Hindered diffusion of proteins in mixture adsorption on porous anion exchangers and impact on flow-through purification of large proteins

Matlschweiger, Alexander; Fuks, Preston; Carta, Giorgio; Hahn, Rainer

doi:10.1016/j.chroma.2018.11.060

Cited by 15 publications

(7 citation statements)

References 40 publications

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“…However, here the chosen resin pore size was bigger than that of the Q Sepharose FF used in the original study and was estimated to lie between 29 and 45 nm . In addition, live protein uptake as well as static binding results confirmed much higher levels of protein absorption and therefore could not be used as our hypothesis …”

Section: Resultsmentioning

confidence: 88%

“…‐v), rejecting the ‘incomplete bead saturation’ hypothesis and suggesting that species other than intact monomers were the cause origin of the ring structure. Another logical reason for ring formation has been suggested to be large protein size and small resin pores . However, here the chosen resin pore size was bigger than that of the Q Sepharose FF used in the original study and was estimated to lie between 29 and 45 nm .…”

Section: Resultsmentioning

confidence: 93%

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Chromatography process development aided by a dye‐based assay

Jasulaityte

Johansson²,

Bracewell

2019

J of Chemical Tech & Biotech

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BACKGROUND: The lifetime of chromatography resins typically averages between 10 and 300 cycles for the manufacture of a therapeutic protein. Developing and establishing the robustness of the method for each separation process represents a significant challenge and is subject to extensive regulatory oversight. This paper presents a novel fluorescence-based assay for residual aggregated proteins to aid the evaluation of the extent of resin regeneration. RESULTS: The versatility of this method was demonstrated by using strong anion and cation exchange agarose resins PraestoQ and SP in conjunction with bovine serum albumin and monoclonal antibody feed materials. The assay entails applying a molecular rotor dye to a sample of free resin and measuring the fluorescence intensity using a plate reader or visualizing under a confocal laser scanning microscope to gain a more detailed characterization. Following five consecutive chromatography cycles, both methods revealed a 10-fold increase in fluorescence intensity along with a proportional reduction in dynamic binding capacity. Furthermore, the use of the assay suggested that fouling was dependent on spatial bead position in the column, bead channel structure and cleaning conditions. CONCLUSION: This work presents a simple assay suitable for use in resin lifetime studies to enhance process understanding.

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Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 93%

Chromatography process development aided by a dye‐based assay

Jasulaityte

Johansson²,

Bracewell

2019

J of Chemical Tech & Biotech

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“…Frontiers in Bioengineering and Biotechnology frontiersin.org 2017), have been developed to increase pore diffusion while reducing steric hindrance (Anspach et al, 1989;Matlschweiger et al, 2019). Some manufacturers have also added porosity data to their documented resin specifications, which previously reported only the (dynamic) binding capacity, approximate particle diameter and average pore size.…”

Section: Figurementioning

confidence: 99%

The use of predictive models to develop chromatography-based purification processes

Bernau

Knödler

Emonts

et al. 2022

Front. Bioeng. Biotechnol.

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Chromatography is the workhorse of biopharmaceutical downstream processing because it can selectively enrich a target product while removing impurities from complex feed streams. This is achieved by exploiting differences in molecular properties, such as size, charge and hydrophobicity (alone or in different combinations). Accordingly, many parameters must be tested during process development in order to maximize product purity and recovery, including resin and ligand types, conductivity, pH, gradient profiles, and the sequence of separation operations. The number of possible experimental conditions quickly becomes unmanageable. Although the range of suitable conditions can be narrowed based on experience, the time and cost of the work remain high even when using high-throughput laboratory automation. In contrast, chromatography modeling using inexpensive, parallelized computer hardware can provide expert knowledge, predicting conditions that achieve high purity and efficient recovery. The prediction of suitable conditions in silico reduces the number of empirical tests required and provides in-depth process understanding, which is recommended by regulatory authorities. In this article, we discuss the benefits and specific challenges of chromatography modeling. We describe the experimental characterization of chromatography devices and settings prior to modeling, such as the determination of column porosity. We also consider the challenges that must be overcome when models are set up and calibrated, including the cross-validation and verification of data-driven and hybrid (combined data-driven and mechanistic) models. This review will therefore support researchers intending to establish a chromatography modeling workflow in their laboratory.

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“…The large pore size of POROS 50 HQ (1726 nm) enhances mass transfer rates due to intraparticle convection and diffusion without compromising the binding capacity ( Yao and Lenhoff, 2006 ). Matlschweiger et al demonstrated that the binding capacity of thyroglobulin (680 kDa) is five times larger when using POROS 50 HQ rather than Q Sepharose FF (60 nm pore size), evidencing the promising balance between transport kinetics and binding capacity of perfusion resins ( Matlschweiger et al, 2019 ).…”

Section: Pegylationmentioning

confidence: 99%

Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

Sánchez‐Trasviña

Flores-Gatica

Enriquez-Ochoa

et al. 2021

Front. Bioeng. Biotechnol.

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Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic’s pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.

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Hindered diffusion of proteins in mixture adsorption on porous anion exchangers and impact on flow-through purification of large proteins

Cited by 15 publications

References 40 publications

Chromatography process development aided by a dye‐based assay

Chromatography process development aided by a dye‐based assay

The use of predictive models to develop chromatography-based purification processes

Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

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