Effects of various pine needle extracts on Chinese hamster ovary cell growth and monoclonal antibody quality.

Chinese hamster ovary (CHO) cells are commonly used as "bio-machines" to pro-duce monoclonal antibodies (mAb) because of their ability to produce very complex proteins. In this study, we evaluated the effects of pine needle water extract (PNWE), pine needle ethanol extract (PNEE), and pine needle polysaccharide extract (PNPE) on the CHO cell growth, mAb production and quality using a Fed-batch culture process. PNPE maintained high VCD and viability, and the titer increase was correlated with its concentration.

Development and Validation of a HPLC-UV Method for Urea and Related Impurities.

Urea is used in biopharmaceutical manufacturing processes for the purification of therapeutic proteins, for cleaning columns, and for refolding proteins after purification. The urea used for such purposes is typically USP grade material obtained from commercial sources and further characterization is required prior to use, such as determination of purity and identity. For this purpose, a robust analytical method is needed that can characterize the known organic impurities of urea.

Monitoring utilizations of amino acids and vitamins in culture media and Chinese hamster ovary cells by liquid chromatography tandem mass spectrometry.

Monitoring amino acids and vitamins is important for understanding human health, food nutrition and the culture of mammalian cells used to produce therapeutic proteins in biotechnology. A method including ion pairing reversed-phase liquid chromatography with tandem mass spectrometry was developed and optimized to quantify 21 amino acids and 9 water-soluble vitamins in Chinese hamster ovary (CHO) cells and culture media. By optimizing the chromatographic separation, scan time, monitoring time window, and sample preparation procedure, and using isotopically labeled (13)C, (15)N and (2)H internal standards, low limits of quantitation (≤0.

Elucidating the role of copper in CHO cell energy metabolism using (13)C metabolic flux analysis.

(13)C-metabolic flux analysis was used to understand copper deficiency-related restructuring of energy metabolism, which leads to excessive lactate production in recombinant protein-producing CHO cells. Stationary-phase labeling experiments with U-(13)C glucose were conducted on CHO cells grown under high and limiting copper in 3 L fed-batch bioreactors. The resultant labeling patterns of soluble metabolites were measured by GC-MS and used to estimate metabolic fluxes in the central carbon metabolism pathways using OpenFlux.

Risk-based Strategy to Determine Testing Requirement for the Removal of Residual Process Reagents as Process-related Impurities in Bioprocesses.

Unlabelled: The purpose of this article is to recommend a risk-based strategy for determining clearance testing requirements of the process reagents used in manufacturing biopharmaceutical products. The strategy takes account of four risk factors. Firstly, the process reagents are classified into two categories according to their safety profile and history of use: generally recognized as safe (GRAS) and potential safety concern (PSC) reagents.

Metabolic markers associated with high mannose glycan levels of therapeutic recombinant monoclonal antibodies.

High mannose (HM) glycan levels on secreted monoclonal antibodies can be influenced by external factors, including osmolality and copper deficiency, and by intrinsic factors determined by different cell lines. In order to identify the metabolic markers associated with HM glycan levels, metabolomics analysis was performed to assess the changes in the extracellular metabolites of recombinant cell lines at different time points during fed-batch production process. Ornithine was identified as the common metabolic marker influenced by both external and intrinsic factors when eight different medium conditions and eight different cell lines exhibiting different levels of HM were compared.

Quantitation of low concentrations of polysorbates in high protein concentration formulations by solid phase extraction and cobalt-thiocyanate derivatization.

A spectrophotometric method was developed to quantify low polysorbate (PS) levels in biopharmaceutical formulations containing high protein concentrations. In the method, Oasis HLB solid phase extraction (SPE) cartridge was used to extract PS from high protein concentration formulations. After loading a sample, the cartridge was washed with 4M guanidine HCl and 10% (v/v) methanol, and the retained PS was eluted by acetonitrile.

Direct determination of residual Pluronic F-68 in in-process samples from monoclonal antibody preparations by high performance liquid chromatography.

A simple and sensitive high performance liquid chromatography (HPLC) method was developed to determine residual Pluronic F-68 (PF-68) in in-process samples of monoclonal antibody (MAb) preparations. The method permits the direct injection of proteinaceous samples after simple sample dilution and is able to quantitate as low as 50 mg/L of PF-68 in the presence of up to approximately 30 g/L of protein. The PF-68 molecule was separated on a restricted access reversed phase column using a step gradient and then measured by an evaporative light scattering detector (ELSD).

Analysis of guanidine in high salt and protein matrices by cation-exchange chromatography and UV detection.

A simple and sensitive HPLC method for quantitative determination of guanidine in high salt and protein matrices was developed. The HPLC system consisted of an Agilent 1100 pump with an online degasser, a UV detector, an autosampler, and Dionex CS 14 cation-exchange guard (4 mm x 50 mm) and analytical (4 mm x 250 mm) columns. The mobile phase was 3.

Development and optimization of organic acid analysis in tobacco with ion chromatography and suppressed conductivity detection.

With the aid of a central composite face-centered design, an ion chromatographic method was developed and optimized for analyzing organic acids in tobacco. A Dionex-100 ion chromatograph with an ion suppressor and a conductivity detector, and a Bio-Rad Aminex HPX-87H column were employed. Only 13 analyses were required to optimize two factors: column temperature and eluent strength.