Using bispecific antibodies in forced degradation studies to analyze the structure–function relationships of symmetrically and asymmetrically modified antibodies

Figures & data

Figure 1. The process for creating asymmetrically oxidized bispecific molecules (BsAb) is presented. (a) Peracetic acid treatment of mAb1 (blue) or mAb2 (peach) resulted in methionine oxidation (illustrated as red lines) throughout the molecule. (b) Combinations of non-oxidized (A) and/or oxidized (B) mAb1 and mAb2 were pooled prior to Fab arm exchange to generate control BsAb1 (AA), asymmetrically oxidized BsAb2 (AB) and BsAb3 (BA), and symmetrically oxidized BsAbs (BB). All BsAbs were purified to the final product prior to analytical characterization.

Table 1. Purity and free thiol levels for the BsAb intermediates and purified final products.

Bispecific product^a	BsAb1		BsAb2		BsAb3		BsAb4
mAb 1	Oxidation (-)		Oxidation (+)		Oxidation (-)		Oxidation (+)
mAb 2	Oxidation (-)		Oxidation (-)		Oxidation (+)		Oxidation (+)
Assay	Post-FAE^b	Final formulation^c	Post-FAE^b	Final formulation^c	Post-FAE^b	Final formulation^c	Post-FAE^b	Final formulation^c
HIC
BsAb	88.3%	99.7%	93.1%	99.2%	90.5%	99.0%	93.1%	98.7%
Parental mAb	11.7%	0.3%	6.9%	0.7%	9.5%	1.0%	6.9%	1.3%
NR-cSDS
Purity	95.9%	96.8%	90.5%	92.4%	87.6%	90.1%	85.1%	88.3%
LMWS	3.9%	3.2%	9.3%	7.6%	12.0%	9.9%	14.5%	11.7%
SEC
Monomer	n/a	99.7%	n/a	99.5%	n/a	99.1%	n/a	99.1%
LMWS	n/a	0.0%	n/a	0.1%	n/a	0.3%	n/a	0.3%
HMWS	n/a	0.3%	n/a	0.4%	n/a	0.6%	n/a	0.6%
Free Thiol^d	n/a	0.7%	n/a	1.1%	n/a	1.0%	n/a	1.0%

^aBispecific Product is synthesized from respective oxidized (+) or unoxidized (-) mAb1 and mAb 2

^bMaterial after Fab-arm exchange reaction prior to downstream purification steps

^cFinal product after purification

^dCalculated as % free thiol/cysteine residue.

All values are expressed as a relative abundance

HMWS = high molecular weight species

LMWS = low molecular weight species

Figure 2. HIC chromatograms of BsAb products. Chromatograms for post-FAE reaction intermediates and final-purified products are shown in the top and bottom panels, respectively.

Figure 3. Subunit MS analysis of mAb and BsAb. (a) The deconvoluted spectra for control and oxidized mAb1 and mAb2 are shown in the left and right panels, respectively. (b) The deconvoluted spectra for BsAb1, BsAb2, BsAb3, and BsAb4 are shown in order from top to bottom. mAb1, mAb2, and methionine oxidation are shown with blue fill, peach fill, and red lines, respectively.

Table 2. Methionine 254 oxidation levels and Fc binding results for BsAb final products.

Duobody® composition^a			Met 254 oxidation (peptide map)^b			Fc binding assay^c
Material	mAb1	mAb2	mAb1	mAb2	Final BsAb product	FcRn	FcγRI	FcγRIIa	FcγRIIIa
BsAb1	Native	Native	3.8%	3.0%	5.7%	100.0%	100.0%	100.0%	100.0%
BsAb2	Native	Oxidized	3.8%	99.9%	56.4%	44.4%	97.6%	94.3%	102.1%
BsAb3	Oxidized	Native	99.9%	3.0%	57.6%	54.3%	120.2%	89.7%	106.4%
BsAb4	Oxidized	Oxidized	99.9%	99.9%	99.4%	8.6%	128.6%	112.6%	125.5%

^aBsAb molecule prepared from native or oxidized parental mAb1 or mAb2.

^bValue expressed as a relative abundance of oxidized Met 254.

^cBinding results are normalized relative to the respective control BsAb1 results.

Figure 4. Protein A affinity chromatography chromatograms for BsAb1, BsAb2, BsAb3, and BsAb4 intact IgG. Protein absorbance was monitored at 280 nm.

Figure 5. The process for creating bispecific antibodies with asymmetric deamidation at Asn330 is presented. (a) mAb1 (blue) and mAb2 (peach) were stressed at 40°C at pH 5.5 to induce selective deamidation at the Asn330 residue in the Fc Region (brown with red lines). Combinations of native or stressed mAb1 and mAb2 were pooled prior to Fab-arm exchange to generate control BsAb5, asymmetrically deamidated BsAb6 and BsAb7, and symmetrically deamidated BsAb8. (b) The bar plot illustrates the levels of FcγRIIIa binding of BsAb6, BsAb7, and BsAb8 relative to the control BsAb5 sample. The HC Asn330 deamidation values for the mAb1 and mAb2 arms of each BsAb are shown above the corresponding bar.