Heterogeneity of Monoclonal Antibodies

Liu, Hongcheng; Gaza-Bulseco, Georgeen; Faldu, Dinesh; Chumsae, Chris; Sun, Joanne

doi:10.1002/jps.21180

Cited by 424 publications

(358 citation statements)

References 253 publications

(169 reference statements)

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“…1,2 When the protein structure is destabilized, parts of the interior is exposed and this may lead to irreversible formation of aggregates. [3][4][5] The mechanisms leading to aggregates are as diverse as the nature of the aggregates themselves-ranging from oligomers and micelles to amorphous aggregates and fibrils 6-9 -and remain a major challenge in protein science. 10,11 Due to minute transient perturbations of the molecular structure, heating may induce aggregation even well below the melting temperature of the protein.…”

Section: Introductionmentioning

confidence: 99%

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

et al. 2010

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Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lagtime behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size-exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.

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Section: Introductionmentioning

confidence: 99%

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

et al. 2010

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“…We are interested in the asparaginelinked glycosylation, which is by far the most frequent in monoclonal IgG antibodies 60 . In these biotherapeutic molecules, N-glycans are linked to the two conserved asparagine residues (Asn 297) in the CH2 domain of the Fc region 61 . A large number of reports describe various impacts of glycosylation on the quality attributes of biologics including in vivo efficacy [62][63][64] , pharmacokinetics (PK) 11 , antibody-dependent cellular cytotoxicity (ADCC) 63,65 and complementdependent cytotoxicity (CDC) activities 66 , stability and overall structure of the molecule 63 , clearance and half-life in vivo 10 as well as immunogenicity 10,67 .…”

Section: Glycosylationmentioning

confidence: 99%

“…Non-fucosylated therapeutic antibodies exhibit 50 to 1,000-fold higher efficacy than their fucosylated counterparts 65 due, in most cases, to enhanced ADCC activity 68 . Given the abundance of research efforts in this area, it is no surprise that many have concluded that glycosylation is one of the main areas requiring development 69 to improve efficacy 70 and safety 10,70 of next generation therapeutics 61 . Hence, the control of glycosylation of recombinant therapeutic molecules expressed in non-human systems is decisive 71 .…”

Section: Glycosylationmentioning

confidence: 99%

“…C-terminal lysine (Lys) and N-terminal glutamine (Gln) charge variants are common for monoclonal antibodies 41,53,61,158 . Even though the mechanism of C-terminal Lys processing has yet to be fully understood, it was suggested that carboxypeptidases cleave the C-terminal Lys residues in a post-translational modification in cultured cells, yielding three antibody species of either 0, 1, or 2 C-terminal Lys residues 41 and lower isoelectric point.…”

Section: C-and N-terminal Modificationsmentioning

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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“…1,2 Currently, production processes for biologics are designed to remove degradation products, 1,2 and the biologics are formulated to increase stability. 1,3-6 Identification of antibodies that are stable throughout expression, purification, formulation, storage, distribution and in vivo is expected to reduce development and production costs, increase product shelf-life and improve potency in vivo if the chemical liability affects target binding or antibody pharmacokinectics.…”

Section: Introductionmentioning

confidence: 99%

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants

DiCara¹,

Andersen²,

Chan³

et al. 2018

mAbs

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Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation. Detailed assessment by LC-MS analysis confirmed the predicted improvement. This work describes both a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlights the value of broad searches of antibody sequence space.

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Heterogeneity of Monoclonal Antibodies

Cited by 424 publications

References 253 publications

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Tailoring recombinant protein quality by rational media design

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants

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