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1.
J Pharm Sci ; 110(2): 771-784, 2021 02.
Article in English | MEDLINE | ID: mdl-33035537

ABSTRACT

Commercial specifications for a new biotherapeutic product are a critical component of the product's overall control strategy that ensures safety and efficacy. This paper describes strategies for setting commercial specifications as proposed by a consortium of industry development scientists. The specifications for some attributes are guided by compendia and regulatory guidance. For other product quality attributes (PQAs), product knowledge and the understanding of attribute criticality built throughout product development should drive specification setting. The foundation of PQA knowledge is an understanding of potential patient impact through an assessment of potency, PK, immunogenicity and safety. In addition to PQA knowledge, the ability of the manufacturing process to consistently meet specifications, typically assessed through statistical analyses, is an important consideration in the specification-setting process. Setting acceptance criteria that are unnecessarily narrow can impact the ability to supply product or prohibit consideration of future convenient dosage forms. Patient-centric specifications enable appropriate control over higher risk PQAs to ensure product quality for the patient, and flexibility for lower risk PQAs for a sustainable supply chain. This paper captures common strategic approaches for setting specifications for standard biotherapeutic products such as monoclonal antibodies and includes considerations for ensuring specifications are patient centric.


Subject(s)
Antibodies, Monoclonal , Patient-Centered Care , Humans
2.
J Pharm Sci ; 108(4): 1442-1452, 2019 04.
Article in English | MEDLINE | ID: mdl-30528942

ABSTRACT

Early-phase specifications are established to ensure that materials used in clinical studies have appropriate product quality, reducing the risk of harm to patients. Currently, guidance is available for specification setting practices at commercial phase. With very limited data and manufacturing experience available, it is not possible to fully align to these expectations at the start of clinical trials. A survey was performed among 19 biopharmaceutical companies to gather information about the current practices for setting specifications in early-phase development. The results indicate that most companies develop platform approaches to support setting specifications at the first-in-human clinical trial stage of development. Based on shared learning across multiple companies, example specification approaches for monoclonal antibodies and antibody-drug conjugates are included. General principles of the example specifications can also be applied to other protein therapeutics and vaccines. Strategies for justification of acceptance criteria are described, along with discussion of considerations for some specific tests. Options for use of non-numerical acceptance criteria are also discussed. While specifications for each molecule must be set considering available molecule-specific information, the presented information leverages shared learning from multiple companies, to provide guidance for early phase specification setting strategies.


Subject(s)
Antibodies, Monoclonal/chemistry , Clinical Trials, Phase I as Topic/standards , Drug Development/standards , Immunoconjugates/chemistry , Technology, Pharmaceutical/standards , Drug Industry/standards , Drug Industry/statistics & numerical data , Humans , Quality Control , Risk Assessment , Surveys and Questionnaires/statistics & numerical data
3.
J Cell Sci ; 127(Pt 23): 5014-26, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-25271059

ABSTRACT

Protein N-glycosylation is a common post-translational modification that produces a complex array of branched glycan structures. The levels of branching, or antennarity, give rise to differential biological activities for single glycoproteins. However, the precise mechanism controlling the glycan branching and glycosylation network is unknown. Here, we constructed quantitative mathematical models of N-linked glycosylation that predicted new control points for glycan branching. Galactosyltransferase, which acts on N-acetylglucosamine residues, was unexpectedly found to control metabolic flux through the glycosylation pathway and the level of final antennarity of nascent protein produced in the Golgi network. To further investigate the biological consequences of glycan branching in nascent proteins, we glycoengineered a series of mammalian cells overexpressing human chorionic gonadotropin (hCG). We identified a mechanism in which galactosyltransferase 4 isoform regulated N-glycan branching on the nascent protein, subsequently controlling biological activity in an in vivo model of hCG activity. We found that galactosyltransferase 4 is a major control point for glycan branching decisions taken in the Golgi of the cell, which might ultimately control the biological activity of nascent glycoprotein.


Subject(s)
Chorionic Gonadotropin/metabolism , N-Acetylglucosaminyltransferases/metabolism , Protein Processing, Post-Translational , Acetylglucosamine/metabolism , Animals , CHO Cells , Chorionic Gonadotropin/chemistry , Chorionic Gonadotropin/genetics , Chorionic Gonadotropin/pharmacology , Computer Simulation , Cricetulus , Glycosylation , HEK293 Cells , Humans , Isoenzymes , Kinetics , Male , Models, Biological , Models, Molecular , N-Acetylglucosaminyltransferases/chemistry , N-Acetylglucosaminyltransferases/genetics , Protein Conformation , Rats , Seminal Vesicles/drug effects , Seminal Vesicles/growth & development , Structure-Activity Relationship , Transfection
4.
BMC Biotechnol ; 11: 95, 2011 Oct 18.
Article in English | MEDLINE | ID: mdl-22008152

ABSTRACT

BACKGROUND: The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. RESULTS: Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. CONCLUSIONS: The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.


Subject(s)
Cell Culture Techniques/methods , Chorionic Gonadotropin/metabolism , Glucose/metabolism , Glutamine/deficiency , Glycolysis , Recombinant Proteins/metabolism , Uridine Diphosphate N-Acetylgalactosamine/analysis , Animals , CHO Cells , Cricetinae , Cricetulus , Female , Glutamine/analysis , Glycosylation , Humans , Hydrogen-Ion Concentration , Nucleotides/metabolism , Polysaccharides/metabolism , Uridine Diphosphate N-Acetylgalactosamine/biosynthesis
5.
Biochemistry ; 42(13): 3979-88, 2003 Apr 08.
Article in English | MEDLINE | ID: mdl-12667089

ABSTRACT

In the past decade, several outbreaks of cholera have been reported to be caused by Vibrio cholerae O139, a strain which differs from the more common O1 strain in that the former is encapsulated. The hexasaccharide repeating subunit has been isolated from the V. cholerae O139 capsular polysaccharide by digestion with a recently discovered polysaccharide lyase derived from a bacteriophage specific for this serogroup. It specifically cleaves at a single position of the 4-linked galacturonic acid producing an unsaturated sugar product in quantities for conformational studies by (1)H and (13)C NMR spectroscopy. We report conformational studies on this oligosaccharide by molecular modeling and NMR spectroscopy including nuclear Overhauser effects and residual dipolar coupling of a sample weakly oriented in liquid crystalline solution. The structure contains a tetrasaccharide epitope homologous to the human Lewis(b) blood group antigen, which adopts a relatively well-defined single conformation. Comparison of these results with those of a previously published study of the intact capsular polysaccharide indicates that the conformations of the epitope in the two cases are identical or at least closely similar. Thus, this epitope, which may be essential for the pathogenicity of this V. cholerae strain, is not a "conformational epitope" requiring a certain critical size for antigenicity as has been reported for several other bacterial capsular antigens.


Subject(s)
Epitopes/chemistry , Polysaccharides, Bacterial/chemistry , Vibrio cholerae O139/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Glycosides/chemistry , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , O Antigens/immunology , Polysaccharide-Lyases/metabolism
6.
Anal Biochem ; 303(2): 176-85, 2002 Apr 15.
Article in English | MEDLINE | ID: mdl-11950217

ABSTRACT

A significant problem in structure determination of complex carbohydrates, especially for bacterial polysaccharides, is determination of the absolute configuration of the component monosaccharides. A number of analytical methods have been used for this purpose but, as a result of the wide variety of chemical properties of sugars found in complex polysaccharides, no single method is universally applicable. High-resolution gas chromatography of volatile derivatives with chiral reagents is the most widely used method. Optical activity, although direct and simple, lacks sensitivity generally requiring a large quantity of pure monosaccharide. We report a combination of high-performance anion-exchange chromatography (HPAEC) with combined electrochemical pulsed amperometric detection and in-line detection of optical rotation with an in-line laser polarimeter for analysis of a number of sugars found in complex polysaccharides. We show that application of the method for analysis of capsular polysaccharides of several gram-positive and gram-negative pathogenic bacteria provides useful information simultaneously on carbohydrate composition and the enantiomeric configuration of component sugars.


Subject(s)
Bacteria/chemistry , Chromatography, Ion Exchange/methods , Polysaccharides/analysis , Hydrogen-Ion Concentration , Molecular Conformation
7.
Carbohydr Res ; 337(4): 335-44, 2002 Feb 18.
Article in English | MEDLINE | ID: mdl-11841814

ABSTRACT

NMR spectroscopy can be used to characterize bacterial polysaccharides such as that of Streptococcus pneumoniae type 1 which is a component of the 23-valent pneumococcal vaccine in clinical use. This particular polysaccharide gives NMR spectra with wide lines apparently due to restricted molecular mobility and chain flexibility which leads to rapid dipolar T(2) relaxation limiting the possibility of detailed spectral analysis. Removal of O-acetyl groups found on approximately two thirds of the repeating subunits of pneumococcal type 1 capsule leads to narrower NMR lines facilitating a complete assignment of the 1H and 13C NMR spectra. Degradation of the polysaccharide by periodate oxidation followed by base treatment leads to an oligosaccharide fragment of approximately three repeating trisaccharide units. This oligosaccharide has narrow NMR lines and 1H and 13C assignments very similar to those of the O-deacetylated polysaccharide. In the native polysaccharide, O-acetyl groups are located on the 2- and 3-positions of the 4-linked galacturonic acid residue providing protection against periodate oxidation. Analysis of NOESY spectra combined with molecular modeling of the oligosaccharide shows that flexibility occurs in certain of the saccharide linkages.


Subject(s)
Polysaccharides, Bacterial/chemistry , Streptococcus pneumoniae/chemistry , Bacterial Vaccines/chemistry , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Oxidation-Reduction
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