Current research approaches in downstream processing of pharmaceutically relevant proteins

Schwaminger, Sebastian P.; Zimmermann, Ines; Berensmeier, Sonja

doi:10.1016/j.copbio.2022.102768

Cited by 12 publications

(8 citation statements)

References 81 publications

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“…It was implemented for the purification of IFN-

2b through ion exchange and size exclusion chromatography [ 44 , 45 ], immobilized metal-ion affinity chromatography, and reverse-phase HPLC [ 46 ]. To overcome some of the packed-bed chromatography limitations, alternative non-chromatographic downstream processes were investigated, namely precipitation, crystallization [ 47 ], membrane-based processes [ 48 ], magnetic fishing [ 49 ], and aqueous biphasic systems [ 50 , 51 ]. The optimization of an efficient and cost-effective purification process is a crucial part of IFN production [ 52 ].…”

Section: Introductionmentioning

confidence: 99%

“…Aqueous biphasic systems (ABSs), also known as aqueous two-phase systems (ATPSs), are liquid–liquid systems prepared by mixing at least two different water-soluble compounds in an aqueous medium, such as polymers, salts, alcohols, carbohydrates, or ionic liquids, which form two liquid phases coexisting in equilibrium above certain concentrations, and can be used in liquid–liquid extraction processes [ 53 , 54 ]. Many ABS have exhibited numerous advantages in biotechnological applications [ 51 , 55 ]. Some of the advantages displayed by ABS include their biocompatibility, mostly due to the presence of high water content in both phases, which can provide a biocompatible and non-denaturing environment for cells, proteins, and other biomolecules.…”

Section: Introductionmentioning

confidence: 99%

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Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems

Carvalho,

Pereiro,

Araújo

2024

IJMS

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Interferon alpha-2b (IFN-α2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN-α2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C2C1Im][C4F9SO3] and [N1112(OH)][C4F9SO3]) vs. mere fluoro-containing IL ([C4C1Im][CF3SO3]), in combination with sucrose or [N1112(OH)][H2PO4] (well-known globular protein stabilizers), or high-charge-density salt K3PO4 were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN-α2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN-α2b high in the enriched IFN-α2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN-α2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN-α2b and BSA with the ABS phase-forming components were assessed by MST, showing the strong interaction between FILs aggregates and both proteins. In view of their biocompatibility, customizable properties, and selectivity, FIL-based ABSs are suggested as an improved purification step that could facilitate the development of biologics.

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“…It was implemented for the purification of IFN-

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems

Carvalho,

Pereiro,

Araújo

2024

IJMS

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“…Elution is achieved by decreasing the salt concentration of the elution buffer [17]. HIC is mostly used as a polishing step to remove protein aggregates [13,[18][19][20][21].…”

Section: Introductionmentioning

confidence: 99%

Aggregate Removal by Responsive Electrospun Membrane based Hydrophobic Interaction Chromatography

Hao,

Chen,

Chiao

et al. 2024

Chemie Ingenieur Technik

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Poly(N‐vinylcaprolactam) (PVCL), a salt responsive ligand, was grafted from electrospun cellulose nanofiber membranes by atom transfer radical polymerization (ATRP). The membranes were used for hydrophobic interaction chromatography. The modified PVCL‐cellulose membranes were used to separate BSA dimers from monomers as well as monoclonal antibody (mAb) aggregates. Fast protein liquid chromatography (FPLC) was used for protein separation and fraction collection. High performance liquid chromatography with a size exclusion column (HPLC‐SEC) analysis confirmed the separation of monomers and aggregates from both BSA and the mAb. The results suggest that use of a responsive ligand that changes its conformation with ionic strength could improve the performance of hydrophobic interaction chromatography enabling the separation of compounds based on slight differences in hydrophobicity.

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“…Monoclonal antibodies (mAbs) are essential biopharmaceuticals for treating numerous diseases. The market demand and annual mAb approvals increase continuously. − Facing the challenge of efficiently producing large amounts of mAbs, multiple upstream processing (USP) advances have resulted in enhanced expression productivity over the last decades. , However, the subsequent downstream processing (DSP) cannot keep pace with the progress in USP, essentially caused by the predominating packed-bed Protein A chromatography capture step. , Although Protein A ligands are suitable as they allow selective binding to various mAb types, , diffusional mass transfer constrains the throughput and the productivity in the packed-bed chromatography operation. , Furthermore, there are capacity and scalability limitations. , Despite excellent yields and purities achieved with Protein A chromatography, efficient alternative capture operations are thus needed and of particular research focus. ,− …”

Section: Introductionmentioning

confidence: 99%

Direct Affinity Ligand Immobilization onto Bare Iron Oxide Nanoparticles Enables Efficient Magnetic Separation of Antibodies

Zimmermann,

Kaveh-Baghbaderani,

Eilts

et al. 2024

ACS Appl. Bio Mater.

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Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.

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Current research approaches in downstream processing of pharmaceutically relevant proteins

Cited by 12 publications

References 81 publications

Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems

Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems

Aggregate Removal by Responsive Electrospun Membrane based Hydrophobic Interaction Chromatography

Direct Affinity Ligand Immobilization onto Bare Iron Oxide Nanoparticles Enables Efficient Magnetic Separation of Antibodies

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