Structural meta-analysis of regular human insulin in pharmaceutical formulations

Abstract

We have studied regular acting, wild-type human insulin at potency of 100 U/mL from four different pharmaceutical products directly from their final finished formulation by the combined use of mass spectrometry (MS), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), and single-crystal protein crystallography (PX). All products showed similar oligomeric assembly in solution as judged by DLS and SAXS measurements. The NMR spectra were compatible with well folded proteins, showing close conformational identity for the human insulin in the four products. Crystallographic assays conducted with the final formulated products resulted in all insulin crystals belonging to the R3 space group with two a dimer in the asymmetric unit, both with the B-chain in the T configuration. Meta-analysis of the 24 crystal structures solved from the four distinct insulin products revealed close similarity between them regardless of variables such as biological origin, product batch, country origin of the product, and analytical approach, revealing a low conformational variability for the converging insulin structural ensemble. We propose the use of MS, SAXS, NMR fingerprint, and PX as a precise chemical and structural proof of folding identity of regular insulin in the final, formulated product.

Introduction

The onset of diabetes mellitus (DM) and loss of control of metabolism affects a large part of the world population [1], [2]. Large effort has been made for the development of new therapeutics for the control of DM as well as in the search for its cure [3], [4], [5], [5], [6], [7], [8], [9], [10], [11].

However, therapeutic reposition of pancreatic hormones – such as insulin and amylin and their analogs – has not been replaced and still remains an important therapy [12], [13], [14] which motivates the worldwide development and revision of process for the production of insulin, new insulin products, analogs, and delivery systems [3], [15], [16], [17], [18], [19], [20] in addition to the large portfolio of insulin products and analogs currently available from different brands.

Along with the inherent difficulties of developing and producing a biological products for therapeutic use, characterization of the final product is also challenging and demands a continuous revision process of analytical methods and quality requirements applied for pharmaceutical ingredients, finished and in-process products.

Protein therapeutics are a class of products which have a complex three dimensional structure in solution whose integrity determines the biological activity, clinical efficacy, and safety. Thus, it is highly desirable that products from this class meet well-defined requirements for structural integrity. Proteolytic footprint has long been the method of choice, generating a peptide mapping which indirectly provides evidences for solution structural arrangement [21]. Previous work have shown that slight changes in excipient used in insulin formulation can result in redistribution of oligomeric forms [22], [23]. However, besides its high analytical precision and sensitivity, this approach is not sufficient to provide the exact three-dimensional structure of these biological entities.

Still remains the question whether the assessment of structural information could be satisfactorily performed in the final finished biological product, providing chemical information about the molecular integrity and the detailed structural information about the exact three-dimensional structure of these biological entities in its final formulations. To address this issue, we have conducted an extensive structural and spectroscopic characterization of regular-acting, wild-type human insulin formulations at same potency of 100 U/mL from four distinct brands.

Insulin is a protein hormone used worldwide in the treatment of diabetes. Its active form is comprised of polypeptide chains A and B, linked by three disulfide bonds. Insulin crystals have been investigated for decades [24], [25], [26], and the insulin high resolution structure has long been known from varying crystallographic techniques such as single crystals [27] and microcrystalline powder [28] diffraction. NMR has also been used in the structural characterization of insulin in its varying oligomeric and conformational states, such as monomers [29], T₆, ${\text{T}}_3{\text{R}}_3^{\text{f}}$ [30] and R₆ hexamer [31] and in comparative studies with diverse mutants [32], [33], [34], [35] and also in diverse solution composition [32], [34], [36]. The choice of the products used in the present study was based on the fact that they are pharmaceutically available for therapeutic use and are produced by four dissimilar methods such as the heterologous recombinant production (in Escherichia coli, Saccharomyces cerevisae or Pichia pastoris) and by the semi-synthetic approach (by the modification of the aminoacid Thr^B30 in porcine insulin), also varying in the excipient composition.