Methods for refolding of recombinant antibodies

What is this patent about?

’205 is related to the field of recombinant protein production and purification, specifically focusing on the structural homogeneity of IgG antibodies. In the production of therapeutic proteins using mammalian cell cultures, such as CHO cells, antibodies often exhibit conformational heterogeneity due to scrambled disulfide bonds or undesirable post-translational modifications like cysteinylation. This structural variance can lead to inconsistent biological activity, reduced stability, and difficulties in protein crystallization, posing significant challenges for pharmaceutical viability.

The underlying idea behind ’205 is that the structural heterogeneity of mammalian-expressed IgG2 antibodies—which typically elute as multiple distinct peaks on RP-HPLC—is caused by different disulfide connectivity patterns in the hinge region. By subjecting these proteins to a controlled redox coupling environment, the invention allows the molecules to reshuffle their disulfide bonds into a more uniform state. The key engineering insight is that the presence or absence of a chaotropic agent during this redox process acts as a conformational switch, allowing the manufacturer to selectively enrich one specific structural variant over others to optimize biological potency.

The claims of ’205 focus on a method for producing an IgG2 antibody preparation that is enriched for a specific structural variant by contacting the recombinantly produced protein with a reduction/oxidation coupling reagent at a pH between 5 and 11. The independent claims specifically define a process where the enrichment is modulated by the use of a denaturant; contacting the antibody with the redox reagent in the absence of a chaotropic agent enriches one structural form, while performing the same contact in the presence of a chaotropic agent enriches a different, distinct structural form.

In practice, the invention utilizes redox pairs such as cysteine/cystine or reduced/oxidized glutathione to facilitate disulfide exchange. When a chaotropic agent like guanidine hydrochloride is added at low concentrations (e.g., 0.5M to 1.5M), it slightly perturbs the antibody's tertiary structure, repositioning the hinge cysteines to favor a specific, highly active conformation. This process effectively converts less active or heterogeneous isoforms into a single, homogeneous population, which can then be isolated using standard chromatographic techniques like ion-exchange or reversed-phase HPLC.

This approach differs from prior art refolding methods which typically involve the complete denaturation and reduction of proteins derived from bacterial inclusion bodies. Instead, ’205 applies a gentle reshuffling to proteins already secreted in a folded state by mammalian cells, targeting only the problematic disulfide linkages. By eliminating variants with lower binding affinity or stability, the method produces a more compact and potent therapeutic product, resulting in up to a three-fold increase in biological activity and improved long-term storage stability compared to untreated antibody preparations.