Two-Dimensional Isoelectric Focusing OFFGEL, Micro-Fluidic Lab-on-Chip Electrophoresis and FTIR for Assessment of Long-Term Stability of rhG-CSF Formulation.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been increasingly recognized from among one of the most abundant families of biosimilars. Upon long-term storage, the rhG-CSF is subject to subtle chemical modifications that rapidly occur and, in particular, produce deaminated variants with divergent charge. Indeed, changes in charge from glutamine deamination may alter the way rhG-SCF will refold and the structure of resulting molecule. To assess this charge heterogeneity, 2-D gel electrophoresis has limited application. Recent micro-fluidic- based technical advances offer a great alternative method to better control liquid volumes on a minute scale. Here, we used IEF OFFGEL-lab-on-chip electrophoresis for 2-D separation of the rhG-CSF peptides according to their isoelectric point (pI) and molecular weight (kDa). We used an rhG-CSF commercial therapeutic formulation, kept refrigerated 24 months after expiry. The samples were analyzed for particulate matter and charge variants. Subsequently, the secondary structure was assessed by FTIR spectroscopy and residual biological activity was recorded. Interestingly, we showed an additional band in the acidic gel area above and below the most intense protein band (fractions 10, 11, and 12 at 22.84s). This observation reveals the presence of the rhG-CSF variant charges without any additional high molecular weight impurity or biological activity decrease. We conclude that after two years of storage, the rhG-CSF solution maintained its native secondary structure with little -sheet deviation, as reflected in the 1622 cm-1 and 1695 cm-1. These data demonstrated that a combined strategy is a more suitable and accurate analytical assessment of the rhG-CSF and recombinant protein-based biosimilars.

Heat treatment of whole milk by the direct joule effect--experimental and numerical approaches to fouling mechanisms.

The development of alternative technologies such as the direct Joule effect to pasteurize and sterilize food products is of great scientific and industrial interest. Our objective was 1) to gain insight into the ability to ensure ultra-high-temperature treatment of milk and 2) to investigate the links among thermal, hydraulic, and electrical phenomena in relation to fouling in a direct Joule effect heater. The ohmic heater [OH; E perpendicular to v (where E is the electrical field and v is the velocity); P (power) = 15 kW] was composed of 5 flat rectangular cells [e (space between the plate and electrode) = 15 mm, w (wall) = 76 mm, and L (length of the plate in plate heat exchanger or electrode) = 246 mm]--3 active cells to ensure heating and 2 (at the extremities) for electrical insulation and the recovery of leakage currents. In the first step, the thermal performance of the OH was investigated vs. the flow regimen [50 < Re (Reynolds number) < 5,000], supplied power (0 < P < 15 kW), and electrical conductivity of fluids (0.1 < sigma(20 degrees C) < 2 S/m) under clean conditions with model fluids. This protocol enabled a global thermal approach (thermal and electrical balance, modeling of the temperature profile of a fluid) and local analysis of the wall temperature of the electrode. An empirical correlation was established to estimate the temperature gradient, T(w)-T(b) (where T(w) is the wall temperature and T(b) is the product temperature) under clean conditions (without fouling) and was used to define operating conditions for pure-volume and direct-resistance heating. In the second step, the ability of OH to ensure the ultra-high-temperature treatment of whole milk was investigated and compared with a plate heat exchanger. Special care was taken to investigate the heat transfer phenomena occurring over a range of temperatures from 105 to 138 degrees C. This temperature range corresponds to the part of the process made critical by protein and mineral fouling. The objectives were 1) to demonstrate the ability of an OH to ensure heat treatment of milk, 2) to study the thermal and hydraulic performance with an increasing power and temperature difference between the inlet and outlet of the OH, 3) to define and validate a criterion to follow heat dissipation efficiency, and 4) to compare the fouling propensity with the different configurations. A heat dissipation coefficient, Rh(CO), was defined and validated to monitor the fouling propensity through global electrical and thermal parameters. Finally, a numerical simulation was developed to analyze heat profiles (wall, deposit, bulk). Because of an increasing Joule effect in the static deposit, the simulation showed how wall overheating would definitively cause fouling to spiral out of control.