Altering the association properties of insulin by amino acid replacement

Abstract

The importance of ProB28 and LysB29 on the self-association of insulin was established by systematically truncating the C terminus of the B chain. The relationship between structure and association was further explored by making numerous amino acid replacements at B28 and B29. Association was studied by circular dichroism, size-exclusion chromatography and ultracentrifugation. Our results show that the location of a prolyl residue at B28 is critical for high-affinity self-association. Removal of ProB28 in a series of C-terminal truncated insulins, or amino acid replacement of ProB28, greatly reduced association. The largest disruption to association was achieved by replacing LysB29 with Pro and varying the amino acid at B28. Several of the analogs were predominantly monomers in solutions up to 3 mg/ml. These amino acid substitutions decreased association by primarily disrupting the formation of dimers. Such amino acid substitutions also substantially reduced the Zn-induced insulin hexamer formation. The formation of monomeric insulins through amino acid replacements was accompanied by conformational changes that may be the cause for decreased association. It is demonstrated that self-association of insulin can be drastically altered by substitution of one or two key amino acids.

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

“…The ␤-strand detachment-driven monomeric behavior in the solution and formation of hexameric crystals of [NMePheB25]-insulin are somehow reflected in the crystal structure (and solution properties) of the [LysB28,ProB29]-insulin, an analogue also with a very substantially diminished (ϳ300ϫ) dimerization capacity (48). Its monomeric behavior did not also prohibit its crystallization as the T 3 R 3 f hexamer in the presence of zinc and phenol (16).…”

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

“…8 The self-associated species can pose manufacturing and delivery challenges (e.g., high viscosity, clogging of the lines and filters 3,9 ) or cause pain to patients upon administration. 10,11 In certain cases, the self-associated species can affect bioactivity and pharmacokinetic properties of biologic drug candidates 12,13 or, depending on the structural and environmental conditions, transform to irreversible aggregates via further covalent linkages. 14 A number of studies have illustrated the diversity of structural mechanisms by which self-association can occur.…”

Ion‐specific modulation of protein interactions: Anion‐induced, reversible oligomerization of a fusion protein

“…The hormone insulin can be stabilized as a reversible hexamer when formulated with zinc. 22,23 However, the hexameric form causes a delayed onset of glucose reduction following subcutaneous injection, which consequently has led to the development of genetically engineered, monomeric, insulin analogues such as HumalogV R and NovologV R . 23 The reversible self-association of some monoclonal antibodies at high concentrations has been implicated in dramatically increasing solution viscosity, leaving significant challenges to be resolved during manufacturing and delivery of the drug product.…”