In basic research, diagnostics, and for therapeutic applications antibodies are indispensable. By genetic engineering it is nowadays possible to manipulate antibodies conveniently in many different ways. For example, antibodies derived from animals can be humanized for therapeutic purposes, antigenic peptides can be added for antigen targeting in vivo, the iso/subtype can be switched, they can be multimerized, or engineered as bispecific or multispecific molecules recognizing two or more different antigenic epitopes simultaneously. Antibodies can be produced in transient or stable mammalian expression systems in huge amounts and in a wide variety of different formats either as whole antibodies or as small antibody derivatives like scFv, nanobodies or Fab-fragments (Brinkmann and Kontermann, 2017).

For certain research applications the use of down-sized antibodies like Fab-fragments is superior to the use of whole antibodies. Due to their smaller size Fab-fragments show improved tissue penetration making them better candidates for immunohistochemistry or the treatment of certain tumors (Beckman et al., 2007). Since Fab-fragments are devoid of the Fc-domain they do not mediate immune effector functions by binding to Fc-receptors or by activation of complement and can replace whole antibodies, wherever immune effector functions are undesirable. Furthermore, because Fab-fragments are monovalent they are best suited for the determination of binding affinities and have proven to be particularly suitable as chaperones for the structure determination of crystallization resistant proteins (Koide, 2009).

Fab-fragments are composed of a light chain (VL + CL, kappa or lambda) linked by a disulfide bond to a shortened heavy chain (VH + CH1), termed the Fd-fragment. The most common method for the generation of Fab-fragments still involves digestion of antibodies with proteolytic enzymes (e.g. papain), separating the Fab-fragment from the Fc-part (Porter, 1959; Brezski and Jordan, 2010). Despite the availability of diverse commercial antibody fragmentation kits generation of sufficient amounts of Fab-fragments by digestion can be very tedious. Because antibodies differ in their sensitivity to proteolytic enzymes, digests are not always complete, resulting in low yield. Therefore, various parameters like amount of enzyme, time of digest, and temperature need to be optimized and after the digest the Fab-fragments have to be separated from the Fc-fragments by an additional purification step.

An alternative method is the genetic engineering and expression of recombinant Fab-fragments in cell-based systems. Due to their small molecule size, E.coli is the organism of choice as expression host, but it has certain disadvantages. Besides the problem of endotoxin contamination, expression of Fab-fragments in E.coli is accompanied by a more or less complicated refolding process, removal of byproducts like misfolded molecules or aggregates, and does not necessarily work for all antibodies (Patil et al., 2022). Expression of Fab-fragments in eukaryotic cells can circumvent these problems leading to correct protein folding and posttranslational modifications as has been described in previous publications (Vazquez-Lombardi et al., 2018).

Whereas the purification of whole IgG antibodies (the most widely used isotype) is a straightforward process, the purification of Fab-fragments can still be improved. Whole IgG antibodies, independent of their species, bind very tightly to staphylococcal protein A or streptococcal protein G via a high affinity binding site located in the Fc-domain (Kato et al., 1995). Therefore, whole IgG antibodies can be purified highly efficiently in a single step by affinity chromatography with easy-to-use protein A or G columns. Since Fab-fragments by definition lack the Fc-domain, purification via protein A or G cannot be considered a generic method for Fab-fragment purification.

Since a common matrix suitable for the purification of all classes of Fab-fragments is not available, various alternative purification matrices and strategies have been developed. For example, Fab-fragments containing variable domains of certain kappa light chain subclasses can be purified by protein L chromatography (Nilson et al., 1992). Columns with affinity ligands specific for subdomains of the light chain (kappa or lambda) or the Fd-fragment (e.g. Capture Select columns from ThermoFisher), are mainly suitable for the purification of human Fab-fragments. Other purification strategies make use of a combination of different purification techniques including ion exchange chromatography and gel filtration. Some of the most common methods are based on the addition of purification tags (e.g. His- or StrepTag) to the C-terminus of the Fd-fragment (Zhao et al., 2009). However, this is unfavorable for downstream experiments, since the influence of tags on the outcome of an experiment is unpredictable, especially when performed in vivo.

In the eighties and nineties of the last century additional contact sites for protein G located in the CH1 domain of IgG antibody heavy chains, conserved across many species and IgG subclasses, have been identified (Erntell et al., 1988; Derrick and Wigley, 1994). These additional contact sites form a low affinity binding surface for protein G that is in the low μM range as compared to the high affinity binding site (KD ~ 10 nM) located within in the Fc-domain (Bailey et al., 2014). However, the almost complete absence of publications in the recent literature on exploiting the CH1-domain for Fab-purification and the plethora of different methods and resins developed for the purification of Fab-fragments indicates that these earlier findings seems to have received little attention.

We speculated that, despite its low affinity, this alternative CH1 binding surface could be used for the routine purification of Fab-fragments via protein G affinity chromatography. To this end we cloned Fab-fragments from six different antibodies of human or murine origin and transiently transfected HEK-cells. All Fab-fragments were produced and could be isolated from the culture supernatants in a highly pure form using protein G columns. Our data suggest, that protein G can be used as a common matrix for the purification of Fab-fragments across different species, making the addition of artificial purification tags and the use of different purification matrices obsolete.