Protein aggregation and bioprocessing

Cromwell, Mary; Hilario, Eric; Jacobson, Fred

doi:10.1208/aapsj080366

Cited by 676 publications

(491 citation statements)

References 22 publications

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“…While generally deleterious to protein function, aggregation is particularly problematic to the biopharmaceutical industry as aggregates have been linked to immunogenic reactions in patients (Büttel et al, 2011) and to shortened therapeutic half‐life (Dobson et al, 2016). In addition, the presence of aggregates during development can lead to a decreased yield and an increase in time to market, due to the need to optimize manufacturing conditions/formulation (Cromwell, Hilario, & Jacobson, 2006; Zurdo et al, 2015). mAb‐based biologics are susceptible to aggregation throughout their lifetime, from over‐expression in the cell (Kramarczyk, Kelley, & Coffman, 2008) and downstream processing (Skamris et al, 2016; Yu et al, 2016) to the final fill‐finish step at high concentration (Cromwell et al, 2006; Rathore & Rajan, 2008).…”

Section: Introductionmentioning

confidence: 99%

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Willis

Kumar

Dobson

et al. 2018

Biotech & Bioengineering

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Monoclonal antibodies (mAbs) currently dominate the biopharmaceutical sector due to their potency and efficacy against a range of disease targets. These proteinaceous therapeutics are, however, susceptible to unfolding, mis‐folding, and aggregation by environmental perturbations. Aggregation thus poses an enormous challenge to biopharmaceutical development, production, formulation, and storage. Hydrodynamic forces have also been linked to aggregation, but the ability of different flow fields (e.g., shear and extensional flow) to trigger aggregation has remained unclear. To address this question, we previously developed a device that allows the degree of extensional flow to be controlled. Using this device we demonstrated that mAbs are particularly sensitive to the force exerted as a result of this flow‐field. Here, to investigate the utility of this device to bio‐process/biopharmaceutical development, we quantify the effects of the flow field and protein concentration on the aggregation of three mAbs. We show that the response surface of mAbs is distinct from that of bovine serum albumin (BSA) and also that mAbs of similar sequence display diverse sensitivity to hydrodynamic flow. Finally, we show that flow‐induced aggregation of each mAb is ameliorated by different buffers, opening up the possibility of using the device as a formulation tool. Perturbation of the native state by extensional flow may thus allow identification of aggregation‐resistant mAb candidates, their bio‐process parameters and formulation to be optimized earlier in the drug‐discovery pipeline using sub‐milligram quantities of material.

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Section: Introductionmentioning

confidence: 99%

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Willis

Kumar

Dobson

et al. 2018

Biotech & Bioengineering

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“…Both selection and engineering of the host cell line, as well as the culture conditions including media composition, shape the protein functionality 7,45 and effect undesired by-products, such as aggregates 46 and low-molecular-weight species (LMW) 47 . The choice of the host cell is decisive, since each of the frequently used cell lines (CHO, NS0 and SP2/0) has a cell-line specific glycosylation fingerprint.…”

Section: Cell Culture Process Optimizationmentioning

confidence: 99%

“…Many authors have published extensive reviews, depicting the current state of the art and strategies, which focus on the cell line 1,7,16,45 and the cell culture parameters 18,51 . Furthermore, through the concentration adjustment of selected media components, and in some cases by supplementing the medium with specific co-factors, it is possible to adjust the glycosylation profile 52 , the charge variants 53 , the aggregation level 46,54 , and the abundance of LMW species 55 . Figure 1.1 outlines the three main strategies allowing to affect cell culture process performance and to tailor the quality attributes of therapeutic molecules.…”

Section: Cell Culture Process Optimizationmentioning

confidence: 99%

“…It results in intracellular aggregation owing to either the interactions of unfolded protein molecules or to inefficient recognition of the nascent peptide chain by molecular chaperones responsible for proper folding. Also, once the protein is secreted into the cell culture media, it may aggregate as a result of adverse environmental conditions 46 .…”

Section: Aggregatesmentioning

confidence: 99%

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Tailoring recombinant protein quality by rational media design

Brühlmann

Jordan

Hemberger³

et al. 2015

Biotechnology Progress

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Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C‐ & N‐terminal modifications), aggregates, low‐molecular‐weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high‐throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015

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“…1 Various stress conditions, such as pH, light, mechanical, and thermal (heat or freeze/thaw), can contribute to aggregate formation. 2,3 These aggregates can have adverse immune responses and may result in reduced efficacy. 4,5 …”

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confidence: 99%

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

Patel

Gospodarek

Larkin³

et al. 2018

mAbs

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For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their Mw using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (Mw increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.

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Protein aggregation and bioprocessing

Cited by 676 publications

References 22 publications

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

Tailoring recombinant protein quality by rational media design

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

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