Altering the Self-Association and Stability of Insulin by Amino Acid Replacement

Brems, David N.; Brown, P. S.; Bryant, Christopher S.; Chance, Ronald E.; DiMarchi, Richard D.; Green, L. Kenney; Howey, Daniel C.; Long, H B; Miller, Alita A.; Millican, Rohn; Pekar, Allen H.; Shields, James E.; Frank, Bruce H.

doi:10.1021/bk-1993-0526.ch019

Cited by 6 publications

(4 citation statements)

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“…Human insulin was the first animal protein to be made by bacteria following a sequence identical to the synthesis of the human pancreatic peptide. Different kinds of insulin analogues were later designed to improve its pharmacokinetic properties (25). The production of Eli Lilly's human insulin requires 31 principal processing steps, 27 of which are associated with product recovery and purification (11).…”

Section: Introductionmentioning

confidence: 99%

Modeling of Preparative Reversed‐Phase HPLC of Insulin

Liu

Kaczmarski

Cavazzini

et al. 2002

Biotechnology Progress

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The adsorption isotherms of three recombinant proteins, human insulin, porcine insulin, and Lispro, were measured by frontal analysis on a YMC-ODS C18 column with an aqueous solution at 31% acetonitrile (0.1% TFA) as the mobile phase. The retention behavior of insulin, its related molecular structure, its conformation, and its aggregation in this phase system are discussed. The experimental isotherm data were fitted to the Langmuir, the Langmuir-Freundlich, and the Toth models. The results allow for a quantitative comparison of the saturation capacities, the equilibrium constants, and the exponents that represent the heterogeneity of the stationary phase obtained for the different insulin variants studied. The Toth model provided the best fit of the experimental data. The overloaded band profiles were calculated using the lumped pore diffusion and the equilibrium-dispersive model of chromatography. An excellent agreement between calculated and experimental profiles was demonstrated.

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Section: Introductionmentioning

confidence: 99%

Modeling of Preparative Reversed‐Phase HPLC of Insulin

Liu

Kaczmarski

Cavazzini

et al. 2002

Biotechnology Progress

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“…Since the approach is based on examining only retention times, it is possible to screen a large number of additives, or even mixtures of additives, over a range of solution conditions within a period of days. SIC can also be used to test mutants specifically engineered to reduce aggregation, such as those developed for insulin (Brange et al, 1989;Brems et al, 1992Brems et al, , 1993. The sensitivity of the SIC technique and the ease of operation and automation make it very attractive as a formulation tool as well as a convenient means to study protein self-association in solution.…”

Section: Discussionmentioning

confidence: 99%

“…A series of single-and double-site Pro"2XLys"'4+XY mutants ot' human insulin were found to disrupt self-association (Brems et al. 1997(Brems et al. , 1993.…”

Section: Protein Aggregatesmentioning

confidence: 99%

“…We have used crystal contact data from the Protein Data Bank to suggest candidate sites. Along similar lines, Brems et al (1992Brems et al ( , 1993 have used information from several crystal forms of insulin to engineer mutants with altered association properties. Successful blocking techniques include site-directed mutagenesis, chemical modification of the site (Przybycien and Bai-ley, 1989), or addition of a competitive binding species (Brems, 1988).…”

Section: Self-interaction Chromatographymentioning

confidence: 99%

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Self-interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments

Patro

Przybycien

2000

Biotechnol. Bioeng.

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We describe a new protein characterization technique called self‐interaction chromatography (SIC), which exploits the specificity of protein–protein interactions that is common to protein aggregates and enables the rapid screening of protein formulation additives as physical stabilizers against aggregation. This technique also enables the identification of specific interaction sites and the determination of their relative importance for self‐association. Mannitol, glycine, and dextran 40 were tested for their stabilizing effect toward the model protein lysozyme. Dextran 40 exhibited a poor stabilizing effect. While mannitol stabilized both the native and acid‐denatured forms of lysozyme, glycine stabilized the native form with respect to the denatured species. These results are in good agreement with findings in the formulation literature. The SIC shows tremendous potential as a rapid formulation development tool. We also screened two putative interaction sites for involvement in the self‐association of lysozyme and estimated the associated binding energies using a binding isotherm model that we developed. The sites screened consisted of residues 41–48 and 125–128 and were selected based on their apparent importance in forming crystal contacts in several different crystal forms of lysozyme. Of the two sites, only residues 125–128 were found to influence self‐association under the conditions we employed. Because the success of this technique depends on the exploitation of self‐interactions between native species, several important applications are also suggested such as separating native from misfolded or variant species and probing site utilization in aggregation versus crystallization phenomena. © 1996 John Wiley & Sons, Inc.

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An Integrated Theory of Adsorption and Partition Mechanism and Each Contribution to Solute Retention in Reversed Phase Liquid Chromatography

Geng

2003

Chin. J. Chem.

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with the combination of the the stoichiometric displacement model for retention (SDM-R) in reversed phase liquid chromatography (RPLC) and the stoichiometric disptacement model for adsorption (SDM-A) in physical chemistry, the total number of mdes of the re-solvated methanol of stationary phase side, N , and that of solute side in the mobile phase, q , c o -* toonemdeofthe desorbiing solute, were separately detennined and referred as the characterization parameters of the contriions of the adsorption m e c h a n i s n a n d p a r t i t i o n~ ' to the solute retention. respectively. A chromatographic system of insulin, using mobile phase conristing of the pseud~homologue of alcohols (methanol, ethanol and Zpropanol)-water and tritluoroacetic acid was employed. The maximum number of the methand layers on the stationary phase d a c e was found to be 10.6, only 3 of which b e i i valid in usual RPLC, traditionally referred as a vdume proces~ in partition mechanisn. However, it still follows the SDM-R. Both of q and N of insulin were found not to be zero, indicating that the retention adsorption mecharusn ' .Whenmethawlisusedastheorganicmod-Xer, the ratio of q/nr was 1.l3, indicating the contriition to insulin retention due to partition mecharusn . being a bit greater than that due to adsorption mecharusn * . A linear relationship between q , or nr and the carbon number of the peudc+homologue in the mobile phase was also found. As a methoddogy for invgtigating the retention m retention and behaviors of biopolymers, a homologue of organic solvents as the organic modifier in mobile phase lw also been explored. mechanisnofinsulin is a mixed mode of partition rrxhumn * a n d Keywords nism, mixed mode, stoichiometric displacement, insulin reversed phase liquid chromatography, retention mecha-*

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Altering the Self-Association and Stability of Insulin by Amino Acid Replacement

Cited by 6 publications

References 0 publications

Modeling of Preparative Reversed‐Phase HPLC of Insulin

Modeling of Preparative Reversed‐Phase HPLC of Insulin

Self-interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments

An Integrated Theory of Adsorption and Partition Mechanism and Each Contribution to Solute Retention in Reversed Phase Liquid Chromatography

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