Analytical characterization of coformulated antibodies as combination therapy

Figures & data

Figure 1. HIC overlays of heat-stressed Combo (profile A) and HIC-fractionated mAb-A (profile C) and mAb-B (profile B). Vertical lines in Profile A indicate fractionation times. Each fraction of HIC-separated Combo (profile A) was reinjected on HIC to verify enrichment (profile B and profile C). Heat-stressed Combo at 40°C for 3 months is shown

Figure 2. Comparison of size variant profiles for (a) mAb-A and (b) mAb-B. Profiles A, mAb-A, and mAb-B; profiles B, mAb-A, and mAb-B fractionated by HIC from Combo; profiles C, Combo by HPSEC and gel electrophoresis (nonreducing and reducing). HC = heavy chain; LC = light chain

Table 1. Deamidation in Combo, mAb-A and mAb-B fractions by HIC, and mAb-A and mAb-B

		Deamidation (%)
Condition	Sample Description	HC Asn-388, Asn-393	HC Asn-329	mAb-B LC Asn-30
25°C	Combo	4.3	5.3	18.6
	mAb-A	3.5	5.6	NA
	mAb-A fraction by HIC	3.8	6.6	NA
	mAb-B	4.2	ND	14.3
	mAb-B fraction by HIC	3.9	ND	13.4
40°C	Combo	8.8	47.4	60.0
	mAb-A	8.7	48.3	NA
	mAb-A fraction by HIC	8.6	48.3	NA
	mAb-B	6.9	8.8	33.6
	mAb-B fraction by HIC	9.2	10.8	45.9

HC = heavy chain; LC = light chain; NA = not applicable; ND = not detected.

Figure 3. Comparison of charge variant profiles for (a) mAb-A and (b) mAb-B. Profiles A, mAb-A and mAb-B; profiles B, mAb-A and mAb-B fractionated by HIC from COMBO; profiles C, Combo by IEC and cIEF. Rectangular area is magnified in inset of (b) IEC profiles

Figure 4. Results of SPR binding assay for detection of coaggregates of mAb-A and mAb-B. Positive control of coaggregates fractionated by HPSEC was obtained from a 1:1 mixture of mAb-A and mAb-B incubated at 50°C for 33 days (black solid line). Shown are aggregate fractions by HPSEC from nonstressed Combo (green solid line) and Combo incubated at 40°C for 3 months (green dotted line)

Table 2. Observed post-translational modifications of Combo, mAb-A and mAb-B under stress conditions

Sample	(a) Deamidation sites under pH stress at 6.5		(b) Methionine oxidation sites under chemical oxidation conditions				(c) Tryptophan oxidation sites under photostress conditions
	HC Asn-388, Asn-393	mAb-B LC Asn-30	HC Met-256	HC Met-432	HC Met-362	mAb-B HC Met-401	mAb-B HC Trp-52	mAb-A HC Trp-102
Combo	8.8	31.0	80.0	38.7	42.2	55.2	12.2	61.2
mAb-A	8.2	NA	79.9	39.7	45.4	NA	NA	63.6
mAb-B	8.8	30.6	60.9	41.3	22.0	56.9	10.9	NA

HC: heavy chain; LC: light chain; NA = not applicable.

Figure 5. Higher-order structure profiles of experimental mAb-B and mAb-A, Combo, and reconstructed Combo by (a) DSC, (b) far UV CD, (c) near UV CD, and (d) FTIR. Profile A, mAb-B (red); profile B, mAb-A (blue); profile C, experimental Combo (black); profile D, reconstructed Combo (gray)

Figure 6. AUC profiles for evaluating intermolecular interactions of mAb-A and mAb-B in Combo. (a) Comparison of AUC profiles of mAb-A, Combo, and a positive control of intermolecular interactions (a:b mixtures of mAb-A and anti-mAb-A antibody). Profile A, mAb-A (red); profile B, Combo (blue); profile C, positive control (a:b mixtures of mAb-A and anti-mAb-A antibody; black). (b) Impact of Combo concentration on ls-g*(s) sedimentation profile. Profile A, Combo at 0.2 mg/mL (red); profile B, Combo at 0.5 mg/mL (blue); profile C, Combo at 2 mg/mL (black). (c) Impact of mAb-A:mAb-B ratio on ls-g*(s) sedimentation profile. Profile A, a:b mixture of mAb-A and mAb-B (red); profile B, 4:1 mixture of mAb-A and mAb-B (blue); profile C, 1:1 mixture of mAb-A and mAb-B (black)

Figure 7. ITC profiles by titration of mAb-B in mAb-A

Table 3. Comparison of hydrodynamic diameters for monomer and dimers in Combo

Theoretical average diameters (nm)			Experimental average diameters (nm)
Combo as monomer	Combo as dimer		mAb-A	mAb-B	Combo
	Minimum	Maximum
D3 = $\left(D1+D2\right)/2$	$D3=\sqrt[3]{2}\left(\left(D1+D2\right)/2\right)$	D3 = D1 + D2	D1	D2	D3
13.4	16.9	26.9	13.5 ± 1.1	13.4 ± 0.5	14.4 ± 1.5

Figure 8. Comparison of average hydrodynamic diameters by DLS with increasing protein concentrations

Figure 9. Representative dose–response curves for (a) mAb-A and (b) mAb-B bioassay of Combo and mAb-A and mAb-B