Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective

Biologic products encounter various types of interfacial stress during development, manufacturing, and clinical administration. When proteins come in contact with vapor–liquid, solid–liquid, and liquid–liquid surfaces, these interfaces can significantly impact the protein drug product quality attributes, including formation of visible particles, subvisible particles, or soluble aggregates, or changes in target protein concentration due to adsorption of the molecule to various interfaces. Protein aggregation at interfaces is often accompanied by changes in conformation, as proteins modify their higher order structure in response to interfacial stresses such as hydrophobicity, charge, and mechanical stress. Formation of aggregates may elicit immunogenicity concerns; therefore, it is important to minimize opportunities for aggregation by performing a systematic evaluation of interfacial stress throughout the product development cycle and to develop appropriate mitigation strategies. The purpose of this white paper is to provide an understanding of protein interfacial stability, explore methods to understand interfacial behavior of proteins, then describe current industry approaches to address interfacial stability concerns. Specifically, we will discuss interfacial stresses to which proteins are exposed from drug substance manufacture through clinical administration, as well as the analytical techniques used to evaluate the resulting impact on the stability of the protein. A high-level mechanistic understanding of the relationship between interfacial stress and aggregation will be introduced, as well as some novel techniques for measuring and better understanding the interfacial behavior of proteins. Finally, some best practices in the evaluation and minimization of interfacial stress will be recommended

Physical characterixation and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size enriched into different size bins by low speed centrifugation or a combination of gravitational sedimentation and Fluorescence-Activated Cell Sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5–10 μm in size displayed elevated cytokine release profiles compared to other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared to controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by Microflow imaging, Transmission Electron Microscopy, and Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in-vitro PBMC studies to rank order the immunogenic potential of various types of mAb particles is discussed

Molecular Biophysics for the Life Sciences

XII, 397 p. 132 illus., 70 illus. in color.onlin

Thermal stability comparison of purified empty and peptide-filled forms of a class I MHC molecule

A secreted form of a class I major histocompatibility complex (MHC) molecule was denatured and renatured in vitro in the absence of peptide. The resulting empty class I heterodimer was immunologically reactive and structurally similar to a heterodimer renatured in the presence of an appropriate restricted peptide. Thermal stability profiles indicated that the two forms of heterodimer differed in their resistance to denaturation by heat but that a significant portion of the empty class I heterodimers had a native conformation at physiological temperatures. Free energies calculated from these data gave a direct measure of the stabilization of the class I MHC molecule that resulted from peptide binding

Reconstitution of cyanobacterial photophosphorylation by a latent Ca+2-ATPase

Photosynthetic membranes derived from sonic extracts of the cyanobacterium contain a latent Ca+2-ATPase which is activated by exposure to trypsin. When sonic membranes are washed with ethylenediaminetetraacetic acid, the ATPase is removed from these membranes with an accompanying loss of photophosphorylation activity. The latent ATPase activity solubilized by washing has been partially purified, and addition of the enzyme to depleted membranes restores photophosphorylation activity to levels approaching 50% of the rates observed in unwashed membranes. These data indicate that this ATPase is the coupling factor responsible for photosynthetic energy transduction in .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23478/1/0000431.pd

Effects of Peptide Length and Composition on Binding to an Empty Class I MHC Heterodimer

Class I major histocompatibility complex (MHC) proteins present peptide antigens to T cells during the immune response against viruses. Peptides are loaded into newly synthesized class I heterodimers in the endoplasmic reticulum such that most or all cell surface class I molecules contain peptides derived from endogenous or foreign proteins. We previously reported the assembly of empty heterodimers of the murine class I MHC molecule H-2K^d, from denatured heavy and light chains from which endogenous peptides had been removed [Fahnestock et al. (1992) Science 258,1658-16621. Here we measure thermal stability profiles of empty versus peptide-filled molecules and compare the effects of human versus murine light chains on the overall stability of the K^d heterodimer. The majority of empty heterodimers are stable at 37 °C regardless of the species of light chain, indicating that our previous report of the unexpectedly high thermal stability was an intrinsic property of the K^d molecule and not due to use of a murine/human chimeric protein. Binding constants arederived for a series of peptides interacting with empty K^d heterodimers. The dissociation constants of four known K^d-restricted peptides range from 2.3 X 10^(-7) to 3.4 X 10^(-8) M. Using a series of 24 analog peptides, the effects of length and peptide composition on binding affinity of one K^d-restricted peptide are explored, and the results are interpreted with reference to the known three dimensional structures of class I MHC protein/peptide complexes

Production and Characterization of an Analog of Acidic Fibroblast Growth Factor with Enhanced Stability and Biological Activity

We have used recombinant DNA methods to produce two forms of bovine acidic fibroblast growth factor (aFGF), one with alanine substituted for the cysteine at position 47 and the other with the Ala47 change plus the substitution of glycine for the naturally occurring histidine at position 93. Both forms were expressed at high levels in Eschtrichia coli and purified to near homogeneity by solubilization of the inclusion bodies containing the aFGF, ion exchange chromatography, refolding of the protein and hydrophobic interaction chromatography. Circular dichroic and infrared spectra suggested that the proteins are similar in secondary and tertsiary structures and contain little or no α-helical conformations. Hydrophobic interaction chromatography showed that aFGF C47A/H93G is slightly more hydrophobic than the aFGF C47A form, suggesting that residue 93 is exposed to the solvent. Half-maximal activity in an in vitro bioassay system was reached at a 10- to 20-fold lower dose for the aFGF C47A/H93G form than for the aFGF C47A form, suggesting that alteration of this residue has an effect on the region responsible for receptor binding. Addition of 50 μg/ml heparin enhanced the in vitro activity of the aFGFs, reducing the half-maximal dose to approximately 100 pg/ml for both forms, comparable to that observed previously for basic FGF with or without heparin in this assay system