| 1. |
- Scheffel, Julia, et al.
(författare)
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ZCa : A protein A-derived domain with calcium-dependent affinity for mild antibody purification
- 2021. - Volume 2178
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Ingår i: Methods in Molecular Biology. - New York, NY : Humana Press Inc.. ; , s. 245-249
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Bokkapitel (refereegranskat)abstract
- Therapeutic antibodies are at the forefront of modern medicine where high purity, which is typically obtained by Protein A-based affinity purification, is of utmost importance. In this chapter, we present a method for neutral and selective purification of antibodies by utilizing an engineered affinity ligand, ZCa, derived from Protein A. This domain displays a calcium-dependent binding of antibodies and has been multimerized and immobilized to a chromatography resin to achieve an affinity matrix with high binding capacity. IgG antibodies can be eluted from the tetrameric ZCa ligand at pH 7 with the addition of EDTA, or at pH 5.5 with EDTA for purification of monoclonal IgG1, which is significantly milder than the low pH (3–4) required in conventional Protein A affinity chromatography. Here, a protocol for selective capture of IgG with elution at neutral pH from a ZCa tetramer ligand immobilized on a chromatography resin is described.
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| 2. |
- von Witting, Emma, et al.
(författare)
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Affinity-Based Methods for Site-Specific Conjugation of Antibodies
- 2021
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 32, s. 1515-1524
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Forskningsöversikt (refereegranskat)abstract
- Conjugation of various reagents to antibodies has long been an elegant way to combine the superior binding features of the antibody with other desired but non-natural functions. Applications range from labels for detection in different analytical assays to the creation of new drugs by conjugation to molecules which improves the pharmaceutical effect. In many of these applications, it has been proven advantageous to control both the site and the stoichiometry of the conjugation to achieve a homogeneous product with predictable, and often also improved, characteristics. For this purpose, many research groups have, during the latest decade, reported novel methods and techniques, based on small molecules, peptides, and proteins with inherent affinity for the antibody, for site-specific conjugation of antibodies. This review provides a comprehensive overview of these methods and their applications and also describes a historical perspective of the field.
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| 3. |
- von Witting, Emma, et al.
(författare)
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Small Bispecific Affinity Proteins for Simultaneous Target Binding and Albumin-Associated Half-Life Extension
- 2021
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 18:1, s. 328-337
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Tidskriftsartikel (refereegranskat)abstract
- Albumin-binding fusion partners are frequently used as a means for the in vivo half-life extension of small therapeutic molecules that would normally be cleared very rapidly from circulation. However, in applications where small size is key, fusion to an additional molecule can be disadvantageous. Albumin-derived affinity proteins (ADAPTs) are a new type of scaffold proteins based on one of the albumin-binding domains of streptococcal protein G, with engineered binding specificities against numerous targets. Here, we engineered this scaffold further and showed that this domain, as small as 6 kDa, can harbor two distinct binding surfaces and utilize them to interact with two targets simultaneously. These novel ADAPTs were developed to possess affinity toward both serum albumin as well as another clinically relevant target, thus circumventing the need for an albumin-binding fusion partner. To accomplish this, we designed a phage display library and used it to successfully select for single-domain bispecific binders toward a panel of targets: TNFα, prostate-specific antigen (PSA), C-reactive protein (CRP), renin, angiogenin, myeloid-derived growth factor (MYDGF), and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex consisting of the ADAPT together with albumin and each of the five targets, TNFα, PSA, angiogenin, MYDGF, and insulin. This simultaneous binding of albumin and other targets presents an opportunity to combine the advantages of small molecules with those of larger ones allowing for lower cost of goods and noninvasive administration routes while still maintaining a sufficient in vivo half-life.
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