by Caitlin Smith
Did you ever stop mid-pipette to wonder where those antibodies came from, invisible in that bit of solution in that tip of yellow? Many researchers have used the highly specific monoclonal antibodies to identify particular proteins, peptides, or epitopes. Most have an idea of how they are produced. Fewer know the recent advances in the purification of their crucial labeling tool. This spotlight will discuss some of the gains made and challenges faced by those who seek to purify antibodies.
Monoclonal antibody purification
The importance of monoclonal antibodies in research and medicine is indisputable. From Western blots to live immunofluorescence to clinical applications, many lines of inquiry rely on the unique specificity of monoclonal antibodies. Produced by hybridomas—lymphocytes cloned from a single cell from an immunized animal, fused to a cancer cell line—monoclonal antibodies are much more specific than polyclonal antibodies, which are isolated from the blood sera of immunized animals.
One important product used for the purification of monoclonal antibodies at production scale (as opposed to laboratory scale) is ceramic hydroxyapatite. Ted Tisch, Bio-Rad’s division marketing manager for process chromatography, explains that, “ceramic hydroxyapatite is calcium phosphate crystals that [are] sintered into spherical beads at high temperature. [It is] used as a polishing step after the protein A capture step. Ceramic hydroxyapatite is very effective at removal suitable for therapeutic drug production of the following contaminates: monoclonal antibody aggregates, nucleic acid, virus, endotoxin, host cell proteins, and leached protein A.” Bio-Rad just released ceramic hydroxyapatite in their Bio-Scale Mini CHT 5 ml cartridge for laboratory (as opposed to production) scale for research and process development.
But this isn’t the only way to purify monoclonals. “The main competition for ceramic hydroxyapatite are cation exchangers and multi-mode separation media,” says Tisch. “Neither of these product classes remove the list of contaminates to the degree of ceramic hydroxyapatite. In addition, ceramic hydroxyapatite reduces the method-development effort to find the optimal conditions for effective removal of contaminates.” This is good news for researchers in the throes of method optimization, considering that balancing product yield and product purity is the major consideration when optimizing.
An example of another type of product for the purification of monoclonal antibodies is GE Healthcare’s Capto Adhere. Intended for use after the initial Protein A capture step—for “polishing” monoclonal antibodies—it’s a new member of GE’s range of Capto ion exchange media. The ligand within Capto adhere is a strong multimodal ion exchanger. It removes contaminants such as DNA, host cell proteins, leached Protein A, viruses, dimers, and aggregates in a single step. Capto adhere can also be followed by anion or cation exchange chromatography for polishing; for this, GE offers their anion exchanger Capto Q, and cation exchanger Capto S.
Phospho-specific antibody purification
Making accurate identifications and estimates of a protein in its phosphorylated and unphosphorylated states is crucial for researchers in cell signaling and other areas. Antibodies that are selective for the phosphorylated form of the protein are a common method for doing this. Kristin Byrne, manager of the immunology department at Quality Controlled Biochemicals (QCB), says that, “feedback from our customers has indicated a need for a fast, low-cost, phospho-specific antibody purification column. To fulfill this need our research and development team has created a proprietary single-step method for phospho-antibody purification. This method provides an enhanced phospho-antibody signal at a reduced cost. This new purification column allows for the immunization of larger peptide sequences for increased antibody production without affecting the phospho-specific antibody purification product.” Their proprietary technology includes a customized phospho peptide conjugated to a thiol-coupling gel, packed in a ready-to-use 10 mm column.
Magnetizing small-scale purification
Anyone with recent purification experience may be acquainted with magnetic beads. Beate Rygg Johnsen, senior product manager at Invitrogen Dynal, says that they “have been around for some time, but their advantages compared to sepharose/agarose have only recently been discovered by a growing number of customers.” Invitrogen Dynal offers magnetic beads (Dynabeads Protein A and G) for the small-scale purification of antibodies (typically the microgram to milligram range). According to Johnsen, “the advantages of using magnetic bead technology for small-scale antibody purification are that it is very fast and convenient (15 - 20 minute protocol and no centrifugation or use of columns). The purified antibody has very low non-specific background due to very efficient washing and rapid reaction kinetics. Also, resin is not lost during the procedure as can occur with the loose pellet of sepharose.”
The road ahead
A major task facing researchers is to solve the problems of capacity and throughput with regard to antibody purification. The past two decades have witnessed large increases in titer concentrations of monoclonal antibodies from cells in culture, from less than 0.1 g/L to currently 2 – 6 g/L, with expectations of up to 10 g/L in the near future. But this also results in a concomitant increase in cell debris and contamination from host cell proteins, DNA, viruses, dimers, and aggregates. “The main challenge is the productivity of the standard monoclonal antibody workflow,” says Tisch. “The increase of monoclonal antibody titers from cell culture ... over the past five years has caused throughput challenges downstream where very large columns, with run for multiple cycles, are required to process the cell culture batch. At this time, there is no projected technology to quickly solve these issues to downstream processing.”
Price tags are also a hurdle. Byrne says that, “customer feedback suggests there is a need for a less expensive alternative to currently available Protein A purification methods. Our R&D team is currently in the process of characterizing the binding capacity of a proprietary protein gel that we hope to launch in early 2008. Our goal is to offer a less expensive Protein A column alternative that will be reliable and reusable compared to other products currently on the market.”
Lastly, Johnsen believes that a worthwhile challenge to be met is small-scale automated antibody purification. “Since Dynabeads are a magnetic resin, protocols involving Dynabeads can be automated on any liquid handling platform (96 or even 384 format),” explains Johnsen. “This would allow for a very convenient system for the purification of small amounts of different antibodies, e.g. for the purification of antibodies for proteoarrays.” Looking to the future, it appears that antibody purification is in for an acceleration.
