The success of an antibody ultimately depends on specificity of detection for the target of interest—and the ability to deliver reproducible, consistent results every time the antibody is used in an experiment.
Recombinant antibodies (rAbs) offer several advantages over those that are more traditionally produced, including the ability to manipulate structure and binding specificity (allowing for humanized antibodies, single-domain or single-chain and other antibody “fragments” and dual-specificity antibodies, for example) as well as the ability to produce a single species in any quantity in perpetuity. When it comes time to scale up production to the milligram or even gram scale—for pre-clinical animal studies, say—it’s no longer a matter of running a few milliliters through a disposable spin column. Here we look at how researchers are processing (often) liters of culture supernatant at a time to generate purified rAbs.
Another advantage of rAbs is that they can be produced in an expression system of choice. If all that’s needed is an antibody for general detection, E. coli may be the best way to go, says John Zhang, president of AbBioSci. In fact, he recommends using bacterial expression as a step to assure that the antibody is still functional and active before scaling up.
Production of properly folded and glycosylated rAbs on the milligram to gram scale calls for expression in a mammalian cell line.
The Toronto Recombinant Antibody Centre (TRAC) uses transiently transfected human embryonic kidney line HEK-293 suspension cells “to rapidly get to the production stage,” says associate director Jarrett Adams. “You’re able to get as much as you can scale up to: a gram of protein can be achieved from somewhere between five and 50 liters, depending on the expression levels of the IgG you’re looking at.”
If someone is making a stable cell line for this scale, “it’s probably because you have some purpose in mind that is commercial in nature and might require something more like GMP-type production,” explains Robert Carnahan, director of the Vanderbilt Antibody and Protein Resource (VAPR) and associate professor of cancer biology at Vanderbilt University. As long as regulatory considerations don’t come into play, it’s easy enough to just transiently transfect more cells to produce another batch of antibody when needed.
Fast and easy
Purifying recombinant IgGs isn’t any different from purifying IgGs from hybridomas. It begins with centrifuging the culture supernatant (supe) to remove cells and debris. The supe can then be further clarified, or even loaded directly onto an affinity column. The antibody is typically eluted from the column by low pH, then the eluent is neutralized and dialyzed, and finally the antibody is concentrated.
Adams runs the supe directly through a gravity-powered column packed with Protein A resin (which binds the Fc region of an antibody). Gravity columns are “simpler, easier to set up, don’t require any expensive infrastructure and are modular,” he says. For a facility like TRAC, which performs hundreds of milligram-scale productions, “it’s probably more painful to try and run these one at a time through a chromatography system than it is to just do it by gravity captures.”
Carnahan’s core takes a different tack. Equipped with three GE Healthcare Life Science’s ÄKTA Protein Purification Systems (often termed fast protein liquid chromatography, or FPLC), the researchers first filter the rAb supe through a 0.22-µm filter and then run that through a GE HiTrap Protein G column (which binds a largely overlapping range of antibodies as Protein A). “The nice thing about those is that they are walk-away,” he says.
For the most part, researchers who are using the rAbs as tools (as opposed to investigating their properties) don’t need them any purer than they would come off the column. If there is such a need, academics may do their own “polishing”—the most common form being ion-exchange chromatography. (The TRAC rAbs are engineered to have a high Tm: “You just heat the antibody up to 60° and watch everything fall out of solution, while your antibody stays in,” Adams says.)
Process economy and regulatory requirements are critical aspects in large-scale chromatography, points out Tomas Bjorkman, senior scientist at GE Healthcare. Protocols are optimized, and data on chemical stability and extractables/leachables are collected to assist with process validation and regulatory submissions.
Tweaking resin so that its binding characteristics and elution profiles are optimal may be essential for larger-scale projects. But in the research realm, scientists “have a lot of little stuff going on, and us spending too much time optimizing any one of those is not a good idea,” says Carnahan. The cost of process development would likely overshadow any savings, “so we would prefer everything to run down the same highway.”
That being said, there are some instances in which optimization is necessary, especially when making “unnatural” antibodies that may not be as stable, or that may not be able to recover from a low-pH elution, for example, says Adams.
There are also some rAbs—especially certain fragment modalities—that may not associate well with Protein A or Protein G. Perhaps the best way to purify these is to add an affinity tag, such as polyhistidine, which allows the fragment to be captured by a metal matrix.
Similarly, although there are good secondary antibodies to detect IgGs, there is a paucity of reagents specific to many antibody fragments. “Adding on an epitope tag like FLAG or MYC off the C-terminal end of the antibody [fragment] allows you to use commercially available secondaries to detect” the fragment, says Adams.
Exceptions aside, “Protein A and Protein G chromatography has really come to the surface as the preferred methodology, because it’s a very simple process. It’s a binding, washing, elution step that is almost universal for antibodies of the same isotype,” Adams adds.
Recombinant antibodies are a viable alternative to traditionally generated antibodies. They offer researchers the ability to tailor or customize the affinity and specificity of the antibody to the target of interest. They can be produced in large quantities, if needed, and they have the potential to provide more consistent results. During the production phases, researchers (and tool providers) are able to monitor, characterize and define standards or criteria for their rAb candidates. Antibodies, whether traditionally generated or recombinant alternatives, will always be a mainstay for researchers interrogating cells to better understand how their targets of interest function.