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1.
J Chromatogr A ; 1218(31): 5195-204, 2011 Aug 05.
Article in English | MEDLINE | ID: mdl-21696747

ABSTRACT

The cation-exchange capture step of a monoclonal antibody (mAb) purification process using single column batch and multicolumn continuous chromatography (MCSGP) was modeled with a lumped kinetic model. Model parameters were experimentally determined under analytical and preparative conditions: porosities, retention factors and mass transfer parameters of purified mAb were obtained through a systematic procedure based on retention time measurements. The saturation capacity was determined through peak fitting assuming a Langmuir-type adsorption isotherm. The model was validated using linear batch gradient elutions. In addition, the model was used to simulate the start-up, cyclic steady state and shut down behavior of the continuous capture process (MCSGP) and to predict performance parameters. The obtained results were validated by comparison with suitable experiments using an industrial cell culture supernatant. Although the model was not capable of delivering quantitative information of the product purity, it proved high accuracy in the prediction of product concentrations and yield with an error of less than 6%, making it a very useful tool in process development.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Cation Exchange Resins/chemistry , Chromatography, Ion Exchange/methods , Immunoglobulin G/isolation & purification , Adsorption , Antibodies, Monoclonal/chemistry , Chromatography, Ion Exchange/instrumentation , Immunoglobulin G/chemistry , Models, Chemical
2.
Biotechnol Bioeng ; 107(6): 974-84, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-20677181

ABSTRACT

A two-step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP-captured material in batch mode. Using multi-modal anion exchange in bind-elute mode, mAb was produced consistently within the purity specification. The benchmark was a state-of-the-art 3-step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2-step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Biotechnology/methods , Immunoglobulin G/isolation & purification , Chromatography, Liquid/methods , Solvents
3.
J Biol Chem ; 276(19): 16146-54, 2001 May 11.
Article in English | MEDLINE | ID: mdl-11278715

ABSTRACT

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3'/RB3") are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific "stathmin-like domain" and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two alpha/beta tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3'. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.


Subject(s)
Microtubule Proteins , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Tubulin/metabolism , Amino Acid Sequence , Animals , Binding Sites , Calcium-Binding Proteins , Carrier Proteins , Intracellular Signaling Peptides and Proteins , Kinetics , Membrane Proteins , Mice , Molecular Sequence Data , Nerve Growth Factors/chemistry , Nerve Growth Factors/metabolism , Protein Structure, Secondary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Stathmin , Surface Plasmon Resonance
4.
Cell ; 102(6): 809-16, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-11030624

ABSTRACT

Phosphoproteins of the stathmin family interact with the alphabeta tubulin heterodimer (tubulin) and hence interfere with microtubule dynamics. The structure of the complex of GDP-tubulin with the stathmin-like domain of the neural protein RB3 reveals a head-to-tail assembly of two tubulins with a 91-residue RB3 alpha helix in which each copy of an internal duplicated sequence interacts with a different tubulin. As a result of the relative orientations adopted by tubulins and by their alpha and beta subunits, the tubulin:RB3 complex forms a curved structure. The RB3 helix thus most likely prevents incorporation of tubulin into microtubules by holding it in an assembly with a curvature very similar to that of the depolymerization products of microtubules.


Subject(s)
Microtubule Proteins , Phosphoproteins/chemistry , Tubulin/chemistry , Amino Acid Sequence , Animals , Binding Sites , Brain Chemistry , Cattle , Crystallography, X-Ray , Dimerization , Microtubules/chemistry , Molecular Sequence Data , Phosphoproteins/isolation & purification , Phosphoproteins/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Stathmin , Tubulin/isolation & purification , Tubulin/metabolism
5.
J Biol Chem ; 275(10): 6841-9, 2000 Mar 10.
Article in English | MEDLINE | ID: mdl-10702243

ABSTRACT

Stathmin is a cytosoluble phosphoprotein proposed to be a regulatory relay integrating diverse intracellular signaling pathway. Its interaction with tubulin modulates microtubule dynamics by destabilization of assembled microtubules or inhibition of their polymerization from free tubulin. The aim of this study was to probe the native structure of stathmin and to delineate its minimal region able to interact with tubulin. Limited proteolysis of stathmin revealed four structured domains within the native protein, corresponding to amino acid sequences 22-81 (I), 95-113 (II), 113-128 (III), and 128-149 (IV), which allows us to propose stathmin folding hypotheses. Furthermore, stathmin proteolytic fragments were mixed to interact with tubulin, and those that retained affinity for tubulin were isolated by size exclusion chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that, to interact with tubulin, a stathmin fragment must span a minimal core region from residues 42 to 126, which interestingly corresponds to the predicted alpha-helical "interaction region" of stathmin. In addition, an interacting stathmin fragment must include a short N- or C-terminal extension. The functional significance of these interaction constrains is further validated by tubulin polymerization inhibition assays with fragments designed on the basis of the tubulin binding results. The present results will help to optimize further stathmin structural studies and to develop molecular tools to target its interaction with tubulin.


Subject(s)
Microtubule Proteins , Phosphoproteins/chemistry , Tubulin/chemistry , Chromatography, Gel , Mass Spectrometry , Microtubules/chemistry , Polymers/chemistry , Solutions , Stathmin
6.
Cell Struct Funct ; 24(5): 345-57, 1999 Oct.
Article in English | MEDLINE | ID: mdl-15216892

ABSTRACT

Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and/or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and/or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological actions: it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3'/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.


Subject(s)
Microtubule Proteins , Phosphoproteins/physiology , Tubulin/metabolism , Animals , Binding Sites , Carrier Proteins , Cell Differentiation/physiology , Cell Division/physiology , Humans , Membrane Proteins , Microtubules/metabolism , Models, Biological , Nerve Growth Factors/chemistry , Nerve Growth Factors/physiology , Neurons/physiology , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Stathmin
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