Purification of Common Light Chain IgG-Like Bispecific Antibodies Using Highly Linear pH Gradients

Beth Sharkey, Sarat Pudi, Ian Wallace Moyer, Lihui Zhong, Bianka Prinz, Hemanta Baruah, Heather Lynaugh, Sampath Kumar, K. Dane Wittrup, Juergen H. Nett 

mAbs 9, 257–268. DOI: 10.1080/19420862.2016.1267090 

Overview 

Monovalent bispecific antibodies (bsAbs) that bind two targets simultaneously have broad potential clinical value, but their manufacture is complicated by the need to correctly pair heavy and light chains. A common light chain (cLC) bispecific format avoids much of this problem because no additional modifications to the antibody are required. However, purifying the desired bsAb from the two parental monospecific antibodies remains challenging. Current methods using salt gradient ion exchange chromatography (IEX) are only effective when the parent antibodies have relatively large differences in isoelectric point (pI). This study introduces the use of linear pH gradients for high-resolution separation of cLC bsAbs. 

Key hypotheses and objectives 

Hypothesis: Previous work has shown that pH gradients achieve higher resolution separation than salt gradients for analytical characterization of monoclonal antibodies. This study proposed that the same principle would extend to preparative-scale purification, and that a linear pH gradient combined with IEX could resolve a cLC bsAb from its two parental homodimers even when their isoelectric points differ by very little, which salt gradients cannot reliably achieve.  

The objectives of the study were to: 

• Demonstrate separation of bsAbs from parental mAbs using pH gradient IEX. 

• Determine how small a pI difference the method can resolve. 

• Confirm compatibility with validated commercially scalable, manufacturing IEX resins. 

Approach and techniques 

Seven cLC bsAbs with parent antibody pI differences ranging from 1.33 to 0.10 pH units were expressed and purified by Protein A chromatography. The resulting mixtures of bispecific and parental homodimer byproducts were then separated using linear pH gradient IEX (both cation and anion exchange) and compared against traditional salt gradient IEX. Heterodimer purity was confirmed by mass spectrometry after IdeS cleavage to remove any heterogeneity that could be introduced by Fc glycans. The method was also tested using IEX resins with larger bead sizes suitable for commercial-scale manufacturing. 

Major findings and results 

• pH gradient IEX chromatography resolved cLC bsAbs from their parental mAbs at pI differences as small as 0.10, including instances where the parent antibodies differed by only a single charged amino acid, outperforming traditional salt gradients. 

• Validated manufacturing-grade IEX resins showed good separation of homodimer from heterodimer, though with some resolution differences compared to the smaller bead analytical resins used for initial screening. 

• The same buffer system worked for both cation and anion exchange, requiring little optimization and no specialized software to control gradient formation. 

Implications for therapeutic antibody development 

This purification method addresses a real bottleneck in bsAb manufacturing by enabling separation of near-identical parental and bispecific species without requiring additional protein engineering to increase the pI difference between the antibodies. Combining IEX with linear pH gradient elution has the potential to become a broadly useful purification and polishing method for bsAbs generally, beyond the cLC format specifically tested in this study.