Determination of protein phase diagrams by microbatch experiments: Exploring the influence of precipitants and pH

Baumgartner, Kai; Galm, Lara; Nötzold, Juliane; Sigloch, Heike; Morgenstern, Josefine; Schleining, Kristina; Suhm, Susanna; Oelmeier, Stefan A.; Hubbuch, Jürgen

doi:10.1016/j.ijpharm.2014.12.027

Cited by 44 publications

(45 citation statements)

References 43 publications

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“…Used buffer substances were citric acid (Merck, Darmstadt, Germany) and sodium citrate (SigmaAldrich, St. Louis, MO, USA) for pH 3, sodium acetate (SigmaAldrich, St. Louis, MO, USA) and acetic acid (Merck, Darmstadt, Germany) for pH 5, MOPSO (AppliChem, Darmstadt, Germany) for pH 7 and bis-tris propane (Molekula, Dorset, UK) for pH 9. Buffers were prepared as described in detail in Baumgartner et al (Baumgartner et al, 2015). The buffer capacity was 100 mM.…”

Section: Preparation Of Buffers and Protein Solutionsmentioning

confidence: 99%

“…Sodium chloride was obtained from Merck (Darmstadt, Germany) and ammonium sulfate was from VWR (Radnor, PA, USA). The precipitant stock solutions were prepared as described in detail by Baumgartner et al (Baumgartner et al, 2015). Phase diagrams for lys hum and lys egg were generated earlier (Baumgartner et al, 2015).…”

Section: Generation Of Protein Phase Diagramsmentioning

confidence: 99%

“…The precipitant stock solutions were prepared as described in detail by Baumgartner et al (Baumgartner et al, 2015). Phase diagrams for lys hum and lys egg were generated earlier (Baumgartner et al, 2015). a-lact concentration in the protein stock solutions at pH 3, 7, and 9 was adjusted to 43.5±1 mg/mL.…”

Section: Generation Of Protein Phase Diagramsmentioning

confidence: 99%

“…Six possible phase states were classified: clear solution, crystallization, precipitation, skin formation, gelation and phase separation. For further details on generation of protein phase diagrams in microbatch experiments see Baumgartner et al (Baumgartner et al, 2015). Figure 2a shows the values of the zeta potential of a-lact, lys hum , and lys egg at pH 3, 5, 7, and 9.…”

Section: Generation Of Protein Phase Diagramsmentioning

confidence: 99%

“…Phase diagrams of a-lact, lys hum , and lys egg at pH 3 (first row), pH 5 (second row), pH 7 (third row), and pH 9 (fourth row) using sodium chloride as precipitant. Data for the phase diagrams of lys hum and lys egg was taken from Baumgartner et al (Baumgartner et al, 2015) Figure 5: Phase diagrams of a-lact, lys hum , and lys egg at pH 3 (first row), pH 5 (second row), pH 7 (third row), and pH 9 (fourth row) using sodium chloride as precipitant. Data for the phase diagrams of lys hum and lys egg was taken from Baumgartner et al (Baumgartner et al, 2015) …”

Section: Figuresmentioning

confidence: 99%

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Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity

Galm

Amrhein

Hubbuch

2017

Biotech & Bioengineering

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The aggregation of proteins became one of the major challenges in the development of biopharmaceu-ticals since the formation of aggregates can affect drug quality and immunogenicity. However, aggregation mechanisms are highly complex and the investigation requires cost, time, and material intensive experi-mental effort. In the present work, the predictive power of protein characteristics for the phase behavior of three different proteins which are very similar in size and structure was studied. In particular, the surface hydrophobicity, zeta potential, and conformational flexibility of human lysozyme, lysozyme from chicken egg white, and α-lactalbumin at pH 3, 5, 7, and 9 were assessed and examined for correlation with experimental stability studies focusing on protein phase behavior induced by sodium chloride and ammonium sulfate. The molecular dynamics (MD) simulation based study of the conformational flexibility without precipitants was able to identify highly flexible protein regions which could be associated to the less regular secondary structure elements and random coiled and terminal regions in particular. Conformational flex-ibility of the entire protein structure and protein surface hydrophobicity could be correlated to differing aggregation propensities among the studied proteins and could be identified to be applicable for predic-tion of protein phase behavior in aqueous solution without precipitants. For prediction of protein phase behavior and aggregation propensity in aqueous solution with precipitants, protein flexibility was further studied in dependency of salt concentration and species by means of human lysozyme. Even though the results of the salt dependent MD simulations could not be shown to be sufficient for prediction of salt depending phase behavior, this study revealed a more pronounced destabilizing effect of ammonium sulfate in comparison to sodium chloride and thus, was found to be in good agreement with theoretical considerations along the Hofmeister series as well as experimentally evaluated phase behavior. Biotechnol. Bioeng. 2017;114: 1170-1183. © 2016 Wiley Periodicals, Inc.

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Section: Preparation Of Buffers and Protein Solutionsmentioning

confidence: 99%

Section: Generation Of Protein Phase Diagramsmentioning

confidence: 99%

Section: Generation Of Protein Phase Diagramsmentioning

confidence: 99%

Section: Generation Of Protein Phase Diagramsmentioning

confidence: 99%

Section: Figuresmentioning

confidence: 99%

See 3 more Smart Citations

Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity

Galm

Amrhein

Hubbuch

2017

Biotech & Bioengineering

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Microbioreactor Systems for Accelerated Bioprocess Development

Hemmerich

Noack

Wiechert

et al. 2018

Biotechnology Journal

138

143

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In recent years, microbioreactor (MBR) systems have evolved towards versatile bioprocess engineering tools. They provide a unique solution to combine higher experimental throughput with extensive bioprocess monitoring and control, which is indispensable to develop economically and ecologically competitive bioproduction processes. MBR systems are based either on down-scaled stirred tank reactors or on advanced shaken microtiter plate cultivation devices. Importantly, MBR systems make use of optical measurements for non-invasive, online monitoring of important process variables like biomass concentration, dissolved oxygen, pH, and fluorescence. The application range of MBR systems can be further increased by integration into liquid handling robots, enabling automatization and, thus standardization, of various handling and operation procedures. Finally, the tight integration of quantitative strain phenotyping with bioprocess development under industrially relevant conditions greatly increases the probability of finding the right combination of producer strain and bioprocess control strategy. This review will discuss the current state of the art in the field of MBR systems and we can readily conclude that their importance for industrial biotechnology will further increase in the near future.

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Model‐Based Investigation on the Mass Transfer and Adsorption Mechanisms of Mono‐Pegylated Lysozyme in Ion‐Exchange Chromatography

Morgenstern

Wang

Baumann

et al. 2017

Biotechnology Journal

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Recent studies highlighted the potential of PEGylated proteins to improve stabilities and pharmacokinetics of protein drugs. Ion-exchange chromatography (IEX) is among the most frequently used purification methods for PEGylated proteins. However, the underlying physical mechanisms allowing for a separation of different PEGamers (proteins with a varying number of attached PEG molecules) are not yet fully understood. In this work, mechanistic chromatography modeling is applied to gain a deeper understanding of the mass transfer and adsorption/desorption mechanisms of mono-PEGylated proteins in IEX. Using a combination of the general rate model (GRM) and the steric mass action (SMA) isotherm, simulation results in good agreement with the experimental data are achieved. During linear gradient elution of proteins attached with PEG of different molecular weight, similar peak heights, and peak shapes at constant gradient length are observed. A superimposed effect of increased desorption rate and reduced diffusion rate as a function of the hydrodynamic radius of PEGylated proteins is identified to be the reason of this anomaly. That is why the concept of the diffusion-desorption-compensation effect is proposed. In addition to the altered elution orders, PEGylation results in a considerable decrease of maximum binding capacity. By using the SMA model in a kinetic formulation, the adsorption behavior of PEGylated proteins in the highly concentrated state is described mechanistically. An exponential increase in the steric hindrance effect with increasing PEG molecular weight is observed. This suggests the formation of multiple PEG layers in the interstitial space between bound proteins and an associated shielding of ligands on the adsorber surface to be the cause of the reduced maximum binding capacity. The presented in silico approach thus complements the hitherto proposed theories on the binding mechanisms of PEGylated proteins in IEX.

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Determination of protein phase diagrams by microbatch experiments: Exploring the influence of precipitants and pH

Cited by 44 publications

References 43 publications

Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity

Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity

Microbioreactor Systems for Accelerated Bioprocess Development

Model‐Based Investigation on the Mass Transfer and Adsorption Mechanisms of Mono‐Pegylated Lysozyme in Ion‐Exchange Chromatography

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