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1.
Mol Omics ; 16(3): 221-230, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32163054

ABSTRACT

Electrospray mass spectrometry (ESI-MS) was used to measure the masses of an intact dimeric monoclonal antibody (Mab) and assess the fucosylation level. The Mab under study was EG2-hFc, a chimeric human-camelid antibody of about 80 kDa (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90). It was obtained from cell culture with and without a fucosylation inhibitor, and treated with EndoS which cleaves between the two core N-acetyl glucosamine (GlcNAc) residues. It is the first time that this combined approach with a unique mass spectrometer was used to measure 146 Da differences as part of a large intact dimeric antibody. Results showed that in the dimer, both heavy chains were fucosylated on the core GlcNAc of the Fc Asn site equivalent to Asn297. In the presence of the fucosylation inhibitor, fucosylation was lost on both subunits. Following reduction, monomers were analyzed and the masses obtained corroborated the dimer results. Dimeric EG2-hFc Mab treated with PNGase F, to deglycosylate the protein, was also measured by MS for mass comparison. In spite of the success of fucosylation level measurements, the experimental masses of deglycosylated dimers and GlcNAc-Fuc bearing dimers did not correspond to masses of our sequence of reference (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90; ; ), which prompted experiments to determine the protein backbone sequence. Digest mixtures from trypsin, GluC, as well as trypsin + GluC proteolysis were analyzed by matrix-assisted laser desorption/ionization (MALDI) MS and MS/MS. A few variations were found relative to the reference sequence, which are discussed in detail herein. These measurements allowed us to build a new "experimental" sequence for the EG2-hFc samples investigated in this work, although there are still ambiguities to be resolved in this new sequence. MALDI-MS/MS also confirmed the fucosylation pattern in the Fc tryptic peptide EEQYNSTYR.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Fucose/metabolism , Acetylglucosamine/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , CHO Cells , Camelidae , Cricetulus , Glycosylation , Humans , Spectrometry, Mass, Electrospray Ionization
2.
J Biotechnol ; 324S: 100015, 2020.
Article in English | MEDLINE | ID: mdl-34154738

ABSTRACT

Core fucosylation of an Fc N-linked glycan affects antibody effector functions, as the absence of fucose increases the antibody dependent cell cytotoxicity (ADCC) response with increased binding to the Fcγ receptors. The work presented here compares two different approaches to incrementally reduce core fucosylation of a camelid heavy chain antibody, EG2-hFc expressed in CHO cells which targets the EGFR receptor. The first method uses a fucosyltransferase (FUT) inhibitor, 2- fluoro peracetylated fucose (2FF), which was added to cell cultures expressing the EG2-hFc antibody in increasing concentrations up to 50µM. At this concentration there was no observed effect on cell growth. Glycan analysis was performed on antibodies collected from culture samples using HILIC-HPLC. The inhibitor reduced total fucosylation from 80% to 17.5% at 20µM 2FF. The second method involved transfecting the EG2-hFc producing cells with a prokaryotic GDP- 6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene in order to deflect the fucose de novo pathway into producing rhamnose which is not incorporated into a glycan. Stable clones from transfected pools were isolated following flow cytometry using the green fluorescent protein (GFP) marker which was co-expressed with the RMD gene. High expressing RMD clones reduced the fucosylation of the antibody glycan to as low as 16%. The addition of 2FF to cultures of these RMD clones reduced the fucosylation level even further to 3% of the antibody glycan. An incremental increase in fucosylation was obtained by step-wise addition of fucose (up to 1 mM) to the RMD cells, in which the fucosylation level increased to a maximum of 87%. We also used ESI-MS to analyze the fucosylation pattern of EG2-hFc with addition of increasing concentrations of 2FF. This showed that 2FF inhibits the addition of fucose in a concentration- dependent and specific manner with the inhibition of fucose occurring one fucose at a time. Control cultures showed the presence of a predominant peak indicating two fucose moieties per antibody. As the 2FF inhibitor concentration was increased peaks corresponding to one fucose per antibody and non-fucosylated antibody predominated with a gradual decrease of the 2 fucose peak to insignificance at 15 µM 2FF.


Subject(s)
Antibodies, Monoclonal , Fucose , Animals , CHO Cells , Cricetinae , Cricetulus , Glycosylation
3.
Immunol Cell Biol ; 97(6): 526-537, 2019 07.
Article in English | MEDLINE | ID: mdl-30680791

ABSTRACT

Human IgG1 and IgG3 antibodies (Abs) can mediate Ab-dependent cellular cytotoxicity (ADCC), and engineering of the Ab Fc (point mutation; defucosylation) has been shown to affect ADCC by modulating affinity for FcRγIIIa. In the absence of a CH 1 domain, many camelid heavy-chain Abs (HCAbs) naturally bear very long and flexible hinge regions connecting their VH H and CH 2 domains. To better understand the influence of hinge length and structure on HCAb ADCC, we produced a series of hinge-engineered epidermal growth factor receptor (EGFR)-specific chimeric camelid VH H-human Fc Abs and characterized their affinities for recombinant EGFR and FcRγIIIa, their binding to EGFR-positive tumor cells, and their ability to elicit ADCC. In the case of one chimeric HCAb (EG2-hFc), we found that variants bearing longer hinges (IgG3 or camelid hinge regions) showed dramatically improved ADCC in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced CH 2 fucosylation. Conversely, an EG2-hFc variant bearing a truncated human IgG1 upper hinge region failed to elicit ADCC. However, there was no consistent association between hinge length and ADCC for four similarly engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC of some HCAbs can be modulated simply by varying the length of the Ab hinge. Although this effect appears to be heavily epitope-dependent, this strategy may be useful to consider during the design of VH H-based therapeutic Abs for cancer.


Subject(s)
Adenocarcinoma/therapy , Antibodies, Monoclonal/metabolism , Breast Neoplasms/therapy , Immunotherapy/methods , Recombinant Fusion Proteins/genetics , Adenocarcinoma/immunology , Animals , Antibodies, Monoclonal/genetics , Antibody Affinity , Antibody-Dependent Cell Cytotoxicity , Breast Neoplasms/immunology , Camelidae , Cell Line, Tumor , ErbB Receptors/immunology , ErbB Receptors/metabolism , Female , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Mutation/genetics , Protein Binding , Protein Engineering
4.
Biotechnol Bioeng ; 116(3): 598-609, 2019 03.
Article in English | MEDLINE | ID: mdl-30080931

ABSTRACT

Refocusing of B-cell responses can be achieved by preserving the overall fold of the antigen structure but selectively mutating the undesired antigenic sites with additional N-linked glycosylation motifs for glycan masking the vaccine antigen. We previously reported that glycan-masking recombinant H5 hemagglutinin (rH5HA) antigens on residues 83, 127, and 138 (g127 + g138 or g83 + g127 + 138 rH5HA) elicited broader neutralizing antibodies and protection against heterologous clades/subclades of high pathogenic avian influenza H5N1 viruses. In this study, we engineered the stably expressing Chinese hamster ovary (CHO) cell clones for producing the glycan-masking g127 + g138 and g83 + g127 + g138 rH5HA antigens. All of these glycan-masking rH5HA antigens produced in stable CHO cell clones were found to be mostly oligomeric structures. Only the immunization with the glycan-masking g127 + g138 but not g83 + g127 + g138 rH5HA antigens elicited more potent neutralizing antibody titers against four out of five heterologous clades/subclades of H5N1 viral strains. The increased neutralizing antibody titers against these heterologous viral strains were correlated with the increased amounts of stem-binding antibodies, only the glycan-masking g127 + g138 rH5HA antigens can translate into more protection against live viral challenges. The stable CHO cell line-produced glycan-masking g127 + g138 rH5HA can be used for H5N1 subunit vaccine development.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus , Influenza A Virus, H5N1 Subtype , Influenza Vaccines , Protein Engineering/methods , Recombinant Proteins , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/chemistry , Antigens, Viral/genetics , Antigens, Viral/immunology , Antigens, Viral/metabolism , CHO Cells , Cricetinae , Cricetulus , Female , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/chemistry , Influenza Vaccines/genetics , Influenza Vaccines/immunology , Influenza Vaccines/metabolism , Mice , Mice, Inbred BALB C , Polysaccharides/chemistry , Polysaccharides/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism
5.
Antiviral Res ; 146: 213-220, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28947234

ABSTRACT

Humans infected with H7N9 avian influenza viruses can result in severe pneumonia and acute respiratory syndrome with an approximately 40% mortality rate, and there is an urgent need to develop an effective vaccine to reduce its pandemic potential. In this study, we used a novel PELC/CpG adjuvant for recombinant H7HA (rH7HA) subunit vaccine development. After immunizing BALB/c mice intramuscularly, rH7HA proteins formulated in this adjuvant instead of an alum adjuvant elicited higher IgG, hemagglutination-inhibition, and virus neutralizing antibodies in sera; induced higher numbers of H7HA-specific IFN-γ-secreting T cells and antibody secreting cells in spleen; and provided improved protection against live virus challenges. Our results indicate that rH7HA proteins formulated in PELC/CpG adjuvant can induce potent anti-H7N9 immunity that may provide useful information for H7N9 subunit vaccine development.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H7N9 Subtype/immunology , Influenza Vaccines/genetics , Influenza Vaccines/immunology , Oligodeoxyribonucleotides/immunology , Adjuvants, Immunologic , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Immunoglobulin G/blood , Influenza A Virus, H7N9 Subtype/genetics , Influenza Vaccines/administration & dosage , Interferon-gamma/metabolism , Mice , Mice, Inbred BALB C , Neutralization Tests , Oligodeoxyribonucleotides/administration & dosage , Orthomyxoviridae Infections/prevention & control , Recombinant Proteins/immunology , Spleen/immunology , T-Lymphocytes/immunology , Vaccination , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology
6.
J Ind Microbiol Biotechnol ; 44(7): 1005-1020, 2017 07.
Article in English | MEDLINE | ID: mdl-28285402

ABSTRACT

The impact of cell culture environment on the glycan distribution of a monoclonal antibody (mAb) has been investigated through a combination of experiments and modeling. A newly developed CHO DUXB cell line was cultivated at two levels of initial Glutamine (Gln) concentrations (0, 4 mM) and incubation temperatures of (33 and 37 °C) in batch operation mode. Hypothermia was applied either through the entire culture duration or only during the post-exponential phase. Beyond reducing cell growth and increasing productivity, hypothermia significantly altered the galactosylation index profiles as compared to control conditions. A novel semi-empirical dynamic model was proposed for elucidating the connections between the extracellular cell culture conditions to galactosylation index. The developed model is based on a simplified balance of nucleotides sugars and on the correlation between sugars' levels to the galactosylation index (GI). The model predictions were found to be in a good agreement with the experimental data. The proposed empirical model is expected to be useful for controlling the glycoprofiles by manipulating culture conditions.


Subject(s)
Antibodies, Monoclonal/metabolism , Cold Temperature , Animals , CHO Cells , Camelidae , Cell Culture Techniques , Cell Proliferation , Cells, Cultured , Cricetulus , Glutamine/metabolism , Glycosylation , Models, Biological , Polysaccharides/metabolism
7.
Eur J Pharm Sci ; 96: 428-439, 2017 Jan 01.
Article in English | MEDLINE | ID: mdl-27721040

ABSTRACT

Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity are well known. However, little is understood about the effect that these modifications have on binding to the high affinity FcγRI receptor. This study analyzed the effect of variable N-glycosylation on a human-llama hybrid mAb (EG2-hFc, 80kDa) binding to FcγRI including a comparison to a full-sized IgG1 (DP-12, 150kDa). This was achieved by the addition of three glycosylation inhibitors (swainsonine, castanospermine, and kifunensine) independently to Chinese hamster ovary (CHO) cell cultures to generate hybrid and high mannose glycan structures. Biophysical analysis by circular dichroism, dynamic light scattering and analytical ultra-centrifugation confirmed that the solution-behaviour of the mAbs remained constant over multiple concentrations and glycan treatments. However, changes were observed when studying the interaction of FcγRI with variously glycosylated mAbs. Both mAbs were observed to have a decreased binding affinity upon treatment with swainsonine which produced hybrid glycans. Following de-glycosylation the binding affinity for EG2-hFc was only marginally reduced (6-fold) compared to a drastic (118-fold) decrease for DP-12. In summary, our data suggest that the relatively low molecular weight of chimeric EG2-hFc may contribute to its enhanced stability against glycan changes making it a highly suitable mAb candidate for therapeutic applications.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Receptors, IgG/metabolism , Animals , Antibodies, Monoclonal/chemistry , CHO Cells , Camelids, New World , Cricetinae , Cricetulus , Glycosylation/drug effects , Humans , Protein Binding/drug effects , Protein Binding/physiology , Protein Structure, Tertiary , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/chemistry
8.
J Biotechnol ; 233: 129-42, 2016 Sep 10.
Article in English | MEDLINE | ID: mdl-27165505

ABSTRACT

Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth and productivity of two recombinant CHO cell lines (CHO-Luc and CHO-hFcEG2) equivalent to or greater than YE-supplemented media. Autolysis of the yeast lysate was not necessary for full activity, suggesting that the active components are present in untreated yeast cells. A bioactive fraction (3KF) of the yeast lysate was isolated from the permeate using a 3kDa molecular weight cut-off (MWCO) filter. Supplementation of this 3KF fraction into the base media supported growth of CHO-Luc cells over eight passages equivalent to YE-supplemented media. The 3KF fraction was fractionated further by a cation exchange spin column using a stepwise pH elution. Metabolomic analysis of a bioactive fraction isolated at high pH identified several arginine and lysine-containing peptides as well as two polyamines, spermine and spermidine, with 3.5× and 4.5× higher levels compared to a fraction showing no bioactivity. The addition of a mixture of polyamines and their precursors (putrescine, spermine, spermidine, ornithine and citrulline) as well as increasing the concentration of some of the components of the original base medium resulted in a chemically-defined (CD) formulation that produced an equivalent viable cell density (VCD) and productivity of the CHO-Luc cells as the YE-supplemented medium. The VCD of the CHO-hFcEG2 culture in the CD medium was 1.9× greater and with equivalent productivity to the YE-supplemented media.


Subject(s)
Biological Products , CHO Cells , Cell Culture Techniques/methods , Culture Media, Conditioned , Saccharomyces cerevisiae/chemistry , Animals , Biological Products/chemistry , Biological Products/pharmacology , CHO Cells/cytology , CHO Cells/drug effects , CHO Cells/metabolism , Cricetinae , Cricetulus , Culture Media, Conditioned/chemistry , Culture Media, Conditioned/pharmacology , Saccharomyces cerevisiae/metabolism
9.
J Virol ; 90(13): 6085-6096, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27099319

ABSTRACT

UNLABELLED: Influenza virus hemagglutinin (HA) N-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response. Considered essential for controlling innate and adaptive immune responses against influenza virus infections, dendritic cells (DCs) trigger proinflammatory and adaptive immune responses in hosts. In this study, we engineered Chinese hamster ovary (CHO) cell lines expressing recombinant HA from pandemic H1, H5, and H7 influenza viruses. rH1HA, rH5HA, and rH7HA were obtained as wild-type proteins or in the presence of kifunensine (KIF) or further with endo-ß-N-acetylglucosaminidase-treated KIF (KIF+E) to generate single-N-acetylglucosamine (GlcNAc) N-glycans consisting of (i) terminally sialylated complex-type N-glycans, (ii) high-mannose-type N-glycans, and (iii) single-GlcNAc-type N-glycans. Our results show that high-mannose-type and single-GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70, and hIL-10 production in human DCs. Significantly higher HLA-DR, CD40, CD83, and CD86 expression levels, as well reduced endocytotic capacity in human DCs, were noted in the high-mannose-type rH1HA and single-GlcNAc-type rH1HA groups than in the complex-type N-glycan rH1HA group. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). The high-mannose-type or single-GlcNAc-type N-glycans of both avian and human HA types are more stimulatory than the complex-type N-glycans. HA-stimulated DC activation was accomplished partially through a mannose receptor(s). These results provide more understanding of the contribution of glycosylation of viral proteins to the immune responses and may have implications for vaccine development. IMPORTANCE: Influenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role in the link between innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5HA, and rH7HA proteins containing distinct N-glycan patterns. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human DCs. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). High-mannose-type and single-GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering mouse and human DC activation and maturation. We believe these results provide some useful information for influenza vaccine development regarding how influenza virus HA proteins with different types of N-glycans activate DCs.


Subject(s)
Dendritic Cells/immunology , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Alkaloids/pharmacology , Animals , Antigens, CD/genetics , B7-2 Antigen/genetics , Birds , CD40 Antigens/genetics , CHO Cells , Cricetinae , Cricetulus , Dendritic Cells/physiology , HLA-DR Antigens/genetics , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Immunoglobulins/genetics , Influenza A Virus, H1N1 Subtype/chemistry , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/chemistry , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H7N9 Subtype/chemistry , Influenza A Virus, H7N9 Subtype/genetics , Influenza in Birds/virology , Influenza, Human/virology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-12/genetics , Interleukin-12/immunology , Interleukin-12 Subunit p40/genetics , Interleukin-12 Subunit p40/immunology , Membrane Glycoproteins/genetics , Pandemics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , CD83 Antigen
10.
J Biotechnol ; 214: 105-12, 2015 Nov 20.
Article in English | MEDLINE | ID: mdl-26387447

ABSTRACT

Glycosylation is a critical quality attribute of many therapeutic proteins, particularly monoclonal antibodies (MAbs). Nucleotide-sugar precursors supplemented to growth medium to affect the substrate supply chain of glycosylation has yielded promising but varied results for affecting glycosylation. Glucosamine (GlcN), a precursor for N-acetylglucosamine (GlcNAc), is a major component of mammalian glycans. The supplementation of GlcN to CHO cells stably-expressing a chimeric heavy-chain monoclonal antibody, EG2-hFc, reduces the complexity of glycans to favour G0 glycoforms, while also negatively impacting cell growth. Although several researchers have examined the supplementation of glucosamine, no clear explanation of its impact on cell growth has been forthcoming. In this work, the glucosamine metabolism is examined. We identified the acetylation of GlcN to produce GlcNAc to be the most likely cause for the negative impact on growth due to the depletion of intracellular acetyl-CoA pools in the cytosol. By supplementing GlcNAc in lieu of GlcN to CHO cells producing EG2-hFc, we achieve the same shift in glycan complexity with marginal impacts on the cell growth and protein production.


Subject(s)
Acetylglucosamine/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Culture Media/metabolism , Glucose/metabolism , Nucleotides/metabolism , Animals , Antibodies, Monoclonal/analysis , CHO Cells , Cricetinae , Cricetulus , Culture Media/chemistry , Glucosamine/metabolism , Glycosylation
11.
MAbs ; 7(3): 571-83, 2015.
Article in English | MEDLINE | ID: mdl-25875452

ABSTRACT

The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and ß1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.


Subject(s)
Antibodies, Monoclonal , Immunoglobulin G , Mutation, Missense , Sialic Acids/metabolism , Amino Acid Substitution , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Antigens, CD/biosynthesis , Antigens, CD/genetics , CHO Cells , Cricetinae , Cricetulus , Humans , Immunoglobulin G/biosynthesis , Immunoglobulin G/genetics , N-Acetyllactosamine Synthase/biosynthesis , N-Acetyllactosamine Synthase/genetics , Sialyltransferases/biosynthesis , Sialyltransferases/genetics
12.
Curr Opin Biotechnol ; 30: 107-12, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25005678

ABSTRACT

Non-human mammalian cells such as CHO have been used predominantly for the production of biopharmaceuticals including monoclonal antibodies (Mabs). Although the glycosylation profile of these products is 'human-like' there is still the possibility of immunogenic epitopes such as α-Gal and Neu5Gc. Human cell lines have now been designed for high productivity of recombinant proteins and ensuring authentic glycosylation patterns. The control of glycosylation on such proteins is important for the efficacy of recombinant biopharmaceuticals as well as the immunogenic properties of viral vaccines such as influenza. We are now starting to understand some of the relationships between the structure of glycans and the function bestowed on the associated protein. This has promoted cell culture technologies for the targeted control of glycosylation to produce pre-determined glycan profiles of secreted products.


Subject(s)
Cell Line , Glycosylation , Recombinant Proteins/metabolism , Animals , CHO Cells , Cell Culture Techniques , Cricetulus , Mammals , Polysaccharides/chemistry , Polysaccharides/metabolism , Recombinant Proteins/chemistry
13.
J Biotechnol ; 186: 98-109, 2014 Sep 30.
Article in English | MEDLINE | ID: mdl-25014402

ABSTRACT

The impact of operating conditions on the glycosylation pattern of humanized camelid monoclonal antibody, EG2-hFc produced by Chinese hamster ovary (CHO) cells has been evaluated by a combination of experiments and modeling. Cells were cultivated under different levels of glucose and glutamine concentrations with the goal of investigating the effect of nutrient depletion levels and ammonia build up on the cell growth and the glycoprofiles of the monoclonal antibody (Mab). The effect of average pH reduction on glycosylation level during the entire culture time or during a specific time span was also investigated. The relative abundance of glycan structures was quantified by hydrophilic interaction liquid chromatography (HILIC) and the galactosylation index (GI) and the sialylation index (SI) were determined. Lower initial concentrations of glutamine resulted in lower glucose consumption and lower cell yield but increased GI and SI levels when compared to cultures started with higher initial glutamine levels. Similarly, reducing the average pH of culture resulted in lower growth but higher SI and GI levels. These findings indicate that there is a tradeoff between cell growth, resulting Mab productivity and the achievement of desirable higher glycosylation levels. A dynamic model, based on a metabolic flux analysis (MFA), is proposed to describe the metabolism of nutrients, cell growth and Mab productivity. Finally, existing software (GLYCOVIS) that describes the glycosylation pathways was used to illustrate the impact of extracellular species on the glycoprofiles.


Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/metabolism , Cell Culture Techniques/methods , Glucose/metabolism , Models, Biological , Animals , CHO Cells , Cricetinae , Cricetulus , Extracellular Space/metabolism , Glutamine/metabolism , Glycosylation , Hydrogen-Ion Concentration
14.
Biotechnol Prog ; 30(3): 584-93, 2014.
Article in English | MEDLINE | ID: mdl-24846804

ABSTRACT

Peptide hydrolysate supplements in mammalian cell cultures provide enhanced growth and productivity. The objective of this study was to compare the bioactivity of ten different commercially available hydrolysates from plant, microbial, and animal sources. The peptide hydrolysates were tested as supplements to cultures of Chinese hamster ovary (CHO) cells that produce human beta interferon (ß-IFN). A soy hydrolysate was shown to support high cell growth but not protein productivity compared to an animal component hydrolysate (Primatone RL). On the other hand, a yeast hydrolysate showed lower cell growth, but comparable productivity of the recombinant protein. Glycosylation analysis showed that the glycan profile of ß-IFN produced in yeast hydrolysate supplemented cultures was equivalent to that from Primatone RL-supplemented cultures. Fractionation of the yeast hydrolysate and Primatone RL produced a similar protein-assayed pattern except for one extra peak at around 1 kDa in the Primatone RL profile. A fraction taken at a molecular weight range of 1.5-1.7 kDa showed the highest growth promoting activity in both samples. However, four other fractions in yeast hydrolysate and two in Primatone RL at lower molecular weights showed some growth promoting activity. In conclusion, the yeast hydrolysates provided a good alternative to the animal sourced Primatone RL for high productivity of ß-IFN from CHO cells with equivalent glycosylation.


Subject(s)
Cell Proliferation/drug effects , Culture Media, Serum-Free/chemistry , Peptides/pharmacology , Protein Hydrolysates/pharmacology , Animals , CHO Cells/drug effects , Cricetulus , Glycosylation , Humans , Interferon-beta/biosynthesis , Peptides/chemistry , Protein Hydrolysates/chemistry , Recombinant Proteins/biosynthesis , Soybean Proteins/chemistry , Soybean Proteins/pharmacology
15.
Biotechnol Bioeng ; 111(6): 1139-49, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24449405

ABSTRACT

Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Chromatography, Liquid/methods , Hydrophobic and Hydrophilic Interactions , Single-Chain Antibodies/isolation & purification , Antibodies, Monoclonal/genetics , Chromatography, Affinity/methods , Humans , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Single-Chain Antibodies/genetics
16.
J Biotechnol ; 170: 17-27, 2014 Jan 20.
Article in English | MEDLINE | ID: mdl-24286971

ABSTRACT

The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25mM) of cultures established at high density (>10(6)ml(-1)) over 24h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation of glucose for the full 24h resulted in a 45% non-glycosylated Mab fraction. Analysis of steady state levels of intracellular lipid-linked oligosaccharides (LLOs) showed that under glucose limitation there was a reduction in the amount of full length LLO (Glc3Man9GlcNac2), with a concomitant increase in the smaller mannosyl-glycans (Man2-5GlcNAc2). Glycan microheterogeneity was quantified by galactosylation and sialylation indices (GI and SI) which showed a direct correlation to the cell specific glucose uptake. The GI increased to 0.83 following media supplementation with a cocktail of uridine, manganese and galactose. This is significantly higher than for a fully humanized antibody (DP12) produced under the similar conditions or for similar antibodies reported in the literature. The high GI of the chimeric antibody (EG2) may be due to its low molecular weight and unusual structure. These findings are important in relation to the low substrate that may occur in fed-batch cultures for Mab production.


Subject(s)
Antibodies, Monoclonal/metabolism , Glucose/metabolism , Lipopolysaccharides/metabolism , Animals , CHO Cells , Cricetulus , Culture Media , Galactose , Glycosylation , Lipopolysaccharides/chemistry , Recombinant Proteins/metabolism
17.
Methods Mol Biol ; 1104: 169-92, 2014.
Article in English | MEDLINE | ID: mdl-24297416

ABSTRACT

The accurate determination of cell growth and viability is pivotal to monitoring a bioprocess. Direct methods to determine the cell growth and/or viability in a bioprocess include microscopic counting, electronic particle counting, image analysis, in situ biomass monitoring, and dieletrophoretic cytometry. These methods work most simply when a fixed volume sample can be taken from a suspension culture. Manual microscopic counting is laborious but affords the advantage of allowing cell viability to be determined if a suitable dye is included. Electronic particle counting is a rapid total cell count method for replicate samples, but some data distortion may occur if the sample has significant cell debris or cell aggregates. Image analysis based on the use of digital camera images acquired through a microscope has advanced rapidly with the availability of several commercially available software packages replacing manual microscopic counting and viability determination. Biomass probes detect cells by their dielectric properties or their internal concentration of NADH and can be used as a continuous monitor of the progress of a culture. While the monitoring of cell growth and viability is an integral part of a bioprocess, the monitoring of apoptosis induction is also becoming more and more important in bioprocess control to increase volumetric productivity by extending bioprocess duration. Different fluorescent assays allow for the detection of apoptotic characteristics in a cell sample.Indirect methods of cell determination involve the chemical analysis of a culture component or a measure of metabolic activity. These methods are most useful when it is difficult to obtain intact cell samples. However, the relationship between these parameters and the cell number may not be linear through the phases of a cell culture. The determination of nucleic acid (DNA) or total protein can be used as an estimate of biomass, while the depletion of glucose from the media can be used as an estimate of cellular activity. The state of cellular viability may be measured by the release of an enzyme such as lactate dehydrogenase or more directly from the intracellular adenylate energy charge from cell lysates. Alternatively, radioactive techniques may be used for an accurate determination of cellular protein synthesis.


Subject(s)
Apoptosis , Cell Proliferation , Molecular Biology/methods , Animals , Biomass , Cell Count/instrumentation , Cell Survival , Colony-Forming Units Assay , Flow Cytometry/methods , Glucose/analysis , Image Processing, Computer-Assisted , In Situ Nick-End Labeling , L-Lactate Dehydrogenase/analysis , L-Lactate Dehydrogenase/metabolism , NAD/analysis , NAD/metabolism , Online Systems , Proteins/analysis , Proteins/genetics , Proteins/metabolism
18.
Drug Des Devel Ther ; 2: 105-14, 2009 Feb 06.
Article in English | MEDLINE | ID: mdl-19920898

ABSTRACT

VB4-845 is a scFv-Pseudomonas exotoxin A fusion construct that targets epithelial cell adhesion molecule (EpCAM). A phase I trial was conducted to determine the maximum tolerated dose (MTD) of VB4-845 when administered as weekly intratumoral (IT) injections to patients with squamous cell carcinoma of the head and neck (SCCHN). Secondary objectives included the evaluation of the safety, tolerability, pharmacokinetic profile, and immunogenicity, and a preliminary assessment of tumor response. Twenty patients with advanced, recurrent SCCHN were treated weekly for four weeks in ascending dose cohorts of 100, 200, 330, 500, 700, and 930 microg. The MTD was established as 930 microg with a dose limiting toxicity of elevated liver enzymes in two of five patients. VB4-845 therapy was well tolerated with common treatment-related adverse events of injection site reactions, fever, gastrointestinal disorders, and elevated liver enzyme levels. All patients developed antibodies to VB4-845 by the end of the study, but only seven patients had neutralizing antibodies. Preliminary efficacy data found 87.5% of EpCAM-positive patients had a positive response to VB4-845 therapy. Noninjected dermal metastases were also resolved in one patient. VB4-845 IT therapy is safe and feasible and warrants further clinical evaluation for the treatment of SCCHN.

19.
Cancer Biother Radiopharm ; 24(4): 477-87, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19694583

ABSTRACT

VB4-845 is a recombinant immunotoxin that is comprised of a truncated form of Pseudomonas exotoxin A (ETA) genetically-linked to a humanized scFv fragment, (4D5MOCB), specific to epithelial cell adhesion molecule (EpCAM). EpCAM is overexpressed on a wide variety of human tumors and thus represents a suitable target antigen for immunotoxin therapy. Preclinical studies were used to evaluate the benefit of locoregional administration of an ETA-based immunotoxin versus systemic delivery. Repeated subcutaneous (s.c.) administration of VB4-845 (up to 77.8 microg/kg) in rats resulted in minimal adverse effects, except for injection-site reactions, while repeated systemic administration elicited symptoms consistent with vascular leak syndrome. S.c. weekly doses of the drug in cynomolgus monkeys resulted in minimal adverse effects limited to injection-site reactions and a transient elevation of liver enzymes in 1 animal. Toxicokinetics showed rapid clearance of the drug, with the development of an immune response by day 14 following repeated injections. These results argue that the local administration of VB4-845 has advantages with respect to safety over systemic administration and may be an effective alternative method for targeting those cancers that are amenable to local routes of administration.


Subject(s)
ADP Ribose Transferases/administration & dosage , Antigens, Neoplasm/immunology , Bacterial Toxins/administration & dosage , Cell Adhesion Molecules/immunology , Exotoxins/administration & dosage , Immunotoxins/administration & dosage , Neoplasms/drug therapy , Neoplasms/immunology , Virulence Factors/administration & dosage , Animals , Cell Line, Tumor , Epithelial Cell Adhesion Molecule , Haplorhini , Humans , Immunotoxins/immunology , Immunotoxins/pharmacokinetics , Immunotoxins/toxicity , Infusions, Subcutaneous , Rats , Rats, Sprague-Dawley , Pseudomonas aeruginosa Exotoxin A
20.
Med Oncol ; 26(3): 257-64, 2009.
Article in English | MEDLINE | ID: mdl-19016010

ABSTRACT

VB4-845 is a novel recombinant fusion protein that targets the epithelial cellular adhesion molecule (EpCAM). This initial clinical trial was conducted to determine the maximum tolerated dose of intratumoral injections in patients with advanced squamous cell carcinoma of the head and neck and to assess pharmacokinetics and immunogenicity. Twenty-four patients with advanced, recurrent squamous cell carcinoma of the head and neck received two cycles of five daily intratumoral VB4-845 injections of 20, 40, 80, 130, 200, or 280 microg. The maximum tolerated dose was established to be 280 microg administered daily for 5 days. Common adverse events were pain due to intratumoral injection and reversibly elevated liver enzymes. Of the 24 patients, 15 had detectable blood levels with a mean drug half-life of 4.0 +/- 0.3 h. VB4-845 reduced or stabilized tumors in 71.4% of epithelial cell adhesion molecule-positive patients. VB4-845 intratumoral injection therapy was well tolerated and feasible.


Subject(s)
Carcinoma, Squamous Cell/drug therapy , Cell Adhesion Molecules/antagonists & inhibitors , Head and Neck Neoplasms/drug therapy , Recombinant Fusion Proteins/administration & dosage , Aged , Antigens, Neoplasm , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Carcinoma, Squamous Cell/metabolism , Drug Administration Schedule , Epithelial Cell Adhesion Molecule , Female , Head and Neck Neoplasms/metabolism , Humans , Injections, Intralesional , Male , Middle Aged , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/pharmacokinetics
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