Recombinant antibodies are manufactured by cloning antibody genes into expression vectors, which are subsequently introduced into expression hosts to provide animal-free antibody production. In addition to reducing the use of animals in research, recombinant antibodies offer exceptional batch-to-batch reproducibility, easier and more rapid production, and can be generated in a multitude of different formats. They also obviate the problems of cell-line drift and mutations that are associated with classical hybridoma production and storage and which can often necessitate cell rescue or re-cloning.
“With recombinant antibodies you already have the antibody sequence in hand,” explains Brady Wu, director of protein production at GenScript, “meaning that you don’t have to worry about the monoclones drifting away from productivity or obtaining a single clone in every batch. Recombinant antibody production also bypasses many of the difficulties of antibody production that are associated with traditional methods.” According to Gary Shiels, vice president of marketing at Active Motif, “the demand for sequence-defined recombinant antibodies is definitely growing, particularly as part of various solutions to address issues associated with data reproducibility within scientific research.”
Ian Wilkinson, CSO at Absolute Antibody, agrees, adding that “issues with antibody reliability and reproducibility have become a high-profile problem in the last few years. Yet while some researchers are beginning to mistrust the quality of antibodies currently on the market, others remain unaware that these antibodies are not always as specific as they are believed to be.” Wilkinson recently co-authored a paper in which analysis of a selection of hybridomas demonstrated frequent expression of additional functional variable regions that impacted negatively on specificity and binding signal. “The only way to be sure of what’s really in a vial of antibody is to have it defined at the amino acid level—i.e. make it recombinantly,” he says.
The number of available format options is almost unlimited.
Recombinant antibodies exist in a multitude of formats. These include full-length immunoglobulins, chimeric and humanized antibodies, fragments like Fab or scFv, and multimeric formats such as diabodies and triabodies. More recently, these have been joined by bispecifics and antibody-drug conjugates. “With recombinant technology, any antibody format can be made available,” says Wilkinson. “The ‘best’ format will depend heavily on the application, and there is no one-size fits all solution. If you’re running an in vivo study in mice, you would choose a mouse IgG. If you then begin performing rat or monkey studies, you will want to switch your antibody into that species. If you’re running a diagnostic assay, you may decide that a human IgG is ideal as a control, or you may consider Fc receptor binding to be a problem and so might want to make use of a Fab. The number of available format options is almost unlimited.”
To provide enhanced flexibility in terms of antibody labeling and purification, Active Motif recently launched AbFlex®. These full-length recombinant antibodies have three distinct molecular tags: 6xHis for use with nickel-based purification systems, avidin for biotin conjugation, and a Sortase recognition motif (LPXTG) for the directed and reproducible covalent attachment of fluorophores, enzymes, peptides, DNA, drugs, or other labels to the antibody using the company’s Sortag-IT™ labeling kits.
“By tagging our AbFlex® antibodies at the end of the constant region of the heavy chain, labeling does not interfere with antigen recognition or antibody functionality,” notes Shiels. “Furthermore, we have observed the performance of AbFlex to equal, or in many cases surpass, the performance of the monoclonal from which the recombinant antibody was derived.” 
Researchers can harness the power of AbFlex through Active Motif’s hybridoma conversion service, offering adaptation of any monoclonal antibody to the AbFlex platform.
Image: Binding performance of AbFlex equals or surpasses monoclonals. Image courtesy of Active Motif.
Also delivering custom recombinant antibody production, GenScript has invested many years in optimizing its cell lines, gene synthesis and plasmid preparation, transfection reagents, and media systems to offer a suite of different services. “Our MamPower™ guaranteed recombinant antibody production service uses a customer-supplied DNA vector or the antibody sequence for rapid recombinant antibody production in either CHO or HEK293 cells,” notes Wu. “Within this service, the desired antibody amount, antibody purity, and endotoxin levels are guaranteed.”
The company also offers platforms promising recombinant antibody production at a high-throughput or large-scale, as well as delivering recombinant antibody fragments including scFv, VHH, VH, and Fab. “Different fragments can have different utilities,” says Wu. “For example, scFv can have some applications in screening the units on the CAR in Car-T therapy, while Fab and VHH may be used in structural biology to stabilize protein subunits and yield good crystals.”
Offering full-length recombinant antibodies, a species and isotype switching service, and partial length antibodies including scFv, Fab, F(ab)2, and bispecifics, BioLegend is highly aware of the growing need shown by researchers to gain access to these products. “Knowing the DNA sequence of an antibody facilitates adjustments or structural modifications for specific applications, as well as aiding electronic data archiving and intellectual property,” explains Dr. Miguel A. Tam, senior manager, product realization and marketing. He reiterates that different antibody fragment types may have advantages over others.
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“Smaller fragments such as scFv may be more effective in penetrating deeper into tissue sections for microscopy applications, whereas other techniques may require antibodies with more than one distinct binding region, such as bispecifics that allow for simultaneous recognition of two target molecules. Further applications may benefit from isotype or species switching, or even altered amino acid chains, which is not feasible with classical hybridomas.” BioLegend’s recombinant antibodies have been widely cited in the literature, including a recent publication in Cellular Immunology during which a sophisticated multicolor flow cytometry panel was generated.
Amanda Turner, product manager custom antibody products at Bio-Rad, describes how the company is utilizing the well-established human combinatorial antibody library (HuCAL®) in conjunction with CysDisplay®, a proprietary method of phage display, to produce recombinant antibodies. “HuCAL is a compilation of human antibody genes, cloned in E. coli phagemid vectors, which cover more than 95% of the structural human immune repertoire,” she says. “CysDisplay is a method to select genes from that library which encode for antibodies binding specifically with high affinity to just about any given antigen.”
“The method of selecting antibodies from highly diverse antibody libraries using phage display technology, rather than through animal immunization and hybridoma technology, offers several advantages,” adds Turner. “In vitro ‘guided selection’ discovers antibodies with rare specificities or properties that are extremely difficult to find with animal-based methods, while in vitro selection using a synthetic naïve library enables generation of antibodies against highly conserved or self-antigens, conformational variants or low immunogenic antigens, which is not possible by immunization of animals. Furthermore, the antibody DNA sequence is secured early in the process, guaranteeing reliable availability of the clone over time and facilitating conversion into different antibody formats or improvement in affinity.”
Recombinant antibodies have come a long way in recent years, with companies such as Bio-Rad and GenScript offering production in a matter of weeks, and Absolute Antibody recently launching a set of bispecific antibodies tailored for immunotherapy research in mice. The latter reportedly represents a world-first for the reagents market and is just one example of what can be achieved with recombinant technology.
Combat flow cytometry pain points with REAfinity antibodies
Although monoclonal antibodies are assumed to be monospecific, hybridomas frequently express additional functional variable regions. These have a detrimental effect on antibody function, as exemplified in a recent study co-authored by Miltenyi Biotec, fueling concerns about antibody reliability and reproducibility.
Recombinant technology facilitates the production of multiple antibody formats, allowing researchers to select the most appropriate format for their chosen application. Miltenyi Biotec’s REAfinity™ antibodies, carefully designed to address the major antibody pain points in flow cytometry, are the only off-the-shelf antibody portfolio specifically tailored to flow applications.
Produced recombinantly in mammalian cell lines under serum-free conditions, REAfinity antibodies offer superior purity and lot-to-lot consistency compared to mouse or rat hybridoma-derived monoclonal antibodies. A mutated Fc region eliminates Fcγ receptor binding, allowing background-free analysis and removing the need to perform Fc receptor blocking steps, while all 10,000 REAfinity antibodies share the same human IgG1 isotype and thus can be used with a universal isotype control for convenience and cost savings.
REAfinity recombinant antibodies from Miltenyi Biotec afford reproducible results through consistent antibody quality and have been expertly optimized for improved flow cytometry.
HuCAL and CysDisplay are trademarks of MorphoSys AG.