Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

Svilenov, Hristo; Winter, Gerhard

doi:10.1016/j.xphs.2020.03.011

Cited by 12 publications

(11 citation statements)

References 46 publications

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“…A strong correlation between k D and B 22 for peptide-12 was observed where repulsive PPIs dominate, which decreased upon increasing attractive PPIs (Figure ), which has also been reported by Svilenov and Winter and Menzen and Friess . Negative B 22 values are typically associated with overall attractive PPIs, increasing the aggregation propensity, which was also observed in our case whereby formulations with the low B 22 parameters included all formulations that showed aggregation formation under thermal stress.…”

Section: Discussionsupporting

confidence: 87%

“…Most of the prepared formulations (∼70%) showed k D and B 22 related to non-repulsive interactions and thus having a certain potential for aggregation at longer time scale. 72,112 Overall, the colloidal stability of peptide-12 under different formulation conditions at pH 6.0 was inferior compared to pH 4.5. At pH 4.5, the addition of ionic tonicity agents shifted the k D and B 22 toward quite low or negative values, resulting in the preference of non-ionic tonicity agents.…”

Section: Influence Of Ph Value and Buffering Agent On Intermolecular ...mentioning

confidence: 98%

“…The use of k D and B 22 as parameters for assessing colloidal stability and the corresponding aggregation risk during storage has been studied in numerous examples for larger proteins and antibodies. ,,− However, there is little reported applying these parameters for the formulation screening of peptides. Examples are the use of self-interaction chromatography for the B 22 analysis of enfuvirtide (a 36-amino acid synthetic peptide) in different solvents and a recently published screening protocol for assessing the peptide stability of plectasin (a 40-amino acid antimicrobial peptide with a distinct tertiary structure that includes three disulfide bridges) .…”

Section: Introductionmentioning

confidence: 99%

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High-Throughput Screening for Colloidal Stability of Peptide Formulations Using Dynamic and Static Light Scattering

et al. 2021

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Selection of an appropriate formulation to stabilize therapeutic proteins against aggregation is one of the most challenging tasks in early-stage drug product development. The amount of aggregates is more difficult to quantify in the case of peptides due to their small molecular size. Here, we investigated the suitability of diffusion self-interaction parameters (k D) and osmotic second virial coefficients (B 22) for high-throughput (HT) screening of peptide formulations regarding their aggregation risk. These parameters were compared to the effect of thermal stress on colloidal stability. The formulation matrix comprised six buffering systems at two selected pH values, four tonicity agents, and a common preservative. The results revealed that electrostatic interactions are the main driver to control colloidal stability. Preferred formulations consisted of acetate and succinate buffer at pH 4.5 combined with glycerol or mannitol and optional m-cresol. k D proved to be a suitable surrogate for B 22 as an indicator of high colloidal stability in the case of peptides as was previously described for globular proteins and antibodies. Formulation assessment solely based on k D obtained by HT methods offers important insights into the optimization of colloidal stability during the early development of peptide-based liquid formulations and can be performed with a limited amount of peptide (∼360 mg).

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Section: Discussionsupporting

confidence: 87%

Section: Influence Of Ph Value and Buffering Agent On Intermolecular ...mentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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High-Throughput Screening for Colloidal Stability of Peptide Formulations Using Dynamic and Static Light Scattering

et al. 2021

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“…Fluorescence spectroscopy is an important method to study the interaction between biological macromolecules and small molecules and ions. The emission peak characteristics, energy transfer, fluorescence lifetime, and other indicators in fluorescence testing can provide useful information on the structure of fluorescent chromophores in protein molecules and their microenvironment (Svilenov & Winter, 2020). J. Li et al.…”

Section: Introductionmentioning

confidence: 99%

Interaction mechanism between soybean protein isolate and citrus pectin

Zhang

et al. 2022

Journal of Food Science

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In this study, citrus pectin (CP) and soybean protein isolate (SPI) were used as raw materials to prepare a complex. The interaction mechanism and structural changes between SPI and CP were deeply studied by fluorescence spectroscopy and Fourier infrared spectroscopy. The results show that CP has a strong quenching effect on SPI's endogenous fluorescence, and with the addition of CP, the endogenous fluorescence intensity of SPI decreased from 13,565.2 to 6067.3. The CP quenching of SPI is static quenching, and the number of combined bits is 1.26. The results of three‐dimensional fluorescence spectra showed that the addition of CP reduced the polarity of SPI amino acid residue microenvironment and changed the protein structure. Hydrophobic interaction exists between CP and SPI. The results of three‐dimensional fluorescence spectra showed that the addition of CP reduced the polarity of the amino acid residue microenvironment of SPI and changed the protein structure. Fourier transform infrared spectroscopy shows that CP could change the secondary structure of SPI by decreasing the α‐helix and β‐sheet, increasing β‐rotation and irregular curl, destroying the ordered structure of SPI and increasing the polarity of the amino acids exposed to the solution. The microstructure analysis shows that SPI–CP composite system has honeycomb structure and dense pores. From the perspective of reaction thermodynamics, it was found that the addition of CP could improve the thermal stability of SPI and increase the denaturation temperature of SPI from 119.73 to 132.97°C. This study can provide a theoretical basis for the preparation of protein–pectin complexes and provides reference for their application in food grade gels and Pickering emulsions.

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“…This offers an excellent opportunity to study the aggregation propensity of the partially unfolded antibodies isothermally at storage temperatures. For example, dilution refolding experiments can provide insights into the aggregation propensity of antibodies in different solution conditions. , Furthermore, dialysis refolding experiments with the ReFOLD assay demonstrated a link between the ability of an antibody to remain monomeric after refolding from urea and the aggregation during long-term storage in different formulations. − …”

Section: Introductionmentioning

confidence: 99%

Combining Unfolding Reversibility Studies and Molecular Dynamics Simulations to Select Aggregation-Resistant Antibodies

Berner

Menzen²,

Winter

et al. 2021

Mol. Pharmaceutics

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The efficient development of new therapeutic antibodies relies on developability assessment with biophysical and computational methods to find molecules with drug-like properties such as resistance to aggregation. Despite the many novel approaches to select well-behaved proteins, antibody aggregation during storage is still challenging to predict. For this reason, there is a high demand for methods that can identify aggregation-resistant antibodies. Here, we show that three straightforward techniques can select the aggregation-resistant antibodies from a dataset with 13 molecules. The ReFOLD assay provided information about the ability of the antibodies to refold to monomers after unfolding with chemical denaturants. Modulated scanning fluorimetry (MSF) yielded the temperatures that start causing irreversible unfolding of the proteins. Aggregation was the main reason for poor unfolding reversibility in both ReFOLD and MSF experiments. We therefore performed temperature ramps in molecular dynamics (MD) simulations to obtain partially unfolded antibody domains in silico and used CamSol to assess their aggregation potential. We compared the information from ReFOLD, MSF, and MD to size-exclusion chromatography (SEC) data that shows whether the antibodies aggregated during storage at 4, 25, and 40 °C. Contrary to the aggregation-prone molecules, the antibodies that were resistant to aggregation during storage at 40 °C shared three common features: (i) higher tendency to refold to monomers after unfolding with chemical denaturants, (ii) higher onset temperature of nonreversible unfolding, and (iii) unfolding of regions containing aggregation-prone sequences at higher temperatures in MD simulations.

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Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

Cited by 12 publications

References 46 publications

High-Throughput Screening for Colloidal Stability of Peptide Formulations Using Dynamic and Static Light Scattering

High-Throughput Screening for Colloidal Stability of Peptide Formulations Using Dynamic and Static Light Scattering

Interaction mechanism between soybean protein isolate and citrus pectin

Combining Unfolding Reversibility Studies and Molecular Dynamics Simulations to Select Aggregation-Resistant Antibodies

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