Comparison of antibody molecules produced from two cell lines with contrasting productivities and aggregate contents.

Cell culture processes that produce therapeutic antibodies with high productivity (titer) and low aggregate content reduce the risk of adverse effects and expense to patients. To elucidate the mechanism of aggregate formation, we compared trastuzumab samples produced from two contrasting cell lines: cell line A, which exhibits high titer and low aggregate content, and cell line B, which exhibits low titer and high aggregate content. Cell line B produced significantly fewer (approximately 1/3) antibodies compared with cell line A and contained higher (approximately 3-fold) percentages of aggregates. The aggregates of antibodies found in the protein A-purified samples of cell line B were associated mostly with noncovalent interactions. Cell line B exhibited a low content of monomers/dimers of light chains in the medium and within cells. Because light chains are essential for the correct folding of heavy chains and secretion of mature antibodies, the characteristics of cell line B may be attributed to low levels of light chain production. In addition, protein A-purified antibodies from cell line B (but not those from cell line A) contained fragments that are expected to expose the hydrophobic CH3 domain, which may serve as nuclei for aggregation.

[Gnathostoma larvae investigation in fresh water fishes in the valley of Chinese Chang River and the morphological characteristics of the larvae].

An investigations were carried out on the prevalence of larval gnathostomes in fresh water fishes in southeast area of the Yang-tze Valley, the people's Republic of China, from October to November. The fishes were collected from five districts which were from Shanghai, Chenchiang, Nanching, Chiuchiang and Nanchang, especially the Shanghai district involved Kunshan, Tienshanhu, Chingpu and Nanhui areas. Species of the fishes collected were Channa argus (110), Siniperca chuatssi (24) and Silurus asotus (39). A total number of fishes examined were 137. Muscles of the fishes were each dissected into small pieces and sliced. These slices were examined under a dissecting microscope. The fish viscera from 4-5 individuals were mixed, homogenized and digested by the artificial gastric juice at 37 degrees C overnight. As a result, four encysted larvae were recovered from the muscles of four fish of C. argus. 34 larvae in total were obtained by the digesting. Those larvae mainly recovered from the viscera. The morphological examination showed those 38 larvae could divide into three groups by their body lengths such as 0.58-0.86 mm (group A), 1.12-2.61 mm (group B) and 4.86 mm (group C). Group A consisted of 5 which belong to the early third-stage parasitologically which usually lives the first intermediate host. The majority (32) belongs to group B. Unfortunately, only one larva was obtained in group C which belongs to the advanced third-stage which usually lives in the second intermediate host. Light and scanning electron microscopy revealed that group A and B had the characteristics of G. hispidum and group C had those of G. spinigerum. Next, we investigated fishes near the tow big lakes, Hongtze-hu and Tai-hu situated along the river. In these lakes 12 genera, 12 species fishes (533 individuals) were captured. From these fishes 18 ganathostome larvae collected. Most of the larvae obtained from C. argus and Monopterus albus. All these larvae belongs group B which identified as G. hispidum. In conclusion, many fishes have only G. hispidum of stage of the early third in their viscera.

A rapid method for simultaneous evaluation of free light chain content and aggregate content in culture media of Chinese hamster ovary cells expressing monoclonal antibodies for cell line screening.

The goal of developing a monoclonal antibody (mAb) production process is high productivity and high quality. Because the productivity and quality of mAbs depend on cell line properties, the selection of cell lines suitable for large-scale production is an important stage in process development for mAb production. The light chain (LC) is important for antibody folding and assembly in the endoplasmic reticulum; cell lines that secrete a large amount of LCs in the medium secrete high-quality antibodies with high productivity. LC contents in culture media have been estimated by western blotting, reverse-phase high-performance liquid chromatography, and enzyme-linked immunosorbent assay. However, these analyses require fine tuning of experimental conditions for each antibody analyzed. Here we report a rapid and simple high-sensitivity size-exclusion chromatography (HS-SEC) method to evaluate the contents of low-molecular weight species (LMWS, mainly consisting of LC monomers and dimers) and high-molecular weight species (HMWS, aggregates) in the media for cell line screening. Because LMWS and HMWS are important indicators of productivity and quality, respectively, for cell line screening, HS-SEC will be useful in the first step of cell line selection needed for large-scale production.

Titer of trastuzumab produced by a Chinese hamster ovary cell line is associated with tricarboxylic acid cycle activity rather than lactate metabolism.

Achieving high productivity and quality is the final goal of therapeutic antibody development, but the productivity and quality of antibodies are known to be substantially dependent on the nature of the cell lines expressing the antibodies. We characterized two contrasting cell lines that produce trastuzumab, namely cell line A with a high titer and a low aggregate content and cell line B with a low titer and a high aggregate content to identify the causes of the differences. We observed the following differences: cell growth (A > B), proportion of defucosylated oligosaccharides on antibodies (A < B), and proportion of covalent antibody aggregates (A > B). Our results suggest that the high monoclonal antibody (mAb) titers in cell line A is associated with the high proliferation and is not caused by the lactate metabolism shift (switching from lactate production to net lactate consumption). Rather, these differences can be accounted for by the following: levels of tricarboxylic acid cycle intermediates (A > B), ammonium ion levels (A ≤ B), and oxidative stress (A > B).

Efficient folding/assembly in Chinese hamster ovary cells is critical for high quality (low aggregate content) of secreted trastuzumab as well as for high production: stepwise multivariate regression analyses.

When developing cell culture processes for therapeutic antibodies, the low content of aggregated proteins is the most critical because administering aggregated antibody molecules might result in adverse effects such as immunogenicity. To characterize cells with high productivity and quality, we determined factors that are closely related to antibody titer, which is a productivity indicator, and the area percentage of high molecular weight species in cultivated media, which is equivalent to aggregate content and is used as a quality indicator. We examined the factors influencing antibody titer and aggregate content using various data from 28 cell lines throughout their culture periods from growth to death phases. Our study using correlation analysis revealed that statistically significant correlations between factors and indicators changes with sampling points, hence we thought that various factors would influence each indicator simultaneously. To understand the relationship between these factors and titer/aggregates contents, we performed stepwise multiple linear regression analyses and deduced a multiple linear model for each indicator. The titer was found to positively associate with specific growth rate and specific production rate and negatively with intracellular heavy chain content. The aggregate content was found to positively associate with protein disulfide isomerase mRNA level and negatively with light chain secreted into culture media, specific production rate, intracellular light chain content, and specific growth rate. Our observations suggest that correct and efficient assembling and/or folding of an antibody molecule in an endoplasmic reticulum are important for high titer and low aggregates contents.