Misincorporation of amino acids in proteins expressed inEscherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA Asn rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions. Many recombinant proteins have been approved as therapeutic drugs by the Food and Drug Administration, and many more are undergoing clinical trial (1). For economic and practical reasons, considerable effort has been made to increase product yield and process efficiency for proteins made in mammalian cell culture. Nowadays, large amounts of proteins can be expressed efficiently in optimized expression systems, with yields from bioreactors having improved more than 100-fold during the past two decades (2). Yields as high as 10 g/liter have been reported for production of monoclonal antibodies in CHO 2 cells (3). These yields are due mainly to improvements in host cell engineering, cell line selection, and culture medium optimization (4). However, it is well known that overexpressing recombinant proteins can lead to nutritional stresses in the host cells and that these stresses can markedly increase the frequency of random translational errors, resulting in a heterogeneous mixture of proteins (5-11). A variety of translational errors have been observed during overexpression of proteins in Escherichia coli, including frame shifts, premature truncation, read-through, leaky stop codons, and amino acid misincorporation (12-16). Nevertheless, there are few such reports for proteins made in mammalian cells, and it is commonly believed that the fidelity of translation in mammalian cells is higher (8, 17). Here we report for the first time that misincorporation, namely of Ser for Asn, can occur in proteins overexpressed in CHO cells under normal recombinant protein production conditions. Further investigation showed that supplementation of the medium with Asn can overcome this problem. Our work demonstrates that protein products should be closely monitored for misincorporation, for example, by molecular mass determination and peptide mapping during optimization of culture conditions. EX...

A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed-batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time-course analysis of cell culture suggest that misincorporation was occurring midway through the fed-batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench-scale culture via controlled supplementation of asparagine-containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales.

In addition to monoclonat immunogtobutin. two kinds of nuctcoproteins. NPI and NP?. were isolated from the supernatants of hybridoma cultures set up in a protein-free medium. As shown by SDS-etectrophorcsis the two nucleoprotcins shared a set of proteins (apparent M, I IO00 to 15000). and differed in the DNA moiety ( 4 150 bp in NP I. -350 bp in NP2). The amino acid composition of the protein moiety confirmed the nucteosomal origin of NPI and NP2. The findings support the view that in hybridoma cultures the cells undergo de& by apoptosis, i.e. a programmed process characterized by initial fragmentation of chromatin.

Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical "ladder" of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.

The iron-rich (500 microM ferric citrate) protein-free supplement was added to six different basal media. Cell growth and monoclonal antibody production of a mouse-mouse hybridoma were investigated in 1.3 1 batch cultures performed in a laboratory bioreactor with automatic control of pH and dissolved oxygen concentration. RPMI 1640 served as the control medium. Fortification of the basal medium by balanced mixtures of amino acids and vitamins showed higher positive effect than daily supplementation by glucose and glutamine. Strongly fortified medium, based on RPMI 1640, was found superior to other basal media. The viability index increased by a factor of 3.04 and the total antibody production by a factor of 2.82, relative to the control.

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC-MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials.

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