Animal cell culture, notably mammalian cell culture, is preferably used for the expression of recombinantly produced, glycosylated proteins for therapeutic and/or prophylactic applications. Glycosylation patterns of recombinant glycoproteins are important, because the oligosaccharide side chains of glycoproteins affect protein function, as well as the intramolecular interactions between different regions of a protein. Such intramolecular interactions are involved in protein conformation and tertiary structure of the glycoprotein. (See, e.g., A. Wittwer et al., 1990, Biochemistry, 29:4175-4180; Hart, 1992, Curr. Op. Cell Biol., 4:1017-1023; Goochee et al., 1991, Bio/Technol., 9:1347-1355; and R. B. Parekh, 1991, Curr. Op. Struct. 1:750-754). In addition, oligosaccharides may function to target a particular polypeptide to certain structures based on specific cellular carbohydrate receptors. (M. P. Bevilacqua et al., 1993, J. Clin. Invest., 91:379-387; R. M. Nelson et al., 1993, J. Clin. Invest., 91:1157-1166; K. E. Norgard et al., 1993, Proc. Natl. Acad. Sci. USA, 90:1068-1072; and Y. Imai et al., 1993, Nature, 361-555-557).
The terminal sialic acid component of a glycoprotein oligosaccharide side chain is known to have an effect on numerous aspects and properties of a glycoprotein, including absorption, solubility, thermal stability, serum half life, clearance from the serum, as well as its physical and chemical structure/behavior and its immunogenicity. (A. Varki, 1993, Glycobiology, 3:97-100; R. B. Parekh, Id., Goochee et al., Id., J. Paulson et al., 1989, TIBS, 14:272-276; and A. Kobata, 1992, Eur. J. Biochem., 209:483-501; E. Q. Lawson et al., 1983, Arch. Biochem. Biophys., 220:572-575; and E. Tsuda et al., 1990, Eur. J. Biochem., 188:405-411).
The amount of sialic acid in glycoproteins is affected by two opposite processes: the intracellular additions of sialic acid by sialyltransferase activity and the extracellular removal of sialic acid by sialidase cleavage.
Intracellular addition of sialic acid is the last stage of the glycosylation process that takes place in the trans-Golgi. This involves the enzymatic transfer of sialic acid from the nucleotide sugar precursor, CMP-sialic acid to an available galactose on the emerging glycan structure that is attached to the newly synthesized protein. Possible limitations to the process that might cause incomplete sialylation include the availability of CMP-sialic acid, the activity of the sialyltransferase enzyme, the amount of the galactose on the emerging glycan structure and the activity of the galactosyltransferase enzyme. Significant amount of research has been focusing on maximizing sialylation through gene over-expression and enzyme activity enhancement of sialyltransferase and glycosyltransferase. Zhang et al. (Biochim Biophys Acta 1425(3):1998, 441-52) reported that expression of human α2,6-sialyltransferase in CHO cells with tissue plasminogen activator (tPA) production enhances the α2,6-sialylation of tPA. Weikert et al (Nat Biotechnol 17(11):1999, 1116-21) reported that coexpression of α2,3-sialyltransferase and β1,4-galactosyltransferase results in greater than 90% sialylation of TNK-tPA and TNFR-IgG. Moreover, supplementation with the proper amount of manganese (Mn2+), a cofactor for β1,4-galactosyltransferase, greatly reduced the amount of rHuEPO in the lower sialylated fraction, increased carbohydrate site occupancy and narrowed carbohydrate branching (Zhang et al. 1998) to bi-antennary structures in these lower sialylated species (Crowell et al. Biotechnol Bioeng 96(3):538-49, 2007).
The amount of sialic acid in glycoproteins is also affected by the extracellular removal of sialic acid by sialidase cleavage. Gramer and Goochee (Biotechnol Prog 9(4):366-73, 1993) have demonstrated an increase of lactate dehydrogenase (LDH), which signified an increase in the cell lysis, correlated with an increase of extracellular sialidase activity in CHO perfusion cultures. Gu et al (Biotechnol Bioeng 55(2):390-8, 1997) also illustrate a remarkable loss of terminal sialic acids of interferon-γ (IFN-γ) along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation.
Consequently, It is essential to delay the onset of cell death and improve cell viability to reduce or avoid this degradation effect.
In general, protein expression levels in mammalian cell culture-based systems are considerably lower than in microbial expression systems, for example, bacterial or yeast expression systems. However, bacterial and yeast cells are limited in their ability to optimally express high molecular weight protein products, to properly fold a protein having a complex steric structure, and/or to provide the necessary post-translational modifications to mature an expressed glycoprotein, thereby affecting the immunogenicity and clearance rate of the product.
As a consequence of the limitations of the culturing of animal or mammalian cells, particularly animal or mammalian cells which produce recombinant products, the manipulation of a variety of parameters has been investigated, including the employment of large-scale culture vessels; altering basic culture conditions, such as incubation temperature, dissolved oxygen concentration, pH, and the like; the use of different types of media and additives to the media; and increasing the density of the cultured cells. In addition, process development for mammalian cell culture would benefit from advances in the ability to extend run times to increase final product concentration while maintaining high product quality. An important product quality parameter is the degree and completeness of the glycosylation structure of a polypeptide product, with sialic acid content commonly used as a measure of glycoprotein quality.
Run times of cell culture processes, particularly non-continuous processes, are usually limited by the remaining viability of the cells, which typically declines over the course of the run. The maximum possible extension of high cell viabilities is therefore desired. Product quality concerns also offer a motivation for minimizing decreases in viable cell density and maintaining high cell viability, as cell death can release sialidases to the culture supernatant, which may reduce the sialic acid content of the protein expressed. Protein purification concerns offer yet another motivation for minimizing decreases in viable cell density and maintaining high cell viability. The presence of cell debris and the contents of dead cells in the culture can negatively impact on the ability to isolate and/or purify the protein product at the end of the culturing run. By keeping cells viable for a longer period of time in culture, there is thus a concomitant reduction in the contamination of the culture medium by cellular proteins and enzymes, e.g., cellular proteases and sialidases that can cause degradation and ultimate reduction in quality of the desired glycoprotein produced by the cells.
Various parameters have been investigated to achieve high cell viability in cell cultures. One parameter involved a single lowering of the culture temperature following initial culturing at 37° C. (for example, Roessler et al., 1996, Enzyme and Microbial Technology, 18:423-427; U.S. Pat. Nos. 5,705,364 and 5,721,121 to T. Etcheverry et al., 1998; U.S. Pat. No. 5,976,833 to K. Furukawa et al., 1999; U.S. Pat. No. 5,851,800 to L. Adamson et al.; WO 99/61650 and WO 00/65070 to Genentech, Inc.; WO 00/36092 to Biogen, Inc.; and U.S. Pat. No. 4,357,422 to Girard et al.).
Other parameters investigated involved the addition of components to the culture. The growth factor inhibitor suramin was shown to prevent apoptosis during exponential growth of CHO K1:CycE cells (Zhangi et al., Biotechnol. Prog. 2000, 16, 319-325). However, suramin did not protect against apoptosis during the death phase. As a result, suramin was capable of maintaining high viability during the growth phase, but did not allow for an extension of culture longevity. The same authors report that for the CHO 111-10PF cell line, dextran sulfate and polyvinyl sulfate could, similarly to suramin, increase day 3 viable cell density and viability relative to the control culture. The effect of dextran sulfate or polyvinyl sulfate during the death phase was however not reported. Suramin, dextran sulfate and polyvinyl sulfate were also reported to be effective at preventing cell aggregation.
The effects of supplementing insect cell culture media with dexamethasone or N-acetylmannosamine on complex glycosylation of proteins, including the addition of terminal sialic acid residues to N-linked oligosaccharides, prepared via baculovirus expression vector system (BEVS) is disclosed in U.S. Pat. No. 6,472,175 to Boyce Thompson Institute For Plant Research, Inc. (Ithaca, N.Y.), 2002.
Protein therapeutics are inherently heterogeneous owing to their size, complexity of structure, and the nature of biological production (Chirino and Mire-Sluis, Nat Biotechnol. 2004; 22:1383-1391). Even in the “pure” protein solution, there will be some percentage of low molecular weight fragments, high molecular weight species, and various degrees of chemical modifications. The formation of high molecular weight species is usually due to protein aggregation, which is a common issue encountered during manufacture of biologics. Typically, the presence of aggregates is considered to be undesirable because of the concern that the aggregates may lead to an immunogenic reaction or may cause adverse events on administration (Cromwell et al, AAPS J. 2006; 8:E572-579). Although some types of aggregates of biologics may function normally, it is still important to maintain consistency in product quality since product consistency is a prerequisite for regulatory approval.
Aggregates of proteins may arise from several mechanisms and occur at each stage during the manufacturing process. In cell culture, secreted proteins may be exposed to the conditions that are unfavorable for protein stability; but more often, accumulation of high amounts of protein may lead to intracellular aggregation owing to either the interactions of unfolded protein molecules or to inefficient recognition of the nascent peptide chain by molecular chaperones responsible for proper folding (Cromwell et al, AAPS J. 2006; 8:E572-579). In the endoplasmic reticulum (ER) of cells, disulfide bond of newly synthesized protein is formed in an oxidative environment. Under normal condition, protein sulfhydryls are reversibly oxidized to protein disulfides and sulfenic acids, but the more highly oxidized states such as the sulfinic and sulfonic acid forms of protein cysteines are irreversible (Thomas and Mallis, Exp Gerontol. 2001; 36:1519-1526). Hyper-oxidized proteins may contain incorrect disulfide bonds or have mixed disulfide bonds with other luminal ER proteins; in either case it leads to protein improper folding and aggregation. It is therefore crucial to maintain a properly controlled oxidative environment in the ER. In this regard, Cuozzo and Kaiser (Nat Cell Biol. 1999; 1:130-135) initially demonstrated that in yeasts glutathione buffered against ER hyperoxidation and later on Chakravarthi and Bulleid (J Biol Chem. 2004; 279:39872-39879) confirmed that in mammalian cells glutathione was also required to regulate the formation of native disulfide bonds within proteins entering the secretory pathway.
With increasing product concentration in the culture, it can be observed in cell culture processes that the product quality decreases, as determined by the measured sialic acid content of the oligosaccharide glycostructure. Usually, a lower limit for an acceptable sialic acid content exists as determined by drug clearance studies. High abundance of a protein produced by cells in culture is optimally accompanied by high quality of the protein that is ultimately recovered for an intended use.
Recombinantly produced protein products that are properly glycosylated are increasingly becoming medically and clinically important for use as therapeutics, treatments and prophylactics. Therefore, the development of reliable cell culture processes that economically and efficiently achieve an increased final protein product concentration, in conjunction with a high level of product quality, such as is determined by sialic acid content, fulfills both a desired and needed goal in the art.
USPTO Backgrounds
USPTO Backgrounds