Preactivation Crosslinking—An Efficient Method for the Oriented Immobilization of Antibodies

Schroeder, Barbara; Xuan, Hoa Le; Völzke, Jule L.; Weller, Michael G.

doi:10.3390/mps2020035

Cited by 18 publications

(16 citation statements)

References 63 publications

(63 reference statements)

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“…Surface chemistry based on organosilanes [18,19] opens up innumerable options for ligand immobilization and surface modification. Protein A [20][21][22] as a binder was examined as a model system. A total capacity of about 4 mg IgG per column was determined.…”

Section: Discussionmentioning

confidence: 99%

Sintered Glass Monoliths as Supports for Affinity Columns

et al. 2021

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A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 min. Due to the glass material’s excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.

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Section: Discussionmentioning

confidence: 99%

Sintered Glass Monoliths as Supports for Affinity Columns

et al. 2021

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“…Surface chemistry based on organosilanes [18,19] opens up innumerable options for ligand immobilization and surface modification. Protein A [20][21][22] as a binder was examined as a model system. The excellent commercial availability of the raw monoliths and the application of 3D printing for the column holder make this approach feasible for many laboratories.…”

Section: Discussionmentioning

confidence: 99%

Sintered Glass Monoliths as New Supports for Affinity Columns

Wilke¹,

Röder²,

Paul³

et al. 2021

Preprint

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A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 minute. Due to the glass material's excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.

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“…The specific and directed immobilization gave higher signals compared to undirected immobilization. Moreover, it enabled the possibility to immobilize antibodies directly from a crude supernatant …”

Section: Affinity Ligands and Their Role In Site-specific Conjugation...mentioning

confidence: 99%

Affinity-Based Methods for Site-Specific Conjugation of Antibodies

2021

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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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Preactivation Crosslinking—An Efficient Method for the Oriented Immobilization of Antibodies

Cited by 18 publications

References 63 publications

Sintered Glass Monoliths as Supports for Affinity Columns

Sintered Glass Monoliths as Supports for Affinity Columns

Sintered Glass Monoliths as New Supports for Affinity Columns

Affinity-Based Methods for Site-Specific Conjugation of Antibodies

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