Nigel Jenkins

Glycosylation of recombinant proteins: Problems and prospects

Glycosylation, the addition of sugar residues to a peptide backbone, is the most extensive posttranslational modifica- tion made to proteins by eukaryotic cells. The majority of recombinant proteins manufactured for human therapy are glycoproteins derived from animal cells, and it is essential to fully characterize and, if possible, control the glycosylation profile of these products. Indeed, the Food and Drug Ad- ministration (FDA) in the United States and the Commit- tee for Proprietary Medical Productions (CPMP) of the European Community are demanding increasingly sophis- ticated carbohydrate analysis on all new glycoproteins des- tined for human therapy. A complete survey of protein glycosylation is beyond the scope of this article, and the reader is referred to the excel- lent reviews recently published on glycoprotein biosyn- thesis, 2-7 regulation of biosynthetic pathways, 1,8-13 biolog- ical functions of glycan structures, 1.14-19 and glycoprotein analysis. 2°-29 This review will focus on the aspects of glyco- protein research that are of direct relevance to the biotech- nology industry, i.e., the reasons and methods for defining the glycosylation patterns of recombinant glycoproteins

Constraints on the transport and glycosylation of recombinant IFN-γ in Chinese hamster ovary and insect cells

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture.

SummaryRecombinant human interferon-λ production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation.

STABILITY OF AMPLIFIED DNA IN CHINESE HAMSTER OVARY CELLS

ABSTRACT A Chinese Hamster Ovary (CHO) cell line containing the human gamma interferon (IFN-∝) gene co-amplified with the gene for dihydrofolate reductase (DHFR), was continuously maintained for 120 days in serum-free medium with 0.1 μ M methotrexate. During this time the IFN-∝ titre fell by 70% due to a reduction in the specific IFN production rate. Cell number was higher in late passage cultures and cell viability was maintained for longer periods. Chromosome studies revealed no significant difference in the overall chromosome frequency and distribution, except for a 16% increase in the modal chromosome number of 18. Southern blot analysis of DHFR showed a 45% decrease in levels over 120 days with IFN-∝ decreasing by 30% over the same time period. Keywords : CHO, stability, amplification, DHFR, interferon, methotrexate.

EFFICIENT HUMAN IFN-GAMMA EXPRESSION IN THE MAMMARY GLAND OF TRANSGENIC MICE

Two hybrid genes (BLG-HuIFN-gamma2 and BLG-HuIFN-gamma3) were constructed on the basis of sheep beta-lactoglobulin (BLG) and human interferon-gamma (HuIFN-gamma) gene sequences. They were used to direct HuIFN-gamma synthesis in the mammary gland of transgenic mice. HuIFN-gamma was efficiently produced in the mammary gland of transgenic mice. BLG-HuIFN-gamma2 transgenic females expressed HuIFN-gamma in the milk at concentrations up to 570 mg/ml, and BLG-HuIFN-gamma3 transgenic females expressed up to 350 mg/ml. All females carrying the BLG-HuIFN-gamma3 gene expressed HuIFN-gamma in their milk. No significant changes were observed in the HuIFN-gamma expression level during the lactation period. Using RT-PCR analysis, ectopic expression for both hybrid genes was found in transgenic mice. Despite ectopic expression of HuIFN-gamma in transgenic mice, their development and pregnancy were normal. The heritability of the HuIFN-gamma expression level in milk was demonstrated up to the F2 generation. This work demonstrates that hybrid genes have the potential to develop in transgenic domestic animals producing HuIFN-gamma in milk.

Control of growth and recombinant protein synthesis by heat-shock in a mutant mammalian cell line

SummaryThe effect of heat-shock on cell growth and the induction of recombinant protein synthesis by a temperature-sensitive (ts) Chinese hamster ovary (CHO) cell line was investigated. An optimal regime of successive 2 hour heat-shocks (42°C) over 72 hours was found to simultaneously arrest cell growth and induce the synthesis of recombinant protein. The enhanced induction achieved from growth arrested cells may find application in the production of cytotoxic proteins.

Analysis of N-Glycans by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

A naturally occurring glycoprotein is made up of a population of individual glycoforms. This molecular diversity arises from variation in oligosaccharide structures at individual glycosylation sites (microheterogeneity) and variable occupation of potential glycosylation sites (variable site occupancy or macroheterogeneity). Whilst there is no analytical method capable of preparatively resolving a glycoprotein into its discrete glycoforms, several methods have been developed in recent years to characterize, to a varying extent, populations of oligosaccharides associated with glycoproteins or glycopeptides (1). Increasingly prevalent among current analytical strategies are mass spectrometric methods. Since the early 1980s, the emergence of “soft” ionization methods for biopolymer characterization by mass spectrometry (MS) such as fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) has provided a formidable weapon in the armoury of the analytical biochemist. Typically, an FAB ion source is accompanied by a double-sector (magnetic and electrostatic) mass analyser, ESI is usually interfaced with a quadrupole analyser and MALDI generally employs simple time-of-flight (TOF) mass analysis (2, 3, 4). All techniques are capable of low picomole to femtomole sensitivity.

Changes in the Glycosylation of Interferon-γ during Culture

We have shown in previous studies that recombinant human interferon-γ (IFN-γ) expressed in CHO cells displays macroheterogeneity caused by variable N-glycosylation (Curling et al., 1990). In this paper, FAB-Mass Spectrometry of IFN-γ glycopeptides was used to assign the N-glycosylation preference of individual asparagine (Asn) sites within the IFN-γmolecule. Whilst both Asn28 and Asn100 N-glycosylation sites were used in doubly-glycosylated IFN-γ, a clear preference for the Asn28 site was apparent in the singly-glycosylated IFN-γ glycoform. The extent of glycosylation at the Asn28 site remained relatively stable during batch culture, whereas the percentage of Asn100 sites glycosylated decreased from 60% to 23% over the same period. These results demonstrate that marked differences exist in the capacity of CHO cells to glycosylate at individual Asn sites during cell culture.

Protein folding in the secretory pathway of animal cells

The exit of newly-synthesized proteins from the lumen of the endoplasmic reticulum (ER) is the rate-determining step in protein secretion. Only correctly-folded and fully-assembled proteins exit the ER and progress along the secretory pathway. Folding and assembly in the ER are mediated by a variety of factors including folding catalysts and molecular chaperones. The properties of these factors, and the nature of their interactions with folding substrates, are beginning to be clarified. Little work has been done to characterize these processes and these factors in cell lines employed for large-scale cell culture. Manipulation of these process may permit improvement in yield or productivity of recombinant proteins by cultured animal cells.

Simultaneous Control of Growth and Productivity Using a Mutant CHO Cell Line

(Keywords: temperature-sensitive mutant, growth arrest, heat shock, induction, recombinant )