Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers.

Knauf, Michael J.; Bell, D. P.; Hirtzer, P.; Luo, Zhen-Ping; Young, John; Katre, Nandini V.

doi:10.1016/s0021-9258(18)68146-3

Cited by 244 publications

(72 citation statements)

References 20 publications

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“…On the one hand, large multimeric complexes are known to be retained owing to the size exclusion, which limits the glomerular filtration removal process in the kidney. It has been reported that proteins smaller than 70 kDa are effectively removed from the circulation by the kidneys [44]. This would imply that glomerular filtration affects dimers and tetramers equally and cannot explain the marked difference in the removal rates of the two forms because both dimeric and tetrameric AChE exceed 70 kDa (the molecular masses of dimeric and tetrameric rHuAChE are approx.…”

Section: Relationship Between N-glycan Sialylation and Oligomerization In Establishing The Residence Time Of Huache In The Circulationmentioning

confidence: 99%

Effect of human acetylcholinesterase subunit assembly on its circulatory residence

et al. 2001

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Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by stably transfected cells, is composed of a mixed population of monomers, dimers and tetramers and manifests a time-dependent circulatory enrichment of the higher-order oligomeric forms. To investigate this phenomenon further, homogeneous preparations of rHuAChE differing in their oligomerization statuses were generated: (1) monomers, represented by the oligomerization-impaired C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT-rHuAChE generated in vitro by complexation with a synthetic ColQ-derived proline-rich attachment domain ('PRAD') peptide. Three different series of each of these three oligoform preparations were produced: (1) partly sialylated, derived from HEK-293 cells; (2) fully sialylated, derived from engineered HEK-293 cells expressing high levels of sialyltransferase; and (3) desialylated, after treatment with sialidase to remove sialic acid termini quantitatively. The oligosaccharides associated with each of the various preparations were extensively analysed by matrix-assisted laser desorption ionization-time-of-flight MS. With the enzyme preparations comprising the fully sialylated series, a clear linear relationship between oligomerization and circulatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740 min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5 min). These observations suggest that multiple removal systems contribute to the elimination of AChE from the circulation. Here we also demonstrate that by the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.

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Section: Relationship Between N-glycan Sialylation and Oligomerization In Establishing The Residence Time Of Huache In The Circulationmentioning

confidence: 99%

Effect of human acetylcholinesterase subunit assembly on its circulatory residence

et al. 2001

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“…The Fc-fusion protein technology is used as an important backbone for drug development (Pechtner et al, 2017). Fc-fusions have a prolonged therapeutic activity due to an increased plasma half-life (Yu et al, 2013;Wang et al, 2014;Strohl, 2015) owing to the (i) Fc interaction with the salvage neonatal Fc-receptor [FcRn; (Roopenian and Akilesh, 2007)], (ii) slower renal clearance due to higher molecular weight (Knauf et al, 1988;Kontermann, 2011), and (iii) reduced immunogenicity (Rath et al, 2015). Moreover, the Fc domain folds independently and can improve the solubility and stability of the partner molecule (Zhang et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

et al. 2021

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The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

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“…The molecular weight of each of the individual blocks is below 3 kDa, well below the renal ltration threshold of 70 kDa. [99][100][101] The carboxylic acid-terminated aniline pentamers (AP) were obtained in analytically pure form using minor modications to the methodology described in the literature (Scheme S1 †). 102 Polyesters 1-4 were synthesized by adaptation of the literature.…”

Section: Resultsmentioning

confidence: 99%

Biodegradable electroactive polymers for electrochemically-triggered drug delivery

et al. 2014

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Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers.

Cited by 244 publications

References 20 publications

Effect of human acetylcholinesterase subunit assembly on its circulatory residence

Effect of human acetylcholinesterase subunit assembly on its circulatory residence

The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

Biodegradable electroactive polymers for electrochemically-triggered drug delivery

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