A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

Saito, Seiji; Namisaki, Hiroshi; Hiraishi, Keiko; Takahashi, Nobuaki; Iida, Shigeru

doi:10.1002/pro.3598

Cited by 29 publications

(28 citation statements)

References 44 publications

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“…A fundamentally different approach would be (re‐)engineering of the product's primary structure to increase its tolerance towards acidic conditions (Ducancel & Muller, ; Saito, Namisaki, Hiraishi, Takahashi, & Iida, ; Skamris et al, ). We have shown that a simple method such as ANS fluorescence could be used to identify candidate molecules that have a low propensity to denature under (mildly) acidic conditions and would thus aggregate to a low extent during subsequent neutralization.…”

Section: Discussionmentioning

confidence: 99%

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Wälchli

Ressurreição

Vogg

et al. 2019

Biotech & Bioengineering

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Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by D-sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields. K E Y W O R D SANS fluorescence, downstream processing, monoclonal antibodies, protein aggregation, protein unfolding, viral inactivation

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Section: Discussionmentioning

confidence: 99%

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Wälchli

Ressurreição

Vogg

et al. 2019

Biotech & Bioengineering

View full text Add to dashboard Cite

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“…Overall, our data shows IgG3 possess superior effector functions over IgG1 that render it well for use in cancer therapy. However, owing to an enhanced susceptibility to proteolysis, shorter half-life than other IgG subclasses and aggregation during bioprocessing, IgG3 has not been used as a therapeutic drug 35 .To circumvent these obstacles engineered human IgG3 anti-CD20 antibodies containing the CH3 constant domain from IgG1 have recently been developed, providing a new arsenal of IgG3 antibodies to explore 36 .…”

Section: Discussionmentioning

confidence: 99%

A three dimensional human immune-tumor cell model reveals the importance of isotypes in antibody-based immunotherapy

Lara

Anania

Virtanen

et al. 2020

Preprint

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AbstractMonoclonal antibodies (mAb) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates in the patients, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a novel pre-clinical human immune – three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer culture, of human CD20+ B-cell lymphoma. We show that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, mediating strong infiltration of monocytic effector cells into 3D spheroids. Hence, the human immune-3D tumor model is an attractive ex vivo system to help filter out mAbs for best efficacy in cancer immunotherapy.

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“…Furthermore, a Lys is present at position 409 in IgG3, but IgG3 is still prone to aggregation under acidic conditions. A recent study has reported that two amino acid mutations in the CH3 domain, N392K and M397V, decreased aggregation during low pH conditions [36]. However, our study only analyzed IgG4 aggregation under low-pH treatment.…”

Section: Plos Onementioning

confidence: 96%

R409K mutation prevents acid-induced aggregation of human IgG4

et al. 2020

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Human immunoglobulin G isotype 4 (IgG4) antibodies are suitable for use in either the antagonist or agonist format because their low effector functions prevent target cytotoxicity or unwanted cytokine secretion. However, while manufacturing therapeutic antibodies, they are exposed to low pH during purification, and IgG4 is more susceptible to low-pH-induced aggregation than IgG1. Therefore, we investigated the underlying mechanisms of IgG4 aggregation at low pH and engineered an IgG4 with enhanced stability. By swapping the constant regions of IgG1 and IgG4, we determined that the constant heavy chain (CH3) domain is critical for aggregate formation, but a core-hinge-stabilizing S228P mutation in IgG4 is insufficient for preventing aggregation. To identify the aggregation-prone amino acid, we substituted the CH3 domain of IgG4 with that of IgG1, changing IgG4 Arg409 to a Lys, thereby preventing the aggregation of the IgG4 variant as effectively as in IgG1. A stabilizing effect was also recorded with other variable-region variants. Analysis of thermal stability using differential scanning calorimetry revealed that the R409K substitution increased the Tm value of CH3, suggesting that the R409K mutation contributed to the structural strengthening of the CH3-CH3 interaction. The R409K mutation did not influence the binding to antigens/human Fcγ receptors; whereas, the concurrent S228P and R409K mutations in IgG4 suppressed Fab-arm exchange drastically and as effectively as in IgG1, in both in vitro and in vivo in mice models. Our findings suggest that the IgG4 R409K variant represents a potential therapeutic IgG for use in low-effector-activity format that exhibits increased stability.

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A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

Cited by 29 publications

References 44 publications

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

A three dimensional human immune-tumor cell model reveals the importance of isotypes in antibody-based immunotherapy

R409K mutation prevents acid-induced aggregation of human IgG4

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