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1.
PDA J Pharm Sci Technol ; 75(1): 24-32, 2021.
Article in English | MEDLINE | ID: mdl-32817319

ABSTRACT

The freezing of biologics has been widely studied from the physical chemistry point of view, for instance in terms of cryo-concentration, excipient crystallization, pH swing, potential protein denaturation, etc. In contrast, considerations on the processing aspects are very limited. For instance, the impact of freezer temperature, container size, freezer load, and freeze chilling capacity on the freezing rate in the most frequent case of freezing in a bottle have not been reported. In this study, the freezing time of either water or buffer solution was measured in various processing conditions. Experimental trials were conducted using containers ranging from 1 to 20 L in two types of freezers: a normal freezer (-30°C set point) and an ultra freezer (-70°C set point). These trials showed that both the container size and the freezer load influenced the freezing times. The current study demonstrated that the use of the well-established Plank model for freezing, coupled with freezer performance characterization, allows the description of the actual freezing kinetics in a very simple and accurate manner. The kinetics can then be modeled to accurately predict both the actual freezer temperature (possibly above the set point) and the freezing time based on freezer load.


Subject(s)
Biological Products , Pharmaceutical Preparations , Freezing , Kinetics , Temperature
2.
Biotechnol Rep (Amst) ; 26: e00465, 2020 Jun.
Article in English | MEDLINE | ID: mdl-32420053

ABSTRACT

For the first time to our knowledge the implementation of a continuous protein A capture process for antibody applications (CoPACaPAnA) embedded in an end-to-end single-use 500 L GMP manufacturing downstream process of a multispecific monoclonal antibody (mAb) using a single-use SMB system was conducted. Throughout the last years, a change concerning the pipelines in pharmaceutical industry could be observed, moving to a more heterogeneous portfolio of antibodies, fusion proteins and nanobodies. Trying to adjust purification processes to these new modalities, a higher degree of flexibility and lower operational and capital expenditure is desired. The implementation of single-use equipment is a favored solution for increasing manufacturing agility and it has been demonstrated that continuous processing can be beneficial concerning processing cost and time. Reducing protein A resin resulted in 59% cost reduction for the protein A step, with additional cost reduction also for the intermediate and polishing step due to usage of disposable technology. The downstream process applied here consisted of three chromatography steps that were all conducted on a single-use SMB system, with the capture step being run in continuous mode while intermediate and polishing was conducted in batch mode. Further, two steps dedicated to virus inactivation/ removal and three filtration steps were performed, yielding around 100 g of drug substance going into clinical phase I testing. Therefore, in this study it has been demonstrated that employing a continuous capture within a GMP single-use downstream processing chain is feasible and worthy of consideration among the biotech industry for future application to modality-diverse pipelines.

3.
Chem Biol ; 12(2): 181-9, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15734645

ABSTRACT

Inhibitors for matrix metalloproteinases (MMPs) are under investigation for the treatment of cancer, arthritis, and cardiovascular disease. Here, we report a class of highly selective MMP-13 inhibitors (pyrimidine dicarboxamides) that exhibit no detectable activity against other MMPs. The high-resolution X-ray structures of three molecules of this series bound to MMP-13 reveal a novel binding mode characterized by the absence of interactions between the inhibitors and the catalytic zinc. The inhibitors bind in the S1' pocket and extend into an additional S1' side pocket, which is unique to MMP-13. We analyze the determinants for selectivity and describe the rational design of improved compounds with low nanomolar affinity.


Subject(s)
Collagenases/chemistry , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemistry , Protease Inhibitors/chemical synthesis , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Drug Design , Matrix Metalloproteinase 13 , Models, Molecular , Molecular Sequence Data , Protease Inhibitors/pharmacology , Protein Structure, Secondary , Water
4.
Bioorg Med Chem Lett ; 14(14): 3715-20, 2004 Jul 16.
Article in English | MEDLINE | ID: mdl-15203149

ABSTRACT

The amidinophenylurea scaffold was earlier shown to provide an excellent template for the synthesis of novel and potent inhibitors of the blood coagulation factor VIIa. In this contribution we describe the structure-based design of potent ligands guided by X-ray crystallography, molecular modeling and docking studies. The design and synthetic efforts were directed towards novel modifications to explore the protease binding region close to the S4 subsite.


Subject(s)
Drug Design , Factor VIIa/antagonists & inhibitors , Fibrinolytic Agents/chemical synthesis , Phenylurea Compounds/chemical synthesis , Binding Sites , Crystallography, X-Ray , Factor VIIa/metabolism , Fibrinolytic Agents/pharmacology , Molecular Structure , Peptide Hydrolases/metabolism , Phenylurea Compounds/pharmacology
5.
Bioorg Med Chem Lett ; 14(11): 2801-5, 2004 Jun 07.
Article in English | MEDLINE | ID: mdl-15125936

ABSTRACT

A series of novel, highly potent, achiral factor Xa inhibitors based on a benzoic acid scaffold and containing a chlorophenethyl moiety directed towards the protease S1 pocket is described. A number of structural features, such as the requirements of the P1, P4 and ester-binding pocket ligands were explored with respect to inhibition of factor Xa. Compound 46 was found to be the most potent compound in a series of antithrombotic secondary assays.


Subject(s)
Benzoates/pharmacology , Factor Xa Inhibitors , Fibrinolytic Agents/chemical synthesis , Benzoates/chemical synthesis , Blood Coagulation Tests , Drug Stability , Fibrinolytic Agents/pharmacology , Humans , Ligands , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Protein Binding , Structure-Activity Relationship
6.
Bioorg Med Chem Lett ; 13(8): 1463-7, 2003 Apr 17.
Article in English | MEDLINE | ID: mdl-12668013

ABSTRACT

Selective inhibition of coagulation factor VIIa has recently gained attraction as interesting approach towards antithrombotic treatment. Using parallel synthesis supported by structure-based design and X-ray crystallography, we were able to identify a novel series of amidinophenylurea derivatives with remarkable affinity for factor VIIa. The most potent compound displays a K(i) value of 23 nM for factor VIIa.


Subject(s)
Factor VIIa/antagonists & inhibitors , Phenylurea Compounds/chemical synthesis , Phenylurea Compounds/pharmacology , Crystallography, X-Ray , Drug Design , Humans , Models, Molecular , Phenylurea Compounds/chemistry , Structure-Activity Relationship
7.
J Med Chem ; 45(14): 2923-41, 2002 Jul 04.
Article in English | MEDLINE | ID: mdl-12086480

ABSTRACT

The family of homodimeric nitric oxide synthases (NOS I-III) catalyzes the generation of the cellular messenger nitric oxide (NO) by oxidation of the substrate L-arginine. The rational design of specific NOS inhibitors is of therapeutic interest in regulating pathological NO levels associated with sepsis, inflammatory, and neurodegenerative diseases. The cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) maximally activates all NOSs and stabilizes enzyme quaternary structure by promoting and stabilizing dimerization. Here, we describe the synthesis and three-dimensional (3D) quantitative structure-activity relationship (QSAR) analysis of 65 novel 4-amino- and 4-oxo-pteridines (antipterins) as inhibitors targeting the H(4)Bip binding site of the neuronal NOS isoform (NOS-I). The experimental binding modes for two inhibitors complexed with the related endothelial NO synthase (NOS-III) reveal requirements of biological affinity and form the basis for ligand alignment. Different alignment rules were derived by building other compounds accordingly using manual superposition or a genetic algorithm for flexible superposition. Those alignments led to 3D-QSAR models (comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA)), which were validated using leave-one-out cross-validation, multiple analyses with two and five randomly chosen cross-validation groups, perturbation of biological activities by randomization or progressive scrambling, and external prediction. An iterative realignment procedure based on rigid field fit was used to improve the consistency of the resulting partial least squares models. This led to consistent and highly predictive 3D-QSAR models with good correlation coefficients for both CoMFA and CoMSIA, which correspond to experimentally determined NOS-II and -III H(4)Bip binding site topologies as well as to the NOS-I homology model binding site in terms of steric, electrostatic, and hydrophobic complementarity. These models provide clear guidelines and accurate activity predictions for novel NOS-I inhibitors.


Subject(s)
Enzyme Inhibitors/chemistry , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/chemistry , Pteridines/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/metabolism , Models, Molecular , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type I , Protein Binding , Pteridines/chemical synthesis , Pteridines/metabolism , Quantitative Structure-Activity Relationship
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