Aggregation and chemical modification of monoclonal antibodies under upstream processing conditions.

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.

Structural basis for the regulation of insulin-like growth factors by IGF binding proteins.

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.

In silico and NMR identification of inhibitors of the IGF-I and IGF-binding protein-5 interaction.

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.

Effects of hedgehog proteins on tissue engineering of cartilage in vitro.

The effects of three derivatives of the N-terminal signaling domain of hedgehog proteins on cartilage engineered in vitro were investigated, with specific focus on the ability to increase tissue growth rate and concentrations of major extracellular matrix components, that is, glycosaminoglycans (GAG) and collagen, and on the effects on morphological appearance of the tissue. Bovine articular chondrocytes were cultured on biodegradable polyglycolic acid (PGA) scaffolds with or without the addition of dipalmitoylated sonic hedgehog (dp-shh), dipalmitoylated indian hedgehog (dp-ihh), or sonic hedgehog dimer (shh-dimer) to medium with either 1% or 10% fetal bovine serum (FBS). All three hedgehog proteins dose-dependently increased construct weights (by up to 1.95-fold, dp-shh at 1,000 ng/mL) and the fraction of GAG over 4 weeks (by up to 2.7-fold, dp-shh at 1,000 ng/mL), as compared to control constructs. Dp-shh and dp-ihh elicited similar responses; a 10-fold higher concentration of nonacylated shh-dimer was necessary to reach comparable results. Positive hedgehog effects were more pronounced in medium containing 1% FBS than in medium containing 10% FBS; however, at either FBS concentration, cartilaginous tissues grown in the presence of hedgehog proteins appeared morphologically more mature. Hedgehog derivatives thus appear as promising candidates to improve the development and composition of engineered cartilage.