Relationship of PEG-induced precipitation with protein-protein interactions and aggregation rates of high concentration mAb formulations at 5 °C

Wälchli, Ruben; Fanizzi, Francesca; Massant, Jan; Arosio, Paolo

doi:10.1016/j.ejpb.2020.03.011

Cited by 18 publications

(13 citation statements)

References 60 publications

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“…In our hands, absorbance measurements of soluble concentration proved more reliable than turbidity measurements, albeit the latter are easier to obtain as they don't require to separate out the precipitate [19][20][21][22]24 . Overall, we recommend using supernatant absorbance as it seems to yield more consistent results.…”

Section: Discussionmentioning

confidence: 83%

“…As long as the incubation time of 48 h is kept constant, the level of evaporation should As a solubility proxy to assess the relative solubility, we chose PEG 1/2 which represents the PEG concentration at which 50% of the protein is still soluble. Other groups have made use of the same approach 19,21,24 , while others used the onset of the sigmoidal curve 22 , which can also readily be obtained from our analysis pipeline. Taken together, these reports indicate that the measurement of PEG 1/2 is suitable for high-throughput solubility screening of protein variants 19 .…”

Section: Discussionmentioning

confidence: 99%

“…The mechanism by which PEG induces protein precipitation is based on excluded volume effects 15 , 16 , 23 , 24 , whereby the addition of polymers leads to an attractive force of entropic nature between proteins 23 – 25 . The association between proteins depends on the balance between the enthalpy gain due to their interaction and the entropy loss due to the formation of a bound state.…”

Section: Introductionmentioning

confidence: 99%

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An open-source automated PEG precipitation assay to measure the relative solubility of proteins with low material requirement

Oeller

Sormanni

Vendruscolo

2021

Sci Rep

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The solubility of proteins correlates with a variety of their properties, including function, production yield, pharmacokinetics, and formulation at high concentrations. High solubility is therefore a key requirement for the development of protein-based reagents for applications in life sciences, biotechnology, diagnostics, and therapeutics. Accurate solubility measurements, however, remain challenging and resource intensive, which limits their throughput and hence their applicability at the early stages of development pipelines, when long-lists of candidates are typically available in minute amounts. Here, we present an automated method based on the titration of a crowding agent (polyethylene glycol, PEG) to quantitatively assess relative solubility of proteins using about 200 µg of purified material. Our results demonstrate that this method is accurate and economical in material requirement and costs of reagents, which makes it suitable for high-throughput screening. This approach is freely-shared and based on a low cost, open-source liquid-handling robot. We anticipate that this method will facilitate the assessment of the developability of proteins and make it substantially more accessible.

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Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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An open-source automated PEG precipitation assay to measure the relative solubility of proteins with low material requirement

Oeller

Sormanni

Vendruscolo

2021

Sci Rep

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“…Schwartz et al (2017) found that polyethylene glycol (PEG) and SNARE induced tethering and fusion, arrested by Qc3, could be restored by added Sec17. PEG not only tethers, but may enhance SNARE-mediated fusion (Zick and Wickner, 2013) through promoting dehydration (Lehtonen and Kinnunen, 1995) and protein association (Wälchli et al, 2020) or, at high levels, induce fusion directly (Burgess et al, 1992). Dimeric GST-PX only acts as a tether.…”

Section: Discussionmentioning

confidence: 99%

Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS

Song

Wickner

2021

eLife

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Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.

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“…31 . Moreover, at least a certain degree of qualitative correlation is observed between native protein-protein interactions (commonly quantified via the second virial coefficient or the diffusion selfinteraction parameter) and solubility 27,32 , liquid-liquid phase separation 33 and viscosity at high protein concentration 34 . However, a reliable prediction of aggregation propensity especially over longer time scale remains a major challenge, and combined with the uncertainties of adverse impact on product quality and safety, protein aggregates continue to be designated by default as critical quality attributes.…”

Section: Protein Aggregation In Biologics Developmentmentioning

confidence: 99%

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

Das

Chou

Jiskoot

et al. 2022

Journal of Pharmaceutical Sciences

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Relationship of PEG-induced precipitation with protein-protein interactions and aggregation rates of high concentration mAb formulations at 5 °C

Cited by 18 publications

References 60 publications

An open-source automated PEG precipitation assay to measure the relative solubility of proteins with low material requirement

An open-source automated PEG precipitation assay to measure the relative solubility of proteins with low material requirement

Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

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Relationship of PEG-induced precipitation with protein-protein interactions and aggregation rates of high concentration mAb formulations at 5 °C

Cited by 18 publications

References 60 publications

An open-source automated PEG precipitation assay to measure the relative solubility of proteins with low material requirement

An open-source automated PEG precipitation assay to measure the relative solubility of proteins with low material requirement

Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

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Relationship of PEG-induced precipitation with protein-protein interactions and aggregation rates of high concentration mAb formulations at 5 °C