Lowering Cost and Improving Quality

A key benefit to using affinity chromatography for purifying proteins is the simplicity of the process, which can often reduce a complex multistep procedure to a single step that results in a high-purity, high-quality product. Step reduction not only reduces production time and associated costs, but decreasing the length of time a protein product is “in process” limits potential degradation or damage, thereby ensuring better final product. Most important, each step of a downstream process reduces final product yield, so it's highly desirable to keep steps to a minimum.

Adenoassociated virus (AAV) is traditionally hard to purify. Over the past decade, it has received increased interest as a potential gene therapy vector. Current, conventional purification processes require multiple purification steps including density-gradient purification, several chromatography steps (ion-exchange, hydrophobic-interaction, heparin, and A-20 affinity), and an associated lengthy cleanroom residence time. With such a multistep process, the typical AAV yield is low. The process also suffers from poor scalability, limiting the commercial feasibility of this promising vector.

To address that challenge, a VHH ligand against AAV was identified and developed. Screening delivered a 14-kDa VHH fragment that was shown to bind AAV subclasses 1, 2, 3, and 5 and provide an eluate with high purity (7). Process steps were reduced from the typical four or five to just one or two steps, with a related reduction in cleanroom time for the recovery process from weeks to days. In this case, step reduction improved product recovery from 30% to >60%. Figure 4 shows the one-step purification of AAV serotype 1 using AVB Sepharose High Performance anti-AAV VHH ligands from GE Healthcare. The VHH ligand produced similar product purity to that of the reference standard.

Contaminant Removal

A highly desirable feature for all purification steps is the ability to remove contaminants such as host-cell proteins (HCPs) and DNA or virus particles. HCP and DNA clearance are good indications of the selectivity of purification media. In an analysis of protein A alternative chromatographic media for antibody purification, VHH ligands were found to be the only alternative that provided comparable HCP removal (10).

Viral clearance studies of the FVIII VHH ligand were performed by McCue, et al. (6). In their studies, clarified cell culture samples were spiked with one of two model viruses: xenotropic murine leukemia virus (X-MLV) to evaluate the adsorbent's ability to remove retroviruses or retrovirus-like particles; and mouse minute virus (MMV) to evaluate the ligand's ability to remove a small nonenveloped virus. Column elution pools were evaluated for virus presence, and polymerase chain reaction (PCR) was used to quantify the amount of virus in both the load and elution fractions. As Table 1 shows, the FVIII adsorbent provided significant clearance of both model viruses (6).

The ability of VHH ligands to remove HCP, DNA, and adventitious or endogenous viruses is expected to be good because of the ligand's complexity. The more complex a ligand is, generally the more selective it is.

A Platform Technology

VHH ligands provide a highly flexible platform technology for purification of almost any target protein with high specificity and selectivity and good contaminant clearance under mild elution conditions. A number of VHH affinity adsorbents are commercially available already and being used for production of CTMs. Many more are being developed for future research and bioprocess use in the future (11).

Use of VHH media as a platform could shorten development times for purification of a given molecule by providing a plug-and-play solution for downstream processing. The simplicity and high specificity of the process reduces overall complexity in multiproduct facilities and ensures a minimum number of process steps. That increases yield while retaining excellent purity and quality of the end product. VHH ligands offer a clear advantage for multiproduct facilities and groups working with typically hard-to-purify molecules.