Applications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments

Abstract

Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (KD ~ 10 nM) for the Fc region of the IgG, but a much lower affinity (KD ~ low εM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 10,000-fold change in affinity from pH 7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment-antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.

A polar ring endows improved specificity to an antibody fragment

“…After sonication and centrifugation, cell free lysate was passed over Ni‐NTA Superflow resin (Qiagen), washed in sonication buffer (50 m M Tris, 500 m M NaCl, 10 m M imidazole, pH 8.0), and eluted with high imidazole buffer (250 m M imidazole). Nickel affinity eluate was passed over an affinity column coupled with Protein‐G‐A1 washed with Tris buffer (50 m M Tris, 150 m M NaCl, 1 m M EDTA, pH 7.4) and eluted with glycine buffer (100 m M , pH 2.6). For SPR analysis, the Fab was dialyzed into EB buffer.…”

Recognition of an α-helical hairpin in P22 large terminase by a synthetic antibody fragment

“…Induction proceeded for 5 h, at which point the cell pellets were harvested. Fabs were purified as described previously using ProteinG-A1 resin for single-step purification (Bailey et al, 2014). To form Fab-TerL complexes, a twofold molar excess of Fab was added to TerL and the mixture was purified on a Superdex 200 16/60 gel-filtration column (GE Healthcare) in ITC buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM MgCl 2 , 5% glycerol).…”

Technology advancements in antibody purification

“…23 The countercurrent movement of the liquid and the stationary phase, called simultaneous moving bed (SMB), is the basic principle in various continuous and semicontinuous countercurrent sequential loading systems that can utilize up to 12 columns simultaneously. 23,43,44 The smaller columns used in these systems reduce affinity resin and buffer consumption by 40%-50%, while sequential loading of the columns with the flow-through from the previous column ensures maximum product capture. 23,43 Also, having all the columns in operation at any given point of time, albeit at different stages of the process, significantly reduces production times.…”

“…However, it binds to the Fab region with much lower affinity (low μM affinity). 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.…”