Identification of parameter interactions influencing the precipitation of a monoclonal antibody with anionic polyelectrolytes

Sieberz, Julia; Stanislawski, Bernd; Wohlgemuth, Kerstin; Schembecker, Gerhard

doi:10.1016/j.seppur.2014.02.033

Cited by 6 publications

(2 citation statements)

References 30 publications

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“…For example, the formation of inter- and intrachain complexes between proteins and polymers prior to coacervation is readily seen in PE–protein systems, but such precursors have been less evident for HP coacervation. Different equilibria in the latter case might explain the appearance of coacervates ,, rather than the precipitates more commonly encountered in the use of polyelectrolytes for protein purification. − In this study we seek (1) to characterize the molecular processes that lead to LLPS in HP systems, analogous to those surmised for PE–protein coacervation, and (2) to expand the current model for HP coacervates by relating the conditions required for coacervation to equilibria among complexes and free proteins.…”

Section: Introductionmentioning

confidence: 99%

Complex Equilibria, Speciation, and Heteroprotein Coacervation of Lactoferrin and β-Lactoglobulin

et al. 2015

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There has been a resurgence of interest in complex coacervation, a form of liquid-liquid phase separation (LLPS) in systems of oppositely charged macroions, but very few reports describe the somewhat anomalous coacervation between acidic and basic proteins, which occurs under very narrow ranges of conditions. We sought to identify the roles of equilibrium interprotein complexes during the coacervation of β-lactoglobulin dimer (BLG2) with lactoferrin (LF) and found that this LLPS arises specifically from LF(BLG2)2. We followed the progress of complexation and coacervation as a function of r, the LF/BLG molar ratio, using turbidity to monitor the degree of coacervation and proton release and dynamic light scattering (DLS) to assess the stoichiometry and abundance of complexes. Isothermal titration calorimetry (ITC) showed that initial complex formation is endothermic, but a large exotherm related to coacervate formation obscured other regions. On the basis of turbidimetry, proton release, and DLS, we propose a speciation diagram that presents the abundance of various complexes as a function of r. Although multiple species could be simultaneously present, distinct regions could be identified corresponding to equilibria among particular protein pairs.

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Section: Introductionmentioning

confidence: 99%

Complex Equilibria, Speciation, and Heteroprotein Coacervation of Lactoferrin and β-Lactoglobulin

et al. 2015

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“…A 25 mL system was used to examine the flocculation of yeast cells and debris, though neither the impact of the solid–liquid separation step nor correlations with larger scale performance were included (Wickramasinghe et al, ). For precipitation scouting, myriad lab‐scale configurations utilizing single vessels have been detailed, ranging from reaction volumes of 1.5 mL up to hundreds of mL (Gagnon, ; Liu et al, ; Sieberz et al, ). These precipitation studies have focused on the assessment of equilibrium conditions and excluded characterization of the particle aging steps and their subsequent impact on downstream operations.…”

Section: Introductionmentioning

confidence: 99%

High throughput screening of particle conditioning operations: I. System design and method development

Noyes

Huffman

Godavarti

et al. 2015

Biotech & Bioengineering

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The biotech industry is under increasing pressure to decrease both time to market and development costs. Simultaneously, regulators are expecting increased process understanding. High throughput process development (HTPD) employs small volumes, parallel processing, and high throughput analytics to reduce development costs and speed the development of novel therapeutics. As such, HTPD is increasingly viewed as integral to improving developmental productivity and deepening process understanding. Particle conditioning steps such as precipitation and flocculation may be used to aid the recovery and purification of biological products. In this first part of two articles, we describe an ultra scale-down system (USD) for high throughput particle conditioning (HTPC) composed of off-the-shelf components. The apparatus is comprised of a temperature-controlled microplate with magnetically driven stirrers and integrated with a Tecan liquid handling robot. With this system, 96 individual reaction conditions can be evaluated in parallel, including downstream centrifugal clarification. A comprehensive suite of high throughput analytics enables measurement of product titer, product quality, impurity clearance, clarification efficiency, and particle characterization. HTPC at the 1 mL scale was evaluated with fermentation broth containing a vaccine polysaccharide. The response profile was compared with the Pilot-scale performance of a non-geometrically similar, 3 L reactor. An engineering characterization of the reactors and scale-up context examines theoretical considerations for comparing this USD system with larger scale stirred reactors. In the second paper, we will explore application of this system to industrially relevant vaccines and test different scale-up heuristics.

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Emerging biomaterials for downstream manufacturing of therapeutic proteins

Stern

Lock

et al. 2019

Acta Biomaterialia

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Identification of parameter interactions influencing the precipitation of a monoclonal antibody with anionic polyelectrolytes

Cited by 6 publications

References 30 publications

Complex Equilibria, Speciation, and Heteroprotein Coacervation of Lactoferrin and β-Lactoglobulin

Complex Equilibria, Speciation, and Heteroprotein Coacervation of Lactoferrin and β-Lactoglobulin

High throughput screening of particle conditioning operations: I. System design and method development

Emerging biomaterials for downstream manufacturing of therapeutic proteins

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