Engineering of novel Staphylococcal Protein A ligands to enable milder elution pH and high dynamic binding capacity

Abstract

We describe novel Staphylococcal Protein A ligands that enable milder elution pH for use in affinity chromatography. The change in elution pH is the result of point mutations to the protein sequence. Two novel ligands are investigated in this study. The first, designated Z(H18S)4, represents a histidine to serine substitution single mutation. The second, designated Z(H18S, N28A)4, is a double mutant comprising histidine to serine and asparagine to alanine mutations. Both are compared against the unmutated sequence, designated Z4, which is currently utilized in a commercially available Protein A stationary phase for the purification of molecules containing Fc domains. The ligands are coupled to a chromatography support matrix and tested against a panel of antibodies and an Fc fusion protein for elution pH, dynamic binding capacity, step-wise elution, and capture from clarified culture media. Results demonstrate that the novel ligands result in milder elution pH, on average >0.5 pH units, when tested in a pH gradient. For step-wise elution at pH 4.0, the Z(H18S, N28A)4 ligand showed on average a greater than 30% increase in yield compared to Z4. Importantly, for the antibodies tested the mutations did not result in a decrease in dynamic binding capacity or other desirable attributes such as selectivity. A potential application of the novel ligands is shown with a pH sensitive molecule prone to aggregation under acidic conditions.

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

“…In the case of Protein L robust synthetic affinity ligands that mimic the action of the natural biological ligand may provide an alternative for both mAb and Ab-fragment processing [122][123][124]. In time it will be interesting to see if the improvements offered by synthetic ligands can match those offered by genetic engineering of already highly evolved affinity proteins, e.g., [125][126][127]. Twenty years of Protein A resin development tends to favor the latter approach, but synthetic ligand development, screening methods, and performance are continually improving.…”

“…Present Protein G resins can be CIP'd with NaOH but are not as chemically stable as Protein A, so an interval of 0.02-0.05 M NaOH is suggested. It is expected that next-generation Protein L resins may benefit from similar recombinant technology [125][126][127].…”

“…Cleaning-in-Place (CIP) studies using the approach noted in Table 3 indicate that MabSelect can survive more than 110 CIP cycles with no significant reduction in performance. More modern ligands derived from Protein A antibody binding domains [125,126] offer enhanced basic pH stability and several hundred CIP cycles using relatively inexpensive 0.1 to 0.5 M NaOH solutions. Present Protein G resins can be CIP'd with NaOH but are not as chemically stable as Protein A, so an interval of 0.02-0.05 M NaOH is suggested.…”

Technology advancements in antibody purification

“…To enable purification of both the Fc and Fab in a single-step process, Bailey et al 44 employed a combinatorial phage display approach to create a Protein-G mutant expressing eight amino acid changes that displayed a 100-fold increase in affinity for Fab, and subsequently, employed a rational approach to create a Protein-G with a pH switch (when moving from pH 7.4 to 4, induces a K D change of 1,000-fold) that could be advantageously used during Fab purification. The need for milder IgG elution conditions during Protein-A chromatography was facilitated by Pabst et al, 46 who engineered a novel SpA ligand that incorporated a double mutant (histidine to serine [H18S] and an asparagine to alanine [N28A]) in the synthetic Z domain (formerly the B domain of the native protein) of SpA. The binding and elution capacity of the SpA containing the double mutant was tested against six antibodies and one Fc-fusion protein and displayed DBC and selectivity similar to that of the parental ligand.…”

“…It successfully facilitated the elution of IgG from SpA under milder conditions (.0.5 pH units), thus enabling the elution of proteins that are sensitive to aggregation under acidic conditions. 46 …”