Catherine Shaffer holds a master's degree in biological chemistry and has worked as a research scientist. She is also an award-winning science fiction author and part-time reporter for local public radio.
Monoclonal antibodies are becoming increasingly important as key therapeutic targets. In 2015, more than 100 antibodies were being evaluated in clinical studies, according to AntibodySociety.org. Pharmaceutical and biotech companies are developing novel antibody-based therapies, and many companies are also working on biosimilars to blockbuster antibody-based drugs like Humira (adalimumab), Lucentis (ranibizumab) and Rituxan (rituximab). New categories of antibody-based therapeutics, such as antibody-drug conjugates, glyco-engineered antibodies and bispecific antibodies, are entering clinical pipelines.
Protein candidates may be discovered via basic research, but further characterization of lead targets for subsequent downstream workflow may require a different skill set when taking a bioprocessing focus.
"Prudent choices made during lead identification and characterization set the stage for a smooth, painless transition to pilot- or clinical-scale antibody production."
Here, we focus on the identification and characterization phases of antibody purification as potential candidates are moved through the workflow.
Process choices are critical for antibodies
In traditional small-molecule drug discovery, potential candidates typically are identified by screening large numbers of compounds or by designing compounds that have activity against the target. Small-molecule lead candidates are synthesized using organic chemistry methods, and scale-up is based on the original synthetic process for that molecule.
For antibody-based therapeutics, lead candidates generally are identified using phage display or hybridoma technologies. Promising candidates are subjected to rigorous identification and characterization, in which not only the antibody structure but also the growth conditions and methods of purification are optimized. Typically, at this point, antibodies are grown in either human embryonic kidney (HEK) 293 cells or Chinese hamster ovary (CHO) cells. Multiple clones are produced and evaluated for expression of the antibody.
Researchers often select multiple candidates that are produced under varying growth conditions when screening for lead antibody candidates. As part of the purification workflow, both the final product and related methodologies are further optimized with a goal of amenability to scale-up procedures.
“You're looking for high expressers. You're asking the cell to do a lot,” says Adam Elhofy, chief science officer for Essential Pharmaceuticals. After a high-expressing clone is selected, media optimization can begin. One of the components of typical cell-growth media is fetal bovine serum—a product phased out of bioproduction years ago for many reasons, including U.S. Food and Drug Administration regulations. Essential Pharmaceuticals makes a chemically defined, animal-origin-free supplement (Cell-Ess) that provides fats and cholesterol for growing cells and scales up to an approvable process for the final product. Alternative media options available from other companies; include PowerCHO Advance™ Media from Lonza, Thermo Fisher Scientific's Chemically Defined Lipid Concentrate and MilliporeSigma’s Ex-Cell Serum Free Media.
The final lead candidates may be further tested based on activity, potency and other physical properties, like isoelectric point and binding on and off rates. Often, there are a number of necessary assays that an antibody candidate must complete before it is selected for further validation.
Ideally, the antibody and production process developed during lead optimization can scale up smoothly to pilot- and clinical-scale production. Choices made during lead optimization and characterization can affect the success of further scale-up operations.
Scale down to scale up
A number of companies provide bioprocessing services. These services range from advice or assay development to complete end-to-end upstream and downstream bioprocessing. Somatek Inc., for example, offers comprehensive bioprocessing services. Somatek president and senior advisor Mark Fitchmun says that using robust, proven technologies and thinking ahead to the scale-up stage are keys to designing a successful bioprocess. “One of the things we do when we design a process for somebody is we look at the facility they have or the facility they're intending to have, and we assess what kind of equipment is already there and what types of technologies their people [are] already comfortable with,” Fitchmun says.
MilliporeSigma also offers an end-to-end bioprocess service that includes its ClinicReady mAb Single-use Template for rapid production of pre-clinical, Phase I and Phase II material.
Instead of trying to scale up a process, Fitchmun recommends visualizing the end process and then following it backwards. “If you're anticipating manufacturing something, it's a mistake to think about scaling up. Imagine where you're going to be, and imagine scaling down. That way, you never use a technique that won't scale up.”
In the lead identification and characterization stages, the scale-down-to-scale-up mind-set could help in choosing among a family of lead candidates that all have similar biological activity. In that case, choosing the candidate that will be faster, easier and more economical to produce makes the most sense. Other considerations at that stage include homogeneity, stability and solubility. “In essence, qualities which lend themselves to pharmaceutical elegance also lend themselves robust, cost-effective manufacturing,” says Fitchmun.
Powerful analytic methods
For analyzing potency and binding properties of antibodies, one of the most powerful and widely used technologies is the Biacore (surface plasmon resonance) instrument from GE Healthcare’s Life Sciences business. Surface plasmon resonance systems are used to analyze antibody interactions, on and off rates and binding specificity of protein targets.
Biacore has been used in the development of two-thirds of antibody therapeutics on the market, according to Jason Schuman, a senior product specialist at GE Healthcare. The instrument enables rapid selection of candidates through primary screening of antibodies from hybridoma lysates; measurement of binding properties that contribute to therapeutic efficacy; and studies required for safety and quality control, such as immunogenicity.
Biacore is an even larger player in the world of biosimilars. Because the original therapy has already been proven effective, there is more dependence on bioanalytical assays for biosimilars, as opposed to cell-based assays, according to Schuman. “More trust is given to the bioanalytical assay,” he says.
Assays remain important for antibody lead characterization. Sartorius Stedim BioOutsource specializes in cell-based bioassays for antibodies, including antibody-dependent cellular toxicity (ADCC) assays, complement-dependent cytotoxicity (CDC) assays, reporter assays and potency assays. The company also offers ELISA, electrochemiluminescence and surface plasmon resonance analysis.
According to Daniel Galbraith, chief scientist for Sartorius Stedim BioOutsource, an antibody molecule may have five to 10 biological functions that it can carry out. That would require five to 10 assays. The advantage Sartorius Stedim BioOutsource offers is having the assays ready, off-the-shelf and validated, with the experience to complete them quickly and accurately.
“The alternative would be to develop those assays in-house and have a team of technicians to run them,” says Galbraith. “From a cost-benefit point of view, it's better to outsource that function than doing it in house.”
The library of available assays and expertise can be important during a difficult bioprocess development. Galbraith says Humira, for example, is a notoriously difficult antibody for manufacturers to copy: “One of its functions is ADCC activity. Trying to replicate that molecule with the same level of activity is difficult for biosimilar manufacturers.” He says a prudent approach is to use a number of his company’s assays at the earliest stage possible, monitoring whether the process used delivers that ideal biosimilar copy.
Smooth workflow
Assays, test systems and methodologies used to identify lead targets, especially antibodies, for potential therapeutic applications must be chosen with pilot testing and manufacturing scale-up in mind to ensure a smooth transition. Robust, proven technologies and methods reduce guesswork and surprises later on. Scaling down to scale up is a worthwhile thought experiment that will avert the error of choosing a nonscalable method. Many products and technologies are available to support antibody lead identification and characterization, and service providers are available to design a bioprocess for your needs.
Image: Shutterstock