Refolding Technologies for Antibody Fragments

Arakawa, Tsutomu; Ejima, Daisuke

doi:10.3390/antib3020232

Cited by 17 publications

(13 citation statements)

References 26 publications

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“…Important to note, assessing the RMY after isothermal unfolding/refolding of the protein for the purpose of formulation development would be quite different compared to unfolding/refolding experiments to increase the monomer yield after protein expression as inclusion bodies. The latter experiments usually include a reduction and new formation of disulphide bonds, as well as extremities in the pH, excipient concentration and the type of excipients used [51][52][53][54][55] . Assessing the aggregation during isothermal unfolding/refolding of the protein as a formulation tool is focused on a pH range which is realistic for long-term storage and administration in patients due to chemical stability and tolerability considerations respectively.…”

Section: Introductionmentioning

confidence: 99%

The ReFOLD assay for protein formulation studies and prediction of protein aggregation during long-term storage

Svilenov

Winter

2019

European Journal of Pharmaceutics and Biopharmaceutics

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The formulation of novel therapeutic proteins is a challenging task which aims at finding formulation conditions that will minimize protein degradation during long-term storage. One particularly important and difficult-to-predict protein degradation pathway is the so-called non-native aggregation. The qualitative and quantitative prediction of the latter has been a subject of extensive research over the past two decades. An increasing body of evidence shows that the widely-used short-term biophysical techniques cannot accurately rank formulation conditions in order of their effect on the aggregation during long-term storage of some therapeutic proteins, e.g. monoclonal antibodies. Here we suggest a novel approach for the selection of formulation conditions that will suppress the formation of protein aggregates during long-term storage. We postulate that conditions (i.e. pH, buffer type, ionic strength) that reduce the isothermal aggregation of various denaturant-induced partially folded protein species will be conditions that impede protein aggregation during long-term storage. To test our hypothesis, we developed an isothermal microdialysis-based unfolding/refolding assay, named ReFOLD, which we use to induce moderate aggregation of partially folded proteins. Next, we assessed the relative monomer yield after isothermal unfolding/refolding of two monoclonal antibodies, each formulated in 12 different conditions. Using the proposed approach, we were able to accurately rank the formulations in order of their effect on the amount of protein aggregates detected after storage for 12 months at 4 ⁰C and 25 ⁰C, while widely-used stability-indicating parameters like protein melting and aggregation onset temperatures failed to provide accurate predictive formulation rankings.

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Section: Introductionmentioning

confidence: 99%

The ReFOLD assay for protein formulation studies and prediction of protein aggregation during long-term storage

Svilenov

Winter

2019

European Journal of Pharmaceutics and Biopharmaceutics

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“…The expression of folded antibody fragments due to the formation of inclusion body and the need for refolding processes is difficult in Escherichia coli cytoplasm (Arakawa, Ejima, 2014). In addition, obtaining high level of soluble protein is often desirable (Fathi-Roudsari, Akhavian-Tehrani, Maghsoudi, 2016).…”

Section: Discussionmentioning

confidence: 99%

The improvement of anti-HER2 scFv soluble expression in Escherichia coli

Farshdari

Ahmadzadeh

Nematollahi

et al. 2020

Braz. J. Pharm. Sci.

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The relationship between the expression of HER2 and malignity of breast tumors has led to the generation of antibodies targeting HER2 + tumors. In addition, the expression of scFvs, as the smallest antigen-binding region of antibody containing two disulfide bonds in Escherichia coli often results in accumulating non-functional protein in the cytoplasm. A redox-modified strain of E. coli such as Origami (DE3) may facilitate the formation of proper disulfide bond in cytoplasm. The present study aimed to optimize the expression of anti-HER2 scFv in Origami and evaluate the influence of induction temperature, and host strain on the solubility of the protein. To this aim, chemically synthesized anti-HER2 scFv of Trastuzumab was cloned in pET-22b (+). The results demonstrated that anti-HER2 scFv is expressed in Origami, purified by using Ni-NTA column, and detected by anti-His antibody in Western blot analysis. The highest anti-HER2 scFv expression in Origami was achieved 24 h after IPTG induction (1 mM) at 37 °C. Further, the total anti-HER2 scFv expression level was higher in BL21, compared to Origami strain. However, the ratio of soluble/insoluble forms of anti-HER2 scFv increased in Origami strain. Furthermore, higher soluble expression was achieved when the culture of recombinant Origami was conducted at lower temperature (25 °C).

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“…Production of antibodies and their derivatives is often given in the form as 'cytoplasmic aggregates (inclusion bodies)' in large quantity, rather than as native forms (soluble forms) in the cytoplasm or periplasm, and the production is divergent depending on the degree of sequence heterogeneity [8,12,13]. Once cytoplasmic aggregates were obtained, several different techniques are employed for refolding, including dilution-based refolding [7], stepwise dialysis refolding [6,[8][9][10], ion-exchange chromatography-based refolding [13], and size-exclusion chromatography-based refolding [14][15][16].…”

Section: Introductionmentioning

confidence: 99%