Protein aggregation: Pathways, induction factors and analysis

Mahler, Hanns‐Christian; Frieß, Wolfgang; Grauschopf, Ulla; Kiese, Sylvia

doi:10.1002/jps.21566

Cited by 753 publications

(667 citation statements)

References 196 publications

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“…A concentration-size chart summarizing the most commonly used methods for sizing aggregates is presented in Figure 8.1 [197,198]. The solid and dashed lines represent boundaries for constant mass of protein particles assuming a spherical density of 1 gm/cc.…”

Section: Introductionmentioning

confidence: 99%

“…Because protein aggregates span a wide range of sizes, and quantification of aggregates depends on the technique employed, there is no one analytical tool that can measure all classes of aggregates [3,199,200], and multiple methods are typically used for cross validation [5,197,199]. The objective of this paper is to evaluate electrospraydifferential mobility analysis (ES-DMA) as another method to add to the suite of protein aggregate characterization tools.…”

Section: Introductionmentioning

confidence: 99%

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Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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The growth of the multibillion dollar bionanoparticle industry has spurred the development of new physical characterization methods. One such method, electrospraydifferential mobility analysis (ES-DMA) constitutes an electrospray for aerosolization of bionanoparticles (such as viruses, gold-nanoparticles, proteins, nanoparticle-protein complexes) and an ion mobility method that operates at atmospheric conditions, and separates bionanoparticles spatially. This dissertation identifies some relevant "problem" areas for ES-DMA by reviewing selected applications.Some such problems are: proteins while passing through ES capillaries are found to interact with it and thus produce time dependent size distributions. Further, it is thought that adsorbed proteins may subsequently desorb and influence size distributions with the ES-DMA which may concomitantly affect quantification of aggregates. These artifacts are studied systematically and it is demonstrated that ES-DMA can quantify adsorption-desorption of complex protein mixtures at high shear rates. Further, it is shown that desorbing proteins do not have a significant effect on size distributions. Another artifact of the ES takes place during the aersolization process. Two units (called monomers) of a bionanoparticle may get encapsulated in the same ES droplet and upon drying of the droplet create artificial dimers thus affecting quantification with ES-DMA. Assuming Poisson distribution, this thesis provides a systematic approach that can be undertaken to eliminate this artifact. A third artifact arises from the low sensitivity of the DMA to size increase. When a ligand (for e.g. protein) adsorbs to a bionanoparticle it creates an increase in the size of the later, which can be used to quantify the amount of ligand adsorbed per bionanoparticle. As ligands can change conformations upon adsorption, using ES-DMA for such applications may be flawed. This issue has been identified and a solution has been provided by integrating a mass analyzer after the ES-DMA.After correcting for these artifacts, this dissertation delves into characterization of different types of bionanoparticles and demonstrates that ES-DMA has several advantages over other traditional techniques such as transmission electron microscopy, size exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and plaque assay and thus has immense potential to become a process analytical technique in biomanufacturing environments. ELECTROSPRAY-DIFFERENTIAL MOBILITY ANALYSIS OF BIONANOPARTICLES

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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“…High molecular weight (HMW) species formation has been described for antibodies under a variety of circumstances, including freeze/thaw, 3,6,9 agitation, 9 and thermal stress. 5,6,9 Additionally, under physiological conditions, antibody light chains and associated variable light chains have been shown to possess the ability to form amyloid, and have been implicated in free light chain amyloidosis, a neurodegenerative disorder.…”

Section: Introductionmentioning

confidence: 99%

“…5,6,9 Additionally, under physiological conditions, antibody light chains and associated variable light chains have been shown to possess the ability to form amyloid, and have been implicated in free light chain amyloidosis, a neurodegenerative disorder. 10 Antibody aggregation is influenced by formulation conditions, such as pH, buffer, and excipient.…”

Section: Introductionmentioning

confidence: 99%

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

et al. 2010

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Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular b-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed 2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.

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“…Iron plays a major role in inhibiting the formation of aggregate forms, as shown in long-term drug substance stability studies, where the abundance of high-molecularweight species was significantly lower at low iron concentrations (0-4 ppm) in the presence of a chelator 174 . Surfactant containing culture media were reported to stabilize the expressed protein and thus reduce aggregation 175,176 . Specifically, the addition of 0.01% (v/v) Polysorbate 80 into chemically defined concentrated feed media reduced overall aggregation levels by 2.6-2.7% of two different CHO cell lines.…”

Section: Aggregatesmentioning

confidence: 99%

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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Protein aggregation: Pathways, induction factors and analysis

Cited by 753 publications

References 196 publications

Electrospray–differential mobility analysis of bionanoparticles

Electrospray–differential mobility analysis of bionanoparticles

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Tailoring recombinant protein quality by rational media design

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