A new scaleable freeze-thaw technology for bulk protein solutions.

A new method of freeze-thaw is described using experimental data obtained from freezing of purified rhGH (recombinant human growth hormone). The method is based on freezing protein solutions in rectangular rather than cylindrical containers. It is hypothesized that the change in container geometry allows for linear scale-up of the freeze-thaw operation based on equivalency of temperature-time profile. The hypothesis is tested using freeze-thaw data from a miniature (30 ml) and a 2.4 litre container. Computational fluid dynamics techniques are used to simulate the freeze process and the simulations are compared with experimental results. Protein quality is assessed as a function of freeze conditions using dynamic light scattering, circular CD, size-exclusion and reverse-phase HPLC measurements. The results demonstrate the applicability of the new approach. Freezing of rhGH solution at concentrations of approx. 30 mg/ml is shown to be possible with no damage to the molecule for up to five cycles of freeze-thaw. A nitrogen blast chest-freezer is designed and evaluated as part of the process. The refrigeration system and the freeze-thaw method can be used to freeze-thaw bulk protein solutions for development work and has the potential for transfer to manufacturing.

Biophysical signatures of noncovalent aggregates formed by a glucagonlike peptide-1 analog: a prototypical example of biopharmaceutical aggregation.

LY307161 is a 31 amino acid analog of glucagonlike peptide-1(7-37)OH susceptible to physical instability associated with pharmaceutical processing. Orthogonal biophysical studies were conducted to explore the origins of this physical instability and to distinguish pharmaceutically desirable states of this aggregating peptide from undesirable ones. Equilibrium sedimentation analysis established that LY307161 exists as a monomer at pH 3, and reversibly self-associates in the pH range 7.5-10.5. Causative factors for physical instability related to lyophilization conditions were investigated. Solution pH, acetonitrile content, and concentration of the peptide prior to lyophilization each impacted physicochemical properties of the resultant powders. A comparative study of two powder samples exhibiting physicochemically disparate properties established that LY307161 forms soluble noncovalent aggregates. FT-IR analyses in the solid and solution states identified a prominent band at 1657-1659 cm(-1) attributed to alpha-helix structure. Noncovalent soluble aggregate exhibited characteristic bands at 1615 and 1698 cm(-1) indicative of intermolecular beta-sheet structure. An agitation-induced, precipitated solid form of LY307161 exhibited a different FT-IR signature indicative of a conformationally distinct species. Circular dichroism and fluorescence spectroscopy, together with dynamic light scattering measurements and dye-aggregate complexation, provided additional insights into the distinctions between aggregated and native LY307161.

Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells.

The neuropathology of Parkinson's Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.

Protein engineering strategies for sustained glucagon-like peptide-1 receptor-dependent control of glucose homeostasis.

OBJECTIVE: We have developed a novel platform for display and delivery of bioactive peptides that links the biological properties of the peptide to the pharmacokinetic properties of an antibody. Peptides engineered in the MIMETIBODY platform have improved biochemical and biophysical properties that are quite distinct from those of Fc-fusion proteins. CNTO736 is a glucagon-like peptide 1 (GLP-1) receptor agonist engineered in our MIMETIBODY platform. It retains many activities of native GLP-1 yet has a significantly enhanced pharmacokinetic profile. Our goal was to develop a long-acting GLP-1 receptor agonist with sustained efficacy. RESEARCH DESIGN AND METHODS: In vitro and in vivo activity of CNTO736 was evaluated using a variety of rodent cell lines and diabetic animal models. RESULTS: Acute pharmacodynamic studies in diabetic rodents demonstrate that CNTO736 reduces fasting and postprandial glucose, decreases gastric emptying, and inhibits food intake in a GLP-1 receptor-specific manner. Reduction of food intake following CNTO736 dosing is coincident with detection of the molecule in the circumventricular organs of the brain and activation of c-fos in regions protected by the blood-brain barrier. Diabetic rodents dosed chronically with CNTO736 have lower fasting and postprandial glucose and reduced body weight. CONCLUSIONS: Taken together, our data demonstrate that CNTO736 produces a spectrum of GLP-1 receptor-dependent actions while exhibiting significantly improved pharmacokinetics relative to the native GLP-1 peptide.

Mixed lineage kinase activity of indolocarbazole analogues.

The MLK1-3 activity for a series of analogues of the indolocarbazole K-252a is reported. Addition of 3,9-bis-alkylthiomethyl groups to K-252a results in potent and selective MLK inhibitors. The in vitro and in vivo survival promoting activity of bis-isopropylthiomethyl-K-252a (16, CEP-11004/KT-8138) is reported.