Like other widely applied bacterial surface receptor proteins for immunoglobulins (Igs), such as protein A and protein L, the Ig-binding domain of protein G (ProtG) has dual binding activity. ProtG can independently associate both with the Fc region of an antibody (mAb) and with its Fab and, thus, provoke cross-linking if applied in solution. Indeed, we observed pronounced precipitation activity when using ProtG equipped with the Azo-tag as a small adapter molecule for the light-controlled affinity purification of mAbs. We demonstrate that this undesired precipitation phenomenon follows the classical Heidelberger-Kendall curve. Furthermore, we describe a mutant of ProtG in which Asn478 at the interface with the Fab is replaced by Arg, which results in the effective loss of this secondary binding activity while maintaining high affinity towards the Ig Fc region. ProtGN478R no longer induces precipitation when mixed with a series of medically relevant mAbs. Hence, Azo-ProtGN478R can be applied as a convenient molecular tool to isolate antibodies from cell culture medium -- even with a high content of albumin -- in a single step via Excitography. In this technique, elution is triggered by trans → cis isomerisation of the Azo-tag upon illumination with mild UV-A light and a harsh pH shift is avoided.
Bacterial immunoglobulin (Ig)-binding proteins, in particular protein A from Staphylococcus aureus and protein G from group G Streptococci, are widely used for the affinity purification of recombinant or monoclonal antibodies (mAbs) both in biomedical research and in the industrial manufacturing of Ig-based biopharmaceuticals. Various robust chromatography resins carrying different covalently immobilized versions of protein A or G are commercially available and allow the rapid isolation of antibodies or Fc-fusion proteins from conditioned cell culture supernatants under standardized conditions. However, one caveat is the use of acid elution buffers to dissociate the protein-protein complex and recover the Ig from the affinity column. Such low pH conditions can promote both protein aggregation and deamidation of Asn and Gln side chains, which is a concern not only for biopharmaceutical manufacturing but also for those areas of fundamental research where homogenous and fully functional protein preparations are crucial.
We have recently developed the Azo-tag, which comprises a cis/trans-isomerizable azobenzene side chain, for the light-controlled affinity chromatography -- dubbed Excitography -- of a wide range of proteins, including antibodies, under native buffer conditions. In this technique, the non-canonical amino acid p-(phenylazo)-L-phenylalanine (Pap) is cotranslationally incorporated into the recombinant protein, as part of the short, 3-4 residue Azo-tag, via amber stop codon suppression using an expanded genetic code. The light-dependent trans → cis isomerization of the Pap side chain is utilized to adsorb and desorb the protein to/from an α-cyclodextrin (α-CD) affinity matrix under different illumination conditions, thus allowing elution simply under mild UV-A radiation in a physiological buffer of choice, directly suitable for subsequent biochemical or cell culture assays.
When applying Excitography to mAbs, instead of incorporating the Azo-tag at the genetic level into the Ig protein a tiny adapter molecule was employed. To this end, the small soluble Ig-binding C2 domain of protein G -- a 56-residues fragment of the natural multi-domain protein anchored in the bacterial membrane -- was equipped with an N-terminal Azo-tag and dubbed Azo-ProtG. When adding Azo-ProtG, produced in an E. coli expression system, to a cell culture medium containing the mAb, the antibody was affinity-purified via Excitography in one step and in a highly efficient manner using a native buffer. However, during this application of ProtG as affinity adapter molecule in solution we observed that the mixing ratio with the antibody was critical to prevent the formation of a precipitate. Interestingly, it turned out that this Ig precipitation behavior upon addition of ProtG, i.e. a single Ig-binding domain of the larger protein G, was reminiscent of the long-known Heidelberger-Kendall curve.
In a series of seminal publications, Heidelberger & Kendall described the quantitative principles of the so-called precipitin reaction, which refers to an antibody forming a precipitate from a mixed solution with its antigen due to multivalent non-covalent complex formation. The resulting precipitin curve describes the relationship between the concentration of the antigen and the amount of precipitate formed in the presence of a constant quantity of antibody, comprising three characteristic zones: (i) in the zone of antibody excess, there is only minor precipitate formation as all binding sites on the antigen are saturated; (ii) in the so-called zone of equivalence, precipitation is maximal owing to multiple intermolecular cross-links between antibodies and antigens upon binding (also known as immune complex); (iii) in the region of high antigen concentration, precipitation is low again as all binding sites of the antibody become saturated.
While this general relationship was originally uncovered in experiments with polyclonal sera and macromolecular antigens displaying multiple epitopes, the same fundamental effect can occur with immunochemical components having less complex composition, such as purified bivalent or bispecific mAbs and oligomeric or oligovalent protein antigens. In this regard, it is noteworthy that a single Ig-binding domain of protein G exhibits two distinct and independent Ig-binding sites: one specific for the Fc portion of IgG and one specific for the Fab -- albeit with lower affinity. Due to the circumstance that the two corresponding interfaces on the ProtG domain do not overlap (Fig. 1), thus allowing binding of two antibody molecules simultaneously, a polymeric complex may be formed when ProtG is mixed with a mAb at suitable ratio (Fig. 2).
Based on the hypothesis that the secondary, Fab-specific binding site of ProtG may effect the crosslinking of antibody molecules (regardless of their antigen specificities) and, thus, lead to the formation of macromolecular immune complexes, we have investigated this phenomenon in greater detail. We report a mutagenesis study to eliminate the binding activity towards the Fab region of antibodies and the development of a truly monovalent Fc-specific ProtG version with improved applicability, in particular with regard to the light-controlled affinity purification of mAbs.