures & data

Figure 1. SEC chromatograms of enriched fractions. IgG1 bulk drug substance starting material contains approximately 4% dimer. Enriched dimer is 92% pure, and enriched monomer is 96% pure.

Table 1. Characterization data for SEC-enriched IgG1 dimer and monomer samples

Enriched IgG1 species	Approximate molar mass by SEC-MALLS (g/mol)	% Purity of Enriched Fraction by SEC	% main peak from non-reduced CE-SDS	Potency by HUVEC
Dimer	300 x 10^3 g/mol	92%	83%	42
Monomer	150 x 10^3 g/mol	96%	90%	100

Figure 2. Fragment-SEC chromatograms of papain-digested dimer and monomer. (A) Papain digest of dimer and monomer. Peak 1 is a Fab/Fab associated species, Peak 2 is intact disulfide-linked Fc, and Peak 3 is the monomeric Fab (Table 2). (B) Papain digest incubation time course study of the dimer. Peak 1 (Fab/Fab species) increases with incubation time, while Peak 3 (Fab) decreases. Peak 2 (intact Fc) remains constant. (C) Papain digest incubation time course study of the monomer. Peak 1 (Fab/Fab species) increases with incubation time, while Peak 3 (Fab) decreases. Peak 2 is unchanged.

Table 2. Characterization data for papain-generated fragments

Papain-generated fragment monitored by SEC	Approximate molar mass by SEC-MALLS (g/mol)	Observed MW by LC-MS (Da)	Assigned SEC Peak Identity
Peak 1	100 x 10^3 g/mol	48206 Da, 47605 Da ^a (mass of Fab: LC + HC 1–230 or LC + HC 1–225)	Fab/Fab non-covalent dimer
Peak 2	55 x 10^3 g/mol	52818 Da (mass of intact disulfide-linked Fc)	Intact disulfide-linked Fc
Peak 3	51 x 10^3 g/mol	48204 Da, 47607 Da ^a (mass of Fab: LC + HC 1–230 or LC + HC 1–225)	Fab

Peaks 1, 2, and 3 were prominently observed in the papain digests of both the dimer and monomer samples, though there is more pre-existing Peak 1 in the dimer sample prior to papain-driven in situ Fab dimerization. ^aNon-specific cleavage of the heavy chain hinge region by papain generates Fab species with two heavy chain fragments differentiated by five residues at their C-terminus. Either Fab form is presumed to be involved in the observed in situ dimerization.

Figure 3. Fragment-SEC chromatograms of FabRICATOR®-digested dimer and monomer. FabRICATOR® digest of dimer and monomer samples. Peak 1 is a Fab^ʹ2/Fab^ʹ2 associated species, Peak 2 is monomeric Fab^ʹ2, and Peak 3 is an Fc/Fc fragment species. Peak 1 is only prominent in the dimer sample. Peak 3 is equally present in both the dimer and monomer sample. Refer to Table 3 for characterization data for FabRICATOR®-generated fragments.

Table 3. Characterization data for FabRICATOR®-generated fragments

FabRICATOR®-generated fragment monitored by SEC	Approximate molar mass by SEC-MALLS (g/mol)	Observed MW by LC-MS (Da)	Assigned SEC Peak Identity
Peak 1 ^a	200 x 10^3 g/mol	98767 Da (mass of Fab^ʹ2: Two disulfide-linked Fab arms)	Fab^ʹ2/Fab^ʹ2 non-covalent dimer
Peak 2 ^b	98 x 10^3 g/mol	98769 Da (mass of Fab^ʹ2: Two disulfide-linked Fab arms)	Fab^ʹ2
Peak 3 ^c	50 x 10^3 g/mol	25232 Da (mass of Fc fragment)	Fc/Fc non-covalent dimer

^a Peak 1 is only significantly present in the FabRICATOR® digest of the dimer sample. ^bPeaks 2 is very prominent in the FabRICATOR® digest of the monomer sample only; it is seen as a small minor peak in the Fabricator digest of the dimer. ^cPeaks 3 is observed equally in the FabRICATOR® digests of both the dimer and monomer sample. The Fc/Fc species is inherent to both samples, and is not indicative of the native dimer’s interface region.

Table 4A. Summary of oxidized heavy chain tryptic peptide data

Heavy Chain Tryptic Peptides	Detected Oxidized Residues^a	Non-normalized Rate Constant Ratio^e (Dimer k:Monomer k)	Normalized Rate Constant Ratio^f (Dimer k:Monomer k)	Protection Level Assignment in Dimer Relative to Monomer^g
1–19	V(12), P(14), S(17)	2.6	1.0	No change
20–38	Y(13), M(5), W(17)	2.1	0.8	No change
44–65	W(4), W(7), I(8), Y(11)	2.0	0.8	No change
68–76	F(3), S(8)	4.5	1.7	Decreased
77–87	Y(4), Q(6), M(7)	1.3	0.5	Increased
88–98	A(1), A(10)	4.0	1.5	Decreased
99–127 ^b	Y(4), H(9), W(10), Y(11), W(15)	-	-	-
128–139^c	F(9), P(6)	-	-	No change
140–153	L(9), V(13)	4.0	1.5	Decreased
154–216^d	Approx. Eight oxidized residues	3.0	1.2	No change
229–254	P(5), L(12), P(10)	3.3	1.3	No change
255–261	M(4)	6.2	2.4	Decreased
262–280	P(2), H(13), P(16)	1.8	0.7	No change
281–294	Y(4), V(8), H(11)	2.6	1.0	No change
299–307	Y(4)	2.6	1.0	No change
308–323	V(2), H(9), W(12)	2.5	1.0	No change
333–340	P(5), E(7)	2.7	1.0	No change
351–361	P(2), Y(5)	2.5	1.0	No change
362–366	M(3)	6.3	2.4	Decreased
367–376	S(4), T(6)	2.0	0.8	No change
377–398	P(4), S(13)	2.6	1.0	No change
399–415	T(1), P(4)	1.8	0.7	No change
423–445	W(1), S(8), M(12)	6.4	2.5	Decreased
446–452	S(5)	3.3	1.3	No change

Underlined peptides indicate CDR-containing peptides. ^aDenotes the detected oxidized amino acid residue, and its numerical position within the peptide. Oxidized residues confirmed by tandem MS data. ^bHC 99–127 non-oxidized parent peptide showed inconsistent recovery in the tryptic digests. Dose response curves were therefore not considered representative, and the peptide was excluded from structural assessment. ^cHC 128–139 displayed rate constants (k) of 0 in both the dimer and monomer samples (see Supplementary Files). There is therefore no difference in protection levels, though a rate constant ratio cannot be calculated. ^dVery large tryptic peptides (40–50 residues) with oxidized forms (+16 Da, +14 Da, +32 Da, etc.) observed by MS data. However, tandem MS data are not able to conclusively confirm the specific sites of oxidation. Based on the average number of oxidized residues observed for other peptides (approximately 2 out of 10 residues are oxidized per peptide), it is assumed that these very large peptides would have approximately 8 oxidized residues. ^eRate constant (k) of dimer divided by the rate constant ratio (k) of the monomer. Mean of all non-normalized ratios: 2.7. Median of all non-normalized ratios: 2.5. ^f Normalization factor: 2.6 (average of the mean and median of the non-normalized ratios). Mean of all normalized ratios: 1.0. Median of all normalized ratios: 1.0. ^gNormalized ratios from 0.6 to 1.4: No change. Normalized ratios ≤ 0.5: increased protection. Normalized ratios ≥ 1.5: decreased protection.

Table 4B. Summary of oxidized light chain tryptic peptide data

Light Chain Tryptic Peptides	Detected Oxidized Residues^a	Uncorrected Rate Constant Ratio^e (Dimer k:Monomer k)	Corrected Rate Constant Ratio^f (Dimer k:Monomer k)	Protection Level Assignment in Dimer Relative to Monomer^g
1–18	Q(3), M(4), S(10), V(15)	0.8	0.3	Increased
19–42	T(4), S(8), Q(9), S(12), W(13)	0.9	0.4	Increased
46–61	Y(4), F(5), V(13), S(15)	2.4	0.9	No change
62–103 ^d	Approx. Eight oxidized residues	2.0	0.8	No change
109–126	P(5), F(8), S(13)	0.4	0.2	Increased
127–142	Y(14), P(15)	1.3	0.5	Increased
146–149	W(3)	3.0	1.1	No change
150–169	L(5), S(7), N(9), V(14)	1.9	0.7	No change
170–183	Y(4), S(13), L(12)	2.0	0.8	No change
191–207	H(8), S(12), P(14)	1.7	0.6	No change

Underlined peptides indicate CDR-containing peptides. ^aDenotes the detected oxidized amino acid residue, and its numerical position within the peptide. Oxidized residues confirmed by tandem MS data. ^bHC 99–127 non-oxidized parent peptide showed inconsistent recovery in the tryptic digests. Dose response curves were therefore not considered representative, and the peptide was excluded from structural assessment. ^cHC 128–139 displayed rate constants (k) of 0 in both the dimer and monomer samples (see Supplementary Files). There is therefore no difference in protection levels, though a rate constant ratio cannot be calculated. ^dVery large tryptic peptides (40–50 residues) with oxidized forms (+16 Da, +14 Da, +32 Da, etc.) observed by MS data. However, tandem MS data are not able to conclusively confirm the specific sites of oxidation. Based on the average number of oxidized residues observed for other peptides (approximately 2 out of 10 residues are oxidized per peptide), it is assumed that these very large peptides would have approximately 8 oxidized residues. ^eRate constant (k) of dimer divided by the rate constant ratio (k) of the monomer. Mean of all non-normalized ratios: 2.7. Median of all non-normalized ratios: 2.5. ^f Normalization factor: 2.6 (average of the mean and median of the non-normalized ratios). Mean of all normalized ratios: 1.0. Median of all normalized ratios: 1.0. ^gNormalized ratios from 0.6 to 1.4: No change. Normalized ratios ≤ 0.5: increased protection. Normalized ratios ≥ 1.5: decreased protection.

Figure 4. Examples of dose response curves and rate constants for oxidized tryptic peptides. Data generated by ProtMap MS software. Linear curves reflect the pseudo first order reaction kinetics of the hydroxyl radical oxidation. Rate constants (k) are also calculated by the software. Blue lines are point-to-point plots of the rate constants (k) at each exposure time. Red lines represent the lines of best fit.

Figure 5. Bar graphs of corrected rate constant ratios (dimer:monomer). (A) Corrected rate constant (k) ratios of heavy chain tryptic peptides (Table 4A). (B) Corrected rate constant (k) ratios of light chain tryptic peptides (Table 4B). For both panels, yellow bars indicate peptides with decreased protection in the dimer. Green bars indicate peptides with increased protection in the dimer. Uncolored bars indicate peptides that show no relative difference in protection levels between the dimer and monomer.

Figure 6. IgG1 homology structure with mapped protection levels. (A) Model of IgG1 constructed using 1BJ1 and 1IGY crystal structures. Colored according to the relative protection levels derived from oxidative labeling. Yellow indicates regions with decreased protection in the dimer. Green indicates regions with increased protection in the dimer. Uncolored areas indicate regions which show no relative difference in protection levels between the dimer and monomer. The blue region represents the Asn-linked Fc glycans typically found in IgG1 therapeutic mAbs. (B) Space-filled cartoon model of the IgG1 with the heavy chains colored purple and light chains colored red. The light chains of an IgG1 are on opposite planes with respect with each other. The heavy chains in the Fab region are also on opposite planes.

Figure 7. Proposed possible orientations of the IgG1 dimer. Cartoon representations of proposed possible dimer orientations based on the hydroxyl radical footprinting data. The bottom mAb has heavy chains in gray and light chains in black. The top mAb has heavy chains in light blue and light chains in dark blue. Head-to-head orientation allows for the light and heavy chains of the dimer to associate with each other in the interface region. (A) Cartoon depiction of a head-to head, a single-arm bound Fab-to-Fab dimer. (B) Cartoon depiction of a head-to head, double arm-bound Fab^ʹ2-to-Fab^ʹ2 dimer.