Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification

Bioresources and Bioprocessing

Table 2 Structures, Molecular weights (MW), pI values, Fc (%), and ∆Fc (%) of bsAb Molecules A, B, and C for monomers and LMW species

Molecule	Category	Protein species	MW (kDa)	pI Value	Fc (%)	∆Fc (%)
A, B, C	Small LMWs	LC-M	23.4	7.76	0%	-40%^A, -29%^B, and -26%^C
A, B, C	Small LMWs	LC-D	46.9	7.98	0%	-40%^A, -29%^B, and -26%^C
A	Target	M	124.6	8.52	40%	0%
	Large LMWs	HH	103.6	8.64	48%	8%
	Large LMWs	M—LC	101.2	8.58	49%	9%
B	Target	M	172.5	8.59	29%	0%
B	Large LMWs	KK	145.5	8.43	35%	6%
C	Target	M	199.8	8.70	26%	0%
	Large LMWs	M—LC	176.4	8.74	29%	3%
	Large LMWs	M—2LC	153.0	8.80	33%	8%

MW values were calculated based on amino acid sequences. pI values were calculated from an Expasy ProtParam tool. Fc (%) and ∆Fc(%) are as defined in Eq. 2 and Eq. 3, respectively. Abbreviations used are listed as followed; D = dimer, M = monomer, HH = hole–hole homodimer; KK = knob–knob homodimer; LC = light chain. ∆Fc (%)A, ∆Fc (%)B, and ∆Fc (%)C of LC-M and LC-D are as compared to monomers of Molecules A, B, C, respectively. The more positive ∆Fc (%) between product-related impurities and monomers may suggest higher effectiveness of impurity removal via CHT-P elution