Influence of pH on Heat-Induced Aggregation and Degradation of Therapeutic Monoclonal Antibodies

Ishikawa, Tomoyoshi; Ito, Takahiko; Endo, Ryosuke; Nakagawa, Keiko; sawa, Eiji; Wakamatsu, Kaori

doi:10.1248/bpb.33.1413

Cited by 62 publications

(56 citation statements)

References 28 publications

(24 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since the stability of IgG1 F c portion (C H 2 and C H 3) depends mainly on environmental stresses like pH and temperature, DSC was used to evaluate the conformational and thermodynamic stability of etanercept. 26,27) The result from a reference of IgG1 was similar to this study. [28][29][30] They exhibited two significant endothermic peaks representing C H 2 and C H 3 from F c portion of IgG1 with similar transition temperatures.…”

Section: Discussionsupporting

confidence: 87%

Effects of pH and Buffer Concentration on the Thermal Stability of Etanercept Using DSC and DLS

Kim

Lim

et al. 2014

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

The protein size, electrical interaction, and conformational stability of etanercept (marketed as Enbrel ® ) were examined by thermodynamic and light scattering methods with changing pH and buffer concentration. As pH of etanercept increased from pH 6.6 to 8.6, electrical repulsion in the solution increased, inducing a decrease in protein size. However, the size changed less in high buffer concentration and irreversible aggregation issues were not observed; in contrast, aggregates of about 1000 nm were observed in low buffer concentration at the pH range. Three significant unfolding transitions (T m ) were observed by differential scanning calorimetry (DSC). Unlikely to T m 1, T m 2 and T m 3 were increased as the pH increased. Higher T m at high buffer concentration was observed, indicating increased conformational stability. The apparent activation energy of unfolding was further investigated since continuous increase of T m 2 and T m 3 was not sufficient to determine optimal conditions. A higher energy barrier was calculated at T m 2 than at T m 3. In addition, the energy barriers were the highest at pH from 7.4 to 7.8 where higher T m 1 was also observed. Therefore, the conformational stability of protein solution significantly changed with pH dependent steric repulsion of neighboring protein molecules. An optimized pH range was obtained that satisfied the stability of all three domains. Electrostatic circumstances and structural interactions resulted in irreversible aggregation at low buffer concentrations and were suppressed by increasing the concentration. Therefore, increased buffer concentration is recommended during protein formulation development, even in the earlier stages of investigation, to avoid protein instability issues.Key words buffer concentration; etanercept; differential scanning calorimetry (DSC); dynamic light scattering (DLS); pH effect; scan rate effect Advances in biotechnology in the last decades have made it possible to produce a number of proteins for therapeutic uses. Protein pharmaceuticals have drawn a lot of attention by virtue of their effectiveness and patient needs. Consequently, therapeutic proteins have overtaken low molecular weight compounds as the objects of novel drug development in the last decade.1,2) However, these proteins have complicated structures, marginal stability, and high sensitivity to degradation. Moreover, the proteins must be stabilized in solution to maintain their efficacy and benefits. 3-6)Protein aggregates are problematic because of possible activity loss and immunogenicity issues.7,8) Therefore, it is important to determine the causes of protein aggregation in order to minimize it to an acceptable level. Mutual interactions between pH and various buffers with lysozyme were investigated using a robust design (RD) method in a previous study.9) Acceptable pH range and buffer were selected based on thermodynamic properties from differential scanning calorimetry (DSC). In addition, selection of suitable buffer system of human epithelial growth factor b...

show abstract

Section: Discussionsupporting

confidence: 87%

Effects of pH and Buffer Concentration on the Thermal Stability of Etanercept Using DSC and DLS

Kim

Lim

et al. 2014

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

show abstract

“…In addition to effector functions and stability and biophysical characteristics, analytical and regulatory aspects are important selection criteria for such monoclonal antibodies (mAbs) destined for clinical use. Further, it is well known that during manufacturing, purification, formulation, and storage, the isotypes behave differently in terms of their sensitivity to pH or temperature, aggregation propensity, and susceptibility to degradation (11)(12)(13). With regard to monitoring or predicting stability, thermal stability is increasingly accepted as a convenient surrogate measure of global stability.…”

mentioning

confidence: 99%

Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability

Peters

Smales

Henry

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: IgG1 and IgG4 have different inter-light chain-heavy chain disulfide bond (DSB) arrangements. Results: IgG4 mutants with an IgG1-like DSB and a S241P hinge mutation showed increased Fab thermal stability and reduced DSB heterogeneity compared with IgG4 WT. Conclusion: Fab domain thermal stability and DSB heterogeneity of IgG4 can be improved. Significance: Such engineered IgG4 molecules offer potential advantages during therapeutic antibody production.

show abstract

“…Degradation of foreign proteins is a significant challenge for their cost-effective production, regardless of the transient expression system used (Hehle et al, 2015). Researchers have noted that optimized cultivation conditions will help to reduce the degradation of recombinant proteins (Ishikawa et al, 2010;Rita Costa et al, 2010). We also aim to address this problem in our future research.…”

Section: Discussionmentioning

confidence: 99%

Expression of recombinant human anti-TNF-α scFv-Fc in Arabidopsis thaliana seeds

Yao¹,

Ai²,

Dong³

et al. 2016

Genet. Mol. Res.

View full text Add to dashboard Cite

ABSTRACT. Recombinant human anti-tumor necrosis factor (TNF)-α scFv-Fc was expressed in TKO mutant Arabidopsis thaliana seeds using plant-specific codons. Immunoblotting using a human IgG1 antibody detected the expression of anti-TNF-α proteins in plants. Results from qRT-PCR analysis demonstrated that the time of harvest significantly affected the protein yield and quality. Our results indicate that the Phaseolus vulgaris β-phaseolin promoter directed anti-TNF-α scFvFc expression in A. thaliana seeds, with a maximum yield obtained at 20-days of development. Although the yield of anti-TNF-α scFvFc protein was not very high, accumulation of recombinant proteins in seeds is an attractive and simple method that can be used to purify biologically active anti-TNF-α scFv-Fc.

show abstract

Influence of pH on Heat-Induced Aggregation and Degradation of Therapeutic Monoclonal Antibodies

Cited by 62 publications

References 28 publications

Effects of pH and Buffer Concentration on the Thermal Stability of Etanercept Using DSC and DLS

Effects of pH and Buffer Concentration on the Thermal Stability of Etanercept Using DSC and DLS

Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability

Expression of recombinant human anti-TNF-α scFv-Fc in Arabidopsis thaliana seeds

Contact Info

Product

Resources

About