Affinity-Based Analytical Purification Of Biotherapeutics For Bioprocess Monitoring
a bioprocess monitoring and analytical technology, applied in the field of biotechnology and the biomanufacturing of recombinant proteins, can solve the problems of inability to transfer into a quality control (“qc”) environment, the technique is not easily able to circumvent the need for product purification, and the light-based technique also suffers from potential signal-to-noise issues
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example 1
Purification of Anti-Enzyme 1 Antibody
[0188]Anti-Enzyme 1 polyclonal antibody was purified from antiserum using two chromatography steps: Protein G capture followed by a second affinity step using a column with Enzyme 1 immobilized on Tosoh Tresyl resin. Multiple elution conditions, including variations in pH and mobile phase modifiers, were screened before finalizing the column method (Table 3). All buffers were adjusted to the final pH using either 2N HCl or 50% NaOH, Minimal recovery (98%) of antibody was Observed using the regeneration buffer as the elution condition.
[0189]ELBA measurements were performed on selected Enzyme 1 column eluates to determine whether the elution conditions affected the ability of the antibody to bind antigen (Table 3). The 500 mM arginine-HCl eluate was assumed to be 100% pure Enzyme 1-specific antibody and was used as the reference standard for the ELISA. All measurements were normalized according to initial sample mass concentration.
[0190]Overall, t...
example 2
Antibody Coupling and Binding Capacity Optimization
[0194]1. Enzyme 1
[0195]A screening study was performed to maximize the coupling reaction yield as well as the binding capacity of the ant Enzyme 1 polyclonal antibody affinity resin. Several variables known to affect coupling yield and binding capacity were studied, including coupling chemistry, coupling buffer (pH and ionic strength), temperature, and ligand density. Coupling yield and static binding capacity were measured and calculated as described in the binding capacity testing section above. As indicated, multiple buffer conditions were studied for the Tosoh Tresyl couplings, while the manufacturer recommended buffers were used exclusively for AminoLink® (pH 7.2), CarboLink™, and SulfoLink® resins. The measured coupling yields were high, generally 70% or greater, while the binding efficiencies were between 2 and 14%. A wide range of binding efficiencies have been reported in the literature for similar affinity constructs, rang...
example 3
Affinity Column Performance in Direct Comparison with Multi-Step Process Train Purification for Enzyme 1
[0212]To test the applicability of the antibody affinity columns, identical starting materials were purified using both the anti-Enzyme affinity column and the multi-step scale-down purification train to enable direct comparisons of the resulting purified materials. Two classes of load materials were studied: (1) drug substance, and (2) clarified harvest fluid for both Enzymes 1 and 2. Drug substance was used as a load to determine whether any product quality attribute, such as glycosylation profile, was changed by the affinity column operation.
[0213]1. Enzyme 1 Direct Comparison Study
[0214]For Enzyme 1, ten lots of harvest material were available for study. These lots were obtained from a large design of experiments (DoE) clone and media selection study, The individual lots were selected to cover a wide range of values for critical quality attributes, such as key glycans or produ...
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