Effects of cysteine, asparagine, or glutamine limitations in Chinese hamster ovary cell batch and fed-batch cultures.

Amino acid availability is a key factor that can be controlled to optimize the productivity of fed-batch cultures. To study amino acid limitation effects, a serum-free chemically defined basal medium was formulated to exclude the amino acids that became depleted in batch culture. The effect of limiting glutamine, asparagine, and cysteine on the cell growth, metabolism, antibody productivity, and product glycosylation was investigated in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine limitation was detrimental to both cell proliferation and productivity for all three CHO cell lines. Glutamine limitation reduced growth but not cell specific productivity, whereas asparagine limitation had no significant effect on either growth or cell specific productivity. Neither glutamine nor asparagine limitation significantly affected antibody glycosylation. Replenishing the CHO-DXB11 culture with cysteine after 1 day of cysteine limitation allowed the cells to partially recover their growth and productivity. This recovery was not observed after 2 days of cysteine limitation. Based on these findings, a fed-batch protocol was developed using single or mixed amino acid supplementation. Although cell density and antibody concentration were lower compared to a commercial feed, the feeds based on cysteine supplementation yielded comparable cell specific productivity. Overall, this study showed that different amino acid limitations have varied effects on the performance of CHO cell cultures and that maintaining cysteine availability is a critical process parameter for the three cell lines investigated.

Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology.

In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO-S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO-S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO-S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO-S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression.

The generation of stable, high MAb expressing CHO cell lines based on the artificial chromosome expression (ACE) technology.

The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming. To improve this process, the ACE System has been developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for the targeted transfection of single or multiple genes and eliminates the need for random integration into native host chromosomes. To illustrate the utility of the ACE System in generating stable, high expressing cell lines, CHO based candidate cell lines were generated to express a human monoclonal IgG1 antibody. Candidate cell lines were generated in under 6 months and expressed over 1 g/L and with specific productivities of up to 45 pg/cell/day under non-fed, non-optimized shake flask conditions. These candidate cell lines were shown to have stable expression of the monoclonal antibody for up to 70 days of continuous culture. The results of this study demonstrate that clonal, stable monoclonal antibody expressing CHO based cell lines can be generated by the ACE System rapidly and perform competitively with those cell lines generated by existing technologies. The ACE System, therefore, provides an attractive and practical alternative to conventional methods of cell line generation.

Expression of melanotransferrin isoforms in human serum: relevance to Alzheimer's disease.

Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimer's disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimer's disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimer's disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G-Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimer's disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimer's disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimer's disease.

Fed-batch CHO cell t-PA production and feed glutamine replacement to reduce ammonia production.

Industrial therapeutic protein production has been greatly improved through fed-batch development. In this study, improvement to the productivity of a tissue-plasminogen activator (t-PA) expressing Chinese hamster ovary (CHO) cell line was investigated in shake flask culture through the optimization of the fed-batch feed and the reduction of ammonia generation by glutamine replacement. The t-PA titer was increased from 33 mg/L under batch conditions to 250 mg/L with daily feeding starting after three days of culture. A commercially available fed-batch feed was supplemented with cotton seed hydrolysate and the four depleted amino acids, aspartic acid, asparagine, cysteine, and tyrosine. The fed-batch operation increased the generation of by-products such as lactate and ammonia that can adversely affect the fed-batch performance. To reduce the ammonia production, a glutamine-containing dipeptide, pyruvate, glutamate, and wheat gluten hydrolysate, were investigated as glutamine substitutes. To minimize the lag phase as the cells adjusted to the new energy source, a feed glutamine replacement process was developed where the cells were initially cultured with a glutamine containing basal medium to establish cell growth followed by feeding with a feed containing the glutamine substitutes. This two-step feed glutamine replacement process not only reduced the ammonia levels by over 45% but, in the case of using wheat gluten hydrolysate, almost doubled the t-PA titer to over 420 mg/L without compromising the t-PA product quality or glycosylation pattern. The feed glutamine replacement process combined with optimizing other feed medium components provided a simple, practical, and effective fed-batch strategy that could be applied to the production of other recombinant therapeutic proteins.

Engineered mammalian chromosomes in cellular protein production: future prospects.

The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly, and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming. To improve this process, the use of engineered chromosomes has been considered. To date, the most successful technique has been based on the artificial chromosome expression or ACE System, which consists of the targeted transfection of cells containing mammalian based artificial chromosomes with multiple recombination acceptor sites. This ACE System allows for the specific transfection of single or multiple gene copies and eliminates the need for random integration into native host chromosomes. The utility of using artificial engineered mammalian chromosomes, specifically the ACE System, is illustrated in several case studies covering the generation of CHO cell lines expressing monoclonal antibodies.

Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells.

The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5­3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a non optimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.

Deletion of the GPI pre-anchor sequence in human p97--a general approach for generating the soluble form of GPI-linked proteins.

Melanotransferrin, also named p97, belongs to the transferrin-like group of iron-binding proteins. Unlike the other members of this family, p97 exists in two forms-one soluble form and one attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The GPI-linked form plays a role in the uptake of iron, while the soluble form of p97 has the unique ability of traversing the blood-brain barrier and may be utilized to deliver drug conjugates into the brain. To investigate these possibilities, a recombinant soluble form of p97 from the GPI-linked p97 protein is required. The approach involved sequential deletions of the p97 GPI pre-anchor sequence (PAS) up to the putative site of cleavage/attachment, releasing p97 from attachment to the GPI-anchor and rendering it soluble. Transfection of the p97 deletion constructs into both the CHO and BHK TK(-) cells was performed with the aim of optimizing the production of p97 by utilizing the cell characteristics unique to each cell line. Altering the GPI PAS resulted in the generation of a recombinant soluble form that was secreted at significantly higher rates than from the full-length expressing cell lines. Increases were from 22 x 10(-9) to 241 x 10(-9)microg/cell/h for expression in the CHO cell system and from 220 x 10(-9) to 4970 x 10(-9)microg/cell/h for the BHK system. Furthermore, there appeared to be differences in the secretion rates between the various deletions suggesting the need for closer examination of the C-terminus in achieving maximum production of the altered proteins. The results of this study are likely applicable for expressing soluble forms of other GPI-linked proteins.

High transcytosis of melanotransferrin (P97) across the blood-brain barrier.

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration- and conformation-dependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.