RESUMO
The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing â¼200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.
Assuntos
Metionina , Racemetionina , Metionina/química , Ácido Edético , Fluxo de Trabalho , Racemetionina/metabolismo , OxirreduçãoRESUMO
One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster ovary (CHO) derived production cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of cells as detected with flow cytometric analysis of intracellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host cell line from which the candidate production cell lines were derived was apoptotic-resistant. This data suggested that unstable cell lines were more prone to apoptosis, which was confirmed by the fact that unstable cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production cell lines at an early stage of the cell line development process, potentially reducing the cost of biotherapeutic development.
Assuntos
Anticorpos Monoclonais/biossíntese , Técnicas de Cultura Celular por Lotes/métodos , Células CHO/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Apoptose , Técnicas de Cultura Celular por Lotes/instrumentação , Reatores Biológicos , Células CHO/citologia , Caspase 3/metabolismo , Separação Celular , Células Clonais/citologia , Células Clonais/metabolismo , Cricetinae , Cricetulus , Citometria de Fluxo , Vetores Genéticos , Instabilidade Genômica , Glutamato-Amônia Ligase/genética , Humanos , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Seleção GenéticaRESUMO
In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide-1-antibody fusion protein (also known as a Glucagon like peptide-1 MIMETIBODY), we have noted that the N-terminal GLP-1 portion of the MIMETIBODY was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality. Product expressed in mouse myeloma host cell lines had a lesser degree of proteolytic degradation and variability in O-linked glycosylation as compared to that expressed in CHO host cell lines. The choice of a specific CHOK1SV derived clone also had an effect on the product quality. In general, molecules that exhibited minimal N-terminal clipping had increased level of O-linked glycosylation in the linker region, giving credence to the hypothesis that O-linked glycosylation acts to protect against proteolytic degradation. Moreover, products with reduced potential for N-terminal clipping had longer in vivo serum half-life. These findings suggest that early monitoring of product quality should be an essential part of production cell line development and therefore, has been incorporated in our process of cell line development for this class of molecules.
Assuntos
Biotecnologia/métodos , Proteínas Recombinantes de Fusão/biossíntese , Animais , Linhagem Celular , Cricetinae , Humanos , CamundongosRESUMO
Therapeutic glycoproteins, for example, antibodies (Abs) and Fc fusion proteins when produced in mammalian cells, such as Chinese hamster ovary (CHO) cells generally exhibit heterogeneity. Both the oligosaccharide moiety and the protein moiety contribute to this phenomenon. Non-enzymatic and enzymatic pathways of protein fragmentation generate heterogeneity in the polypeptide backbone. In the non-enzymatic pathway, physical and chemical events such as light, oxidation, and others can cause the protein moiety to become unstable leading to its fragmentation. Intracellular and secreted proteases are involved in the enzymatic degradation of proteins. This degradative process is modulated by the oligosaccharide moiety of the glycoprotein as well as glycosidases, including sialidases that are secreted in the culture medium. This review focuses on the factors that modulate heterogeneity of the protein moiety especially by the enzymatic methods. Availability of the CHO genome database will facilitate the development of host cell lines with minimal degradative properties.
Assuntos
Proteólise , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Animais , Células CHO , Engenharia Celular , Cricetulus , Glicoproteínas/metabolismo , Controle de Qualidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genéticaRESUMO
Manufacturing cell line development at Centocor involves transfection of antibody genes into host cell lines and isolating primary transfectomas that upon subcloning yield high expressing cell lines for the desired antibody. In an attempt to increase productivity of these cell lines, we set out to identify the rate-limiting step in the process of antibody expression and secretion. For this purpose, 30 antibody expressing cell lines with variable antibody expression levels were analyzed for heavy-chain and light-chain mRNA expression levels. Results suggested that the increase in antibody titer of the subclones (compared to their primary clones) was partly due to an increase in heavy-chain and light-chain mRNA levels; higher expressers were associated with approximately 1.0 x 10(7) and 1.5 x 10(7) copies of heavy-chain and light-chain per 10 nanogram of cDNA, respectively. Generally, the level of light-chain mRNA was higher compared to the level of heavy-chain mRNA in a majority of the cell lines, and the difference in their levels was not due to their differential stability. The data generated from all the cell lines tested in this study suggested that there was a correlation of light-chain and heavy-chain transcript levels to antibody productivity, with the coefficient of correlation being 0.59 for light chain and 0.81 for heavy chain. We conclude that transcription of heavy chain and to a lesser extent light chain could be one of the rate-limiting steps in the antibody expression pathway. Hence, methods that would increase these mRNA levels could be beneficial in the attempt to improve the antibody expression level of production cell lines.
Assuntos
Anticorpos Monoclonais/biossíntese , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , RNA Mensageiro/biossíntese , Animais , Anticorpos Monoclonais/genética , Formação de Anticorpos , Linhagem Celular Tumoral , Dosagem de Genes , Humanos , Hibridomas/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Mieloma Múltiplo , RNA Mensageiro/genéticaRESUMO
Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.