Molecular engineering strategies and methods for the expression and purification of IgG1-based bispecific bivalent antibodies

Dimasi, Nazzareno; Fleming, Ryan; Wu, Herren; Gao, Changshou

doi:10.1016/j.ymeth.2018.08.004

Cited by 19 publications

(19 citation statements)

References 31 publications

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“…Trastuzumab and NIP228 antibodies were transiently expressed in Chinese hamster ovary (CHO) cells with yields of 300 mg L −1 after 14 days of transient expression. Expression levels were quantified by using a protein A quantification method . Purification was carried out by protein A affinity chromatography, which resulted in 99 % monomeric content for both antibodies (Figure a).…”

Section: Resultsmentioning

confidence: 99%

“…SEC of the purified Fabs showed monomeric content for both Fabs of approximately 80 %, with the presence of high‐molecular‐weight aggregates, undigested antibodies and fragments (Figure b). For the transient expression, the Fabs were cloned into a MedImmune mammalian expression vector, and expression was carried out in CHO cells for 14 days, resulting in 250 and 150 mg L −1 for trastuzumab Fab and NIP228 Fab, respectively. The Fabs were purified from the culture medium by anti‐kappa affinity chromatography.…”

Section: Resultsmentioning

confidence: 99%

“…Expression levels were quantified by using ap rotein Aq uantification method. [39,40] Purification was carried out by protein Aa ffinity chromatography,w hich resulted in 99 %m onomeric content for both antibodies (Figure 2a). Fabs were prepared by papain digestion of antibodies.…”

Section: Preparation Of Fabs From Trastuzumab and From An Isotype Conmentioning

confidence: 99%

“…After approximately 80 %d igestion, both trastuzumab and NIP228 Fabs were purified by anti-kappa affinity chromatography.S EC of the purified Fabs showed monomeric contentf or both Fabs of approximately 80 %, with the presence of high-molecularweighta ggregates, undigested antibodies and fragments (Figure 2b). For the transient expression, the Fabs were cloned into aM edImmune mammalian expression vector, [39,40] and expression was carriedo ut in CHO cells for 14 days, resulting in 250 and 150 mg L À1 for trastuzumab Faba nd NIP228 Fab, respectively.T he Fabs were purified from the culture mediumb y anti-kappaa ffinity chromatography.B oth recombinant Fabs had am onomeric content of 95 %, withm inor amounts of aggregates and fragments ( Figure 2c). The Fabs prepared by papaind igestion and by transient expression were used directly for rebridging withoutfurtherpurification.…”

Section: Preparation Of Fabs From Trastuzumab and From An Isotype Conmentioning

confidence: 99%

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Characterization of Disulfide Bond Rebridged Fab–Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload

Ruddle

Fleming

et al. 2019

ChemMedChem

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We describe the characterization of antigen binding fragments (Fab)–drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolobenzodiazepine dimers, which are DNA cross‐linkers, are a class of payloads used in antibody–drug conjugates (ADCs). SG3710 was designed to rebridge two adjacent cysteines, such as those that form the canonical interchain disulfide bond between the light and heavy chain in Fab fragments. The rebridging generated homogenous Fab conjugates, with a drug‐to‐Fab ratio of one, as demonstrated by the preparation of rebridged Fabs derived from the anti‐HER2 trastuzumab antibody and from a negative control antibody both prepared using recombinant expression and papain digestion. The resulting anti‐HER2 trastuzumab Fab‐rebridged conjugate retained antigen binding, was stable in rat serum, and demonstrated potent and antigen‐dependent cancer cell‐killing ability. Disulfide rebridging with SG3710 is a generic approach to prepare Fab–pyrrolobenzodiazepine dimer conjugates, which does not require the Fabs to be engineered for conjugation. Thus, SG3710 offers a flexible and straightforward platform for the controlled assembly of pyrrolobenzodiazepine dimer conjugates from any Fab for oncology applications.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Preparation Of Fabs From Trastuzumab and From An Isotype Conmentioning

confidence: 99%

Section: Preparation Of Fabs From Trastuzumab and From An Isotype Conmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of Disulfide Bond Rebridged Fab–Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload

Ruddle

Fleming

et al. 2019

ChemMedChem

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show abstract

“…Expression was carried out for 14 days using an AstraZeneca proprietary culture medium. The expression level in the culture supernatant was determined using a protein A binding method [35,37]. After expression, the cells were pelleted, discarded after centrifugation, and the culture medium was filtered with 0.2-micron polyethersulfone Rapid-FlowTM 75mm Filter Units (Thermo Scientific, Waltham, MA, USA).…”

Section: Transient Expression Of Parental Antibodiesmentioning

confidence: 99%

Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media

Steinhardt

Fleming

et al. 2019

Pharmaceutics

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Bispecific antibody (bsAb) applications have exponentially expanded with the advent of molecular engineering strategies that have addressed many of the initial challenges, including improper light chain pairing, heterodimer purity, aggregation, and pharmacokinetics. However, the lack of high-throughput methods for the generation of monovalent bsAbs has resulted in a bottleneck that has hampered their therapeutic evaluation, as current technologies can be cost-prohibitive and impractical. To address this issue, we incorporated single-matched point mutations in the CH3 domain to recapitulate the physiological process of human IgG4 Fab-arm exchange to generate monovalent bsAbs. Furthermore, we utilized the substitutions H435R and Y436F in the CH3 domain of IgG1, which incorporates residues from human IgG3, thus ablating protein A binding. By exploiting this combination of mutations and optimizing the reduction and reoxidation conditions for Fab arm exchange, highly pure monovalent bsAbs can be rapidly purified directly from combined culture media using standard protein A purification. This methodology, reported herein for the first time, allows for the high-throughput generation of monovalent bsAbs, thus increasing the capacity for evaluating monovalent bsAb iterations for therapeutic potential.

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Methods for transient expression and purification of monoclonal antibodies in mammalian cells

Kamle

Lee

et al. 2022

Advances in Protein Molecular and Structural Biology Methods

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Molecular engineering strategies and methods for the expression and purification of IgG1-based bispecific bivalent antibodies

Cited by 19 publications

References 31 publications

Characterization of Disulfide Bond Rebridged Fab–Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload

Characterization of Disulfide Bond Rebridged Fab–Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload

Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media

Methods for transient expression and purification of monoclonal antibodies in mammalian cells

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