Recombinant human granulocyte-macrophage colony-stimulating factor: effect of glycosylation on pharmacokinetic parameters
Keywords: glycosylation, pharmacokinetic parameters, rhGM-CSF.
The pharmacokinetic behaviour of the non-glycosylated, bacterially-derived recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and the glycosylated mammalian product was studied after intra and extra vascular administration of a single dose in rodents. Each route of administration gave a different rhGM-CSF concentration-time profile. After extra vascular administration of equivalent doses, a higher peak concentration and faster elimination were observed in the group treated with the E. coli-derived cytokine. The faster elimination resulted in a return to pre-treatment plasma levels after 12 hrs, versus 48 hrs following the administration of glycosylated rhGM-CSF. After intravascular administration, clearance of rhGM-CSF was significantly decreased by the presence of carbohydrates. Non-significant differences in the terminal phase of the biphasic kinetics were found, but the distribution phase was significantly longer for the glycosylated form.
Colony-stimulating factors are a family of glycoproteins that modulate the haematopoietic process and control survival, proliferation, differentiation, and functional ability of mature haematopoietic progenitors, with frequent overlapping activities (Armitage, 1998). The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) belongs to this family. It is produced by a variety of cells including lymphocytes, endothelial cells, fibroblasts, monocytes, as well as some malignant cells (Fleetwood et al. 2005). Human GM-CSF cDNA codes for a protein with 144 amino acids, with a leading peptide of 17 amino acids, two N-linked glycosylation sites, four potential O-linked glycosylation sites, and two disulfide bridges (Kaushansky et al. 1987). Due to the glycosylation, the apparent molecular mass of the protein varies from 18 to 32 kDa. It is classified as a multilineage colony-stimulating factor because it stimulates the proliferation and differentiation of haematopoietic progenitor cells of neutrophil, eosinophil, and monocyte origin. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is approved by the US Food and Drug Administration (FDA) for the acceleration of myeloid recovery following autologous bone marrow transplantation and in patients who fail to engraft after bone marrow transplantation. Evidence also supports the use of GM-CSF in many other clinical settings in which patients are neutropenic (Trindade et al. 1998; Bilgin et al. 2001).
Three synthetic human GM-CSFs have been produced by using recombinant DNA technology, employing different expression systems: bacteria, yeast, and mammalian cells (Hussein et al. 1995). The selection of the host cell to produce the recombinant protein is important because these preparations are not identical and present different characteristics, such as the amino acid sequence, the size of the protein, and the degree of glycosylation. In the case of rhGM-CSF, the degree of glycosylation can influence pharmacokinetics, biologic activity, antigenicity, and toxicity (Wadhwa et al. 1996; Armitage, 1998; Wadhwa et al. 1999). Although the product from mammalian cells is more similar to the natural protein, only commercial preparations produced in E. coli or S. cerevisiae are presently available for clinical application.
The main objective of the current study was to analyze the influence of the glycosylation of rhGM-CSF on kinetic parameters such as clearance, distribution, elimination half-life, and maximum plasma concentration, employing rats and mice as model systems. Therefore, both glycosylated rhGM-CSF produced in Chinese hamster ovary (CHO) cells in our laboratory and non-glycosylated rhGM-CSF produced in a bacterial system (Growgen, Bioprofarma S.A., Argentina) were employed in order to compare their pharmacokinetics.
obtain glycosylated rhGM-CSF, a recombinant CHO.K1 cell line adapted
to grow in suspension was used (Bollati Fogolín et al.
2004). These cells were inoculated at a concentration of 1 x 105
cells/mL in spinner flasks (Techne,
UK) with a
and lactate were determined routinely with an YSI Model 2000 glucose/lactate
analyzer (Yellow Springs Instruments,
glycoprotein that was present in the micro filtrated cell culture
supernatants was purified by immunoaffinity chromatography using the
M7E10 anti-GM-CSF monoclonal antibody (Forno et al.
2004). Elution was performed using
administration. Two groups (64 mice per group) of two-month-old
male and female BALB/c mice (Comisión Nacional de Energía Atómica, Argentina)
were injected intraperitonealy (ip) with 0.41 nmol of glycosylated
or non-glycosylated protein in a total volume of 200 µL, respectively. Mice
were housed in a temperature-controlled room at
administration. Two groups (8 rats per group) of two-month-old
male and female Wistar rats (Comisión
Nacional de Energía Atómica, Argentina) were injected intravenously
(iv) into the tail vein with 3.4 nmol of glycosylated or non-glycosylated
protein in a total volume of 250 µL. Animals were housed in a temperature-controlled
All animal experimental protocols were in accordance with the “Principles of Laboratory Animal Care” (NIH publication 85-23, and all subsequent revisions thereof).
of rhGM-CSF in plasma. Blood samples were centrifuged at 2,000
rpm for 10 min at
Determination of pharmacokinetic parameters. Plots of rhGM-CSF concentration versus time were constructed, and pharmacokinetic parameters were calculated from these plots (Shargel et al. 2005). Maximum plasma concentration (Cmax), the time to reach the Cmax (tmax), constants of the initial phase (A and α) constants of the elimination phase (B and β), distribution/post-absorption half-life (t1/2 α), terminal half-life (t1/2 β), and total plasma clearance (CL) were calculated with Microcal Origin software version 5.0 (Microcal Software, USA). Differences between the two preparations were evaluated by a paired Student’s test. Probabilities lower than were considered significant.
Glycosylation, the addition of sugar residues to a peptide backbone, is a significant post-translational modification in eukaryotic cells and plays important roles in defining the properties of glycoproteins, including biological activity, immunogenicity, pharmacokinetics, solubility, and protease resistance (Rudd et al. 2001; Mitra et al. 2006). Since it is important to understand the influence of glycosylation on the pharmacokinetic behaviour of rhGM-CSF, we have studied glycosylated and non-glycosylated rhGM-CSF to gain insight into how the native glycosylated human protein might behave.
was produced by recombinant CHO.K1 cells adapted to grow in suspension using
spinner flasks as described in Materials and Methods.
The maximum viable cell density was 2.6 x 106 cells/mL
which was reached after 6 days, and the GM-CSF concentration was in
the range 1,000 - 3,000 ng/mL. The ammonium concentration in the medium
The purification of the secreted rhGM-CSF was achieved by immunoaffinity chromatography employing a monoclonal antibody (M7E10). Preparations were judged to be more than 95% pure, based on densitometry of SDS-PAGE protein bands. They exhibited significant molecular weight microheterogeneity with multiple closely-spaced bands of apparent molecular masses between 18-32 kDa (Figure 1), in agreement with previous results from our laboratory (Forno et al. 2004).
The pharmacokinetic profiles of glycosylated and non-glycosylated rhGM-CSF were determined following ip or iv administration of a single dose of protein as described below.
Intraperitoneal administration. Figure 2 shows the mean plasma concentration of rhGM-CSF following ip administration in mice. There was no apparent difference in the rhGM-CSF level between males and females (data not shown). The maximal level was 6.4-fold higher for non-glycosylated rhGM-CSF and it was reached 20 min post-injection, whereas the concentration of glycosylated rhGM-CSF continued rising until 30 min after injection. The Cmax values were 0.018 ± 0.001 nmol/mL and 0.0028 ± 0.0005 ng/mL for non-glycosylated and glycosylated rhGM-CSF, respectively.
After the drug absorption phase, the elimination pattern for each protein was characterized by an initial post-absorption phase followed by an elimination phase as it is described by Shargel et al. (2005), and analyzed in terms of its circulating half-life. The glycosylated form exhibited a significantly longer t1/2 value than the non-glycosylated protein for both post-absorption and elimination phases. Thus, the glycosylated rhGM-CSF showed a post-absorption phase (t1/2 α) of 60 min, followed by a half-life of elimination phase (t1/2 β) of 1,500 min. This slow decline in the level of the glycosylated protein in blood was consistent with its low clearance value (0.09 mL/min). For the non‑glycosylated protein, t1/2 α and t1/2 β values were around 6 times lower than those for the glycosylated rhGM-CSF (Table 1), reflecting a faster clearance for the E. coli-derived rhGM-CSF (0.26 mL/min).
Intravenous administration. Figure 3 shows the plasma concentration of rhGM-CSF following iv administration in rats. There was no apparent difference in the rhGM-CSF level between males and females (data not shown). The mean plasma concentration of rhGM-CSF declined bi-exponentially after injection, and the concentration of the non-glycosylated and glycosylated rhGM-CSF were similar to basal values at 15 and 30 min post-dose, respectively. Curve fitting and calculation of the A, B, α, and β values allowed us to determine half-lives for both clearance phases in the case of each form of rhGM-CSF, as shown in Table 2.
For t1/2 β, no significant differences were detected between the non-glycosylated and glycosylated forms of rhGM-CSF. In contrast, the non-glycosylated form of rhGM-CSF demonstrated an increased rate of initial distribution, as indicated by its significantly reduced t1/2 α. This value is consistent with a faster clearance for the E. coli-derived rhGM-CSF (2.83 mL/min) as compared to mL/min for the glycosylated protein.
It is often difficult to accurately forecast the pharmacokinetic behaviour of native proteins, even when their structural properties are well characterized. In the case of glycosylated proteins, it is known that this modification frequently affects glomerular filtration, stability, immunogenicity, tissue localization, cell uptake, liver excretion, etc. As a heavily glycosylated member of the haematopoietic cytokine family, reports support the key role of the carbohydrate chains of hGM‑CSF in terms of toxicity (Denzlinger el al. 1993; Dorr, 1993) and immunogenicity (Gribben et al. 1990; Revoltella et al. 1997).
This study explores the clearance of two rhGM-CSF preparations produced by different host cells. We used a mammalian cell expression system to produce glycosylated rhGM-CSF that structurally resembles the natural molecule (Cebon et al. 1990). The glycosylated rhGM-CSF was produced from a recombinant CHO.K1 cell line that was grown in suspension culture. A single immunoaffinity isolation procedure was applied, resulting in a purity of ≥95%. Following purification, the CHO cell-expressed rhGM-CSF was compared to a commercially-available, bacterially-expressed, non-glycosylated version.
When ip administration was studied in mice, we found that the non-glycosylated protein had a more rapid absorption, with an earlier and higher Cmax than the glycosylated protein.
After maximum levels for both rhGM-CSF molecules were reached, a two compartment pharmacokinetic model was followed. Half-lives α and β were longer for the glycosylated protein, in accordance with a decrease in the clearance rate. This pharmacokinetic profile of glycosylated rhGM-CSF resulted in a relatively stable plasma concentration, which could be therapeutically more effective than the high peaks and troughs following injections of a molecule with a relatively short half-life such as the non-glycosylated cytokine. Similar behaviour was found following single subcutaneous administration of yeast-derived rhGM-CSF in patients (Stute et al. 1995).
Following iv administration, we showed that the clearance of the rhGM-CSF was significantly decreased by the addition of carbohydrates. We did not find a significant difference in the terminal phase of the biphasic pharmacokinetics but, in contrast, the t1/2 α value was significantly higher for the glycosylated form.
N-linked and O-linked glycosylation are common modifications of plasma proteins. Although in many instances enzymatic deglycosylation or generation of a non-glycosylated recombinant form results in the maintenance of its in vitro activity, the in vivo behaviour of the protein can be significantly altered. rhGM-CSF exemplifies this general finding, in which a non-glycosylated recombinant protein increases its rate of clearance in mice and rats. The same effect was observed for other glycoproteins used as therapeutics (Chitlaru et al. 2002; Koury, 2003).
Several mechanisms that account for this in vivo behaviour have been suggested. An increase in molecular size can reduce clearance via glomerular filtration. In the case of the glycosylated rhGM-CSF the more acidic glycoforms exhibit a molecular weight of 35,000 Da based on migration on SDS-PAGE larger than that observed for the non-glycosylated form (14,000 Da). This property has been employed to design new therapeutics with improved efficacy. PEGylation increases the plasma half-life of a protein by decreasing the rate of systemic clearance, which reduces the frequency of dosing. Moreover, several PEGylated interferons have gained regulatory approval since 2000 (Walsh, 2003).
Another major factor responsible for the extended elimination half-life of glycoproteins is the presence of sialic acids on the glycans, which inhibit binding to hepatic asialoglycoprotein clearance receptors that have specificity for terminal galactose or N-acetylgalactosamine residues, and which vary markedly among mammals (Park and Baenziger, 2004).
This also has been exploited to create new variants of existing products. For example, Aranesp (darbepoetin alfa) is an analogue of recombinant human erythropoietin that was approved in 2001. It has an increased sialic acid content, due to the introduction of two additional glycosylation sites in the EPO backbone, resulting in a significantly longer half-life than the native molecule (Egrie and Browne, 2001).
The thorough characterization of the glycosylation pattern of the mammalian derived cytokine used in this work showed that it has a high degree of branching where more than 90% of the galactose of the N-glycans capped with sialic acid residues. Microheterogeneity of the protein structure of rhGM-CSF by translational glycosylation processing results in differences in the degree of sialylation and different branching of the carbohydrate moieties, which give rise to about 16 isoforms with different pI values due to the presence of predominantly tri-and tetra-antennary N-linked oligosaccharide chains (Forno et al. 2004).
Additionally, receptor-mediated removal appears to be the main pathway of metabolism for many proteins, thus, the increased half-lives of glycosylated proteins might be function of decreased receptor affinity and decreased receptor-mediated elimination from circulation (Koury, 2003).
Our findings show that glycosylation improved the pharmacokinetic parameters of rhGM-CSF. It is likely that the lower clearance of glycosylated rhGM-CSF will confer a clinical advantage over the non-glycosylated product by potentially allowing less frequent dosing in patients.
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