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Purpose: To investigate antibody stability and formation of modified species under upstream processing conditions. Methods: The stability of 11 purified monoclonal human IgG1 and IgG4 antibodies, including an IgG1-based bispecific CrossMab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Analytical techniques include size exclusion chromatography (SEC), turbidity measurements, cation exchange chromatography (cIEX), dynamic light scattering (DLS) and differential scanning calorimetry (DSC). Results: Sensitivity in downscale stress models varies among antibodies and results in formation of high molecular weight (HMW) aggregates. Stability is increased in cell culture medium and in bioreactors. Media components stabilizing the proteins were identified. Extensive chemical modifications were detected both in stress models as well as during production of antibodies in cell culture fermentations. Conclusions: Protective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality. [ABSTRACT FROM AUTHOR]

Summary: Insulin-like growth factor binding proteins (IGFBPs) control the extracellular distribution, function, and activity of IGFs. Here, we report an X-ray structure of the binary complex of IGF-I and the N-terminal domain of IGFBP-4 (NBP-4, residues 3–82) and a model of the ternary complex of IGF-I, NBP-4, and the C-terminal domain (CBP-4, residues 151–232) derived from diffraction data with weak definition of the C-terminal domain. These structures show how the IGFBPs regulate IGF signaling. Key features of the structures include (1) a disulphide bond ladder that binds to IGF and partially masks the IGF residues responsible for type 1 IGF receptor (IGF-IR) binding, (2) the high-affinity IGF-I interaction site formed by residues 39–82 in a globular fold, and (3) CBP-4 interactions. Although CBP-4 does not bind individually to either IGF-I or NBP-4, in the ternary complex, CBP-4 contacts both and also blocks the IGF-IR binding region of IGF-I. [Copyright &y& Elsevier]

Insulin‐like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti‐apoptotic actions depend primarily on their availability to bind to the cell surface IGF‐I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF‐binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 Å crystal structure of the complex of IGF‐I bound to the N‐terminal IGF‐binding domain of IGFBP‐5 (mini‐IGFBP‐5), a prototype interaction for all N‐terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF‐I and the IGFBP‐5 fragment and a surrounding network of polar interactions. A solvent‐exposed hydrophobic patch is located on the IGF‐I pole opposite to the mini‐IGFBP‐5 binding region and marks the IGF‐I receptor binding site. [ABSTRACT FROM AUTHOR]

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1­104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel -sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs. [ABSTRACT FROM AUTHOR]

Recently we have determined the crystal structure of the insulin-like growth factor-I (IGF-I) in complex with the N-terminal domain of the IGF-binding protein-5 (IGFBP-5). Here we report results of computer screening for potential inhibitors of this interaction using the crystal coordinates. From the compounds suggested by in silico screens, successful binders were identified by NMR spectroscopic methods. NMR was also used to map their binding sites and calculate their binding affinities. Small molecular weight compounds (FMOC derivatives) bind to the IGF-I binding site on the IGFBP-5 with micromolar affinities and thus serve as potential starting compounds for the design of more potent inhibitors and therapeutic agents for diseases that are associated with abnormal IGF-I regulation.