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Downstream processing of biopharmaceuticals is costly and time-consuming, often involving multiple steps with significant time and energy expended on maximizing product quality and yield. Affinity chromatography is one of the simplest and most effective methods for purifying protein and peptide therapeutics, offering reduced process steps and therefore higher yields than nonaffinity methods can provide. Protein A is widely used for affinity purification of monoclonal antibodies (MAbs), Fc fragments, and Fc fusion proteins. But it is a challenge to find a protein A equivalent for nonantibody targets.
To be useful in bioprocess applications, an affinity medium must provide the specificity required to purify a product effectively while allowing elution conditions that preserve product structure and activity. It is also important that an affinity medium can withstand cleaning processes required for effective adsorbent cleaning to meet regulatory standards in therapeutics production.
To address these challenges, BAC derived a class of affinity ligands from unique heavy-chain antibodies found in genus Camelidae (trade named CaptureSelect). Heavy-chain antibodies are devoid of the entire light chain and CH1 domain found in conventional antibodies (Figure 1). Because of that absence, antigens are bound only by the variable domain of the heavy chain (VHH) without any less binding affinity than conventional antibodies. Derivative VHH ligands are ~12-kD single-domain fragments that make up the antigen-binding domain. They are efficiently produced by the yeast Saccharomyces cerevisiae in a system that is completely free of animal components. This new class of ligand has been shown to provide not only a suitable alternative to protein A (1), but also an equivalent for a wide range of nonantibody targets, a number of which are being used in manufacturing clinical trial materials (CTMs).
PRODUCT FOCUS: NON-MA
The unique suitability of the VHH domain for use as an affinity ligand lies in the stability of its structure as a fragment. It is the smallest known functional antigen-binding antibody fragment that shows high affinity and stability. The Fv domain is its equivalent fragment in conventional antibodies that consists of a heavy-chain variable domain (VH) and a light-chain variable domain (VL) held together by a noncovalent bond. If that bond is broken, all antigen-binding functionality is lost. Although the method of synthetic production — linking the two variable domains together into a single chain — retains the antigen binding functionality of scFv fragments, they often show less affinity than their parent antibody. Because the VHH domain exists as a single polypeptide chain, it is an extremely stable and easily produced fragment that retains the full binding activity of its parent heavy-chain antibody.
VHH fragments have been used successfully as affinity ligands in chromatography processes for purifying many types of protein, including antibodies (2,3,4,5), blood factors (6), and adenoassociated viruses (7,8). Ligands for a specific target molecule are identified from expression libraries that represent the heavy-chain antibody repertoire of an immunized llama. Coming from a highly variable mammalian immune system, these expression libraries provide an opportunity to select VHH molecules that meet very specific requirements for specificity, affinity, elution profile, and cleaning conditions. Thus, VHH affinity ligands can be created to match exactly the processes for which they are intended. Immobilization onto resins suitable for large-scale bioprocess applications make these ligands good tools for downstream processing.
Platform Technology for Multiproduct FacilitiesVHH affinity media enable a “plug-and-play” platform approach providing a highly specific primary capture step that can be slotted into existing downstream processes. Figure 2 illustrates how VHH media can be incorporated into any “generic” biopharmaceutical purification process for comparable results with current MAb purification platforms using protein A as a primary capture step.
Many biotherapeutics are produced in multiproduct contract manufacturing facilities. It would be significantly convenient if purification processes for most/all products could be made similar — for example, if nonantibody products could be purified by a similar process to the protein A purification of MAbs. To that aim, the use of a VHH ligand for primary capture of recombinant Factor VIII (FVIII) was investigated as an alternative to a traditional multistep process involving chromatographic separation and precipitation techniques.
The VHH adsorbent was found to be highly selective for recombinant FVIII. It provided good viral clearance and significantly improved host-cell DNA and protein clearance (6). Of importance, FVIII elution was achieved at a neutral pH because the protein is relatively unstable, and yields exceeded 70%. In addition, the adsorbent retained capacity following 25 cycles of cleaning and reuse, and it produced consistent results across the range from laboratory to manufacturing scale. That demonstrated the suitability of the VHH ligand for use in FVIII commercial manufacturing. Table 1 summarizes the key features of the anti-FVIII adsorbent.
Table 1: Summary of FVIII adsorbent analysis (6)
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