Spatially Resolved Effects of Protein Freeze-Thawing in a Small-Scale Model Using Monoclonal Antibodies

Protein freeze-thawing is frequently used to stabilize and store recombinantly produced proteins after different unit operations in upstream and downstream processing. However, freeze-thawing is often accompanied by product damage and, hence, loss of product. Different effects are responsible, including cold denaturation, aggregation effects, which are caused by inhomogeneities in protein concentration, as well as pH and buffer ingredients, especially during the freeze cycle. In this study, we tested a commercially available small-scale protein freezing unit using immunoglobin G (IgG) as monoclonal antibody in a typical formulation buffer containing sodium phosphate, sodium chloride, and Tween 80. Different freezing rates were used respectively, and the product quality was tested in the frozen sample. Spatially resolved tests for protein concentration, pH, conductivity, and aggregation revealed high spatial differences in the frozen sample. Usage of slow freezing rates revealed high inhomogeneities in terms of buffer salt and protein distribution, while fast rates led to far lower spatial differences. These protein and buffer salt inhomogeneities can be reliably monitored using straight forward analytics, like conductivity and photometric total protein concentration measurements, reducing the need for HPLC analytics in screening experiments. Summarizing, fast freezing using steep rates shows promising results concerning homogeneity of the final frozen product and inhibits increased product aggregation.

EFFECTS OF BUFFER SALT CONCENTRATION ON THE DOMINATED DEPOSITION MECHANISM AND OPTICAL CHARACTERISTICS OF CHEMICALLY DEPOSITED CADMIUM SULFIDE THIN FILMS

Effects of buffer salt concentration on the rate of deposition, dominated deposition mechanism and subsequently the structural, morphological, and optical properties of cadmium sulfide (CdS) thin films deposited by chemical bath deposition (CBD) on glass substrate were investigated. The precursors were chosen to be cadmium chloride (CdCl2) as the cadmium source, thiourea (CS(NH[Formula: see text]) as the sulfur source, ammonium nitrate (NH4NO3) as the buffer salt and ammonia as the complexing agent and the pH controller. The influence of the NH4NO3 concentration on the structure, morphology, film uniformity, stoichiometry and optical properties of CdS thin films was also studied by X-ray diffractometer (XRD), field emission scanning electron microscope (FE-SEM), energy dispersive X-ray (EDX) spectroscope, uv–visible and photoluminescence (PL) spectroscopes. The XRD studies revealed that all the deposited films exhibited a (002)h/(111)c preferred orientation. The crystallite size was increased from 20[Formula: see text]nm to 30[Formula: see text]nm by the increase of concentration of NH4NO3 from 0.5[Formula: see text]M to 2.5[Formula: see text]M. The morphology of CdS thin films were agglomerated spherical particles consisted of smaller particles. The surface of thin films deposited at the NH4NO3 concentration of 0.5[Formula: see text]M was compact and smooth. The increase of the concentration of NH4NO3 decreased the packing density of the films. The optical band gap was in the range of 2.25–2.4[Formula: see text]eV, which was decreased by the decrement of packing density. The PL spectra showed two peaks centered at 400[Formula: see text]nm and 500[Formula: see text]nm which are attributed to violet and band-to-band emissions, respectively.

Differential extraction of proteoglycans from cartilage tissue matrix compartments in isotonic buffer salt solutions and commercial tissue-culture media.

Small tissue blocks of native rat growth plate cartilage were incubated for short periods in one of several generally used isotonic buffer salt solutions or commercial tissue-culture media. The total percentage (approximately 12) of [35S]-labeled proteoglycans (PG) extracted from cartilage matrix under these conditions was not significantly influenced by either the chemical composition of the medium or the presence of a protease inhibitor. Morphological examination of incubated tissue after fixation in the presence of ruthenium hexamine trichloride (RHT) (included to preserve PG in situ) revealed, however, that the PG staining profiles across cartilage matrix varied with the composition of the incubation medium used. The various susceptibilities exhibited by PG within the different matrix compartments to selective extraction was estimated semi-quantitatively. The observed effects may prove useful in extracting these molecules differentially from cartilage matrix compartments.