Identification and characterization of rabbit scFv antibodies suitable for immuno-affinity separation of recombinant human kynureninase from Escherichia coli cell lysate.
J Biosci Bioeng
; 137(4): 298-303, 2024 Apr.
Article
en En
| MEDLINE
| ID: mdl-38296747
ABSTRACT
In this study we successfully developed an on-demand affinity chromatographic resin for manufacturing non-Fc-based biopharmaceuticals. Affinity chromatography columns with immobilized rabbit single-chain variable fragments (scFvs) were used for directly purifying the recombinant human kynureninase (KYNase) as a model target therapeutic protein from Escherichia coli cell lysates. Among the 38 different anti-KYNase scFv clones identified, four unique clones were selected as candidates for further characterization owing to their relatively low KYNase binding affinity at pH 4.0, thereby facilitating enzyme elution. Subsequently, all four clones were successfully produced and purified, followed by covalent coupling to NHS-activated HiTrap HP columns. While KYNase was specifically adsorbed to all four scFv-immobilized columns and was eluted at pH 4.0, the respective levels of static binding capacity (SBC) and recovery among the four scFv clones were different at this elution pH. That is, the scFv-immobilized columns captured KYNase with SBC ranging from 1.15 to 2.68 mg/cm3-bed with clone R2-47 exhibiting the highest level of SBC, with a ligand utilization of 39.4 %. Moreover, using the scFv column of R2-47, 90.7 % of the captured human KYNase was recovered in the first elution step at pH 4.0, and approximately 67 % of enzymatic activity was retained. In summary, high-purity human KYNase was obtained from the E. coli cell lysate by one-step affinity purification, and 89.7 % of KYNase was recovered in the first elution step. The methodology demonstrated in the current study could be applied for the purification and development of various therapeutic proteins.
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Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Anticuerpos de Cadena Única
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
J Biosci Bioeng
Asunto de la revista:
ENGENHARIA BIOMEDICA
/
MICROBIOLOGIA
Año:
2024
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Japón