Biomedical subjects

W Jiskoot

Publications and source records attributed to W Jiskoot.

29 records · Page 2Linked to original sources

PubMed1992

The influence of sucrose, dextran, and hydroxypropyl-beta-cyclodextrin as lyoprotectants for a freeze-dried mouse IgG2a monoclonal antibody (MN12).

The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated. MN12 was freeze-dried in different formulations [without lyoprotectant or in the presence of sucrose, dextran, or hydroxypropyl-beta-cyclodextrin (HP beta CD)] and under varying conditions (with or without secondary drying). Subsequently, the monoclonal antibody was stored for 18 or 32 days at various temperatures (4, 37, or 56 degrees C). For comparison, solutions of MN12 were stored under the same conditions. Regardless of the lyoprotectant used, precipitation and a concomitant reduction of the antigen-binding capacity by about 10% were observed upon reconstitution of lyophilized MN12. HP beta CD proved to be the most effective stabilizer to prevent degradation of lyophilized MN12 during storage. Compared with MN12 solutions, HP beta CD-containing lyophilized MN12 cakes were more resistant to heat-induced charge alterations and loss of antigen-binding capacity.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1992

Acid-induced structural changes of a mouse IgG2a monoclonal antibody (MN12) studied by transient electric birefringence measurement.

Acid-induced structural changes of a mouse IgG2a monoclonal antibody (MN12) as indicated by Jiskoot et al. (Eur. J. Biochem. 201,223-232 (1991)) were studied by measuring the transient electric birefringence of MN12 in aqueous solution and in glycerol-water mixtures at different pH conditions. A multi-exponential analysis program, DISCRETE (Provencher,S.W., Biophys.J.16,27-41 (1976)), and a constrained inverse Laplace transform program, CONTIN (Provencher, S.W., Comp. Phys. Comm. 27, 213-227 (1982)) have been used to determine the number of exponentials needed to represent the data and their decay times. Measurement of the time-resolved electric field induced birefringence makes it possible to study rotational processes on a timescale from several tens of nanoseconds to microseconds. This enabled us to monitor the segmental flexibility and the rotational motion of single antibody molecules as well as the occurrence of aggregates. The results show an increase in hydrodynamic dimensions of MN12 upon lowering the pH from 6.6 to 2.7. Additionally, the original segmental flexibility, which could be monitored for the samples in glycerol-water mixtures, is altered at low pH. The results have been interpreted as swelling of MN12 followed by dimerization.

Animals↗

PubMed1991

Non-random conformation of a mouse IgG2a monoclonal antibody at low pH.

The pH dependence of the conformation of a mouse IgG2a, kappa monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near-ultraviolet and far-ultraviolet CD, and electric-field-induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH region between pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid-induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16-0.18 microseconds) was observed for MN12 by electric-field-induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23 microseconds; a further increase to 0.30 microseconds was observed after 24 h. The combined results suggest an acid-induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.

Animals↗

PubMed1991

Preparation and application of a fluorescein-labeled peptide for determining the affinity constant of a monoclonal antibody-hapten complex by fluorescence polarization.

A simple and rapid method for determining the affinity constant of a monoclonal antibody-peptide complex under equilibrium conditions is presented. A peptide corresponding to sequence 178-185 of meningococcal strain MC50 class 1 outer membrane protein, which is recognized by monoclonal antibody MN12 (mouse IgG2a), was synthesized. After fluorescein was coupled to the peptide, the peptide-fluorescein conjugate was used for binding studies with MN12, employing fluorescence polarization of the fluorescein label to probe the bound fraction of the peptide. Scatchard analysis showed that the affinity constant was pH dependent. Storage of MN12 under alkaline conditions resulted in a loss of antigen-binding sites, but did not alter the affinity constant. Sips plots showed a homogeneity index of unity.

Amino Acid Sequence↗

PubMed1991

Purification and stabilisation of a poorly soluble mouse IgG3 monoclonal antibody.

The anti-T cell monoclonal antibody (Mab) RIV9 (mouse IgG3, kappa) has been developed for clinical use in the treatment of allograft rejection. In order to obtain a clinical grade Mab preparation, RIV9 was purified from cell culture supernatants by protein A affinity and anion exchange chromatography. Reasonable yields of highly purified product could only be obtained if stabilising compounds were added and Tween 80 was used in all stages of the purification process. Prior to anion exchange chromatography, dextran sulphate (MW 5000) was added to keep the Mab in solution. Many other additives were tested but did not solubilise RIV9 under the low salt strength conditions required for ion exchange chromatography. The complex character of the solubility-determining factors was demonstrated by the influence of buffer composition, buffer concentration, pH, and sodium chloride concentration on the solubility of RIV9.

Animals↗

PubMed1991

Preparation of clinical grade monoclonal antibodies from serum-containing cell culture supernatants.

Three mouse monoclonal antibodies (Mab), RIV6, MN12, and WT31, were purified from cell culture supernatants containing foetal bovine serum (FBS) by two-step purification protocols, involving protein A affinity and ion exchange chromatography. Provided that the purification conditions were adapted to the physico-chemical properties of the individual Mab, clinical grade products could be obtained. The residual levels of bovine IgG originating from FBS were below 1% on a protein basis. Endotoxin levels were below 1 ng/ml. The contents of other serum proteins, DNA, and protein A were below or near the detection limits. The final products met the requirements for therapeutic Mab. Special attention was paid to the behaviour of foetal bovine IgG in the different purification steps. Large variations in the IgG contents of different batches of FBS were observed. However, the properties of the IgG fractions of the batches were very similar. A major IgG fraction with a low affinity for protein A and with components with relatively acidic isoelectric points (pIs) was distinguished from a minor fraction exhibiting a high affinity for protein A and a more diverse pI pattern. The impact of these findings on the purification strategy used for the Mab is discussed.

Animals↗

PubMed1990

Analytical approaches to the study of monoclonal antibody stability.

The stability of two purified monoclonal antibodies, MN12 and WT31, was investigated. The monoclonal antibodies were incubated for 32 days at different pH values (ranging from 3.0 to 10.0) at 4 and 37 degrees C. Various analytical methods were used to assess changes in physicochemical properties of the proteins. The monoclonal antibodies were more susceptible to degradation at 37 degrees C than at 4 degrees C. At low pH irreversible precipitation occurred. Decomposition of the proteins was enhanced at increasing pH values in the alkaline range. This was concluded from mouse IgG-specific and antigen-specific enzyme-linked immunosorbent assays, flow cytometry, analytical gel permeation chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and immunoblotting. No substantial change in the apparent affinity constant of MN12 was observed, as determined by an affinity enzyme-linked immunosorbent assay. Fluorescence spectra, fluorescence polarization values, and fluorescence quenching parameters of MN12 and WT31 were not substantially affected, indicating that no major irreversible conformational changes had occurred. It was concluded that each of the techniques used has only limited value for stability assessment of monoclonal antibodies and, hence, that the application of several analytical techniques is essential to gain insight into monoclonal antibody stability.

Animals↗

PubMed1990

A purification strategy for the production of clinical grade monoclonal antibodies.

A strategy for the purification of murine IgG monoclonal antibodies (MAbs) is described. It consists of a combination of protein A affinity chromatography and ion exchange chromatography. The method was used for the purification of two MAbs, WT31 and MN12. After in vitro cultivation in the presence of foetal bovine serum, the medium components were efficiently removed from the MAbs, yielding clinical grade products. Automated purification under aseptic conditions was effectuated by employing a computer-controlled four-column system.

Animals↗

PubMed1989

Two-step purification of a murine monoclonal antibody intended for therapeutic application in man. Optimisation of purification conditions and scaling up.

The murine hybridoma cell line WT31, which produces a monoclonal antibody (Mab) of the IgG1 isotype with specificity for the human T cell receptor, was grown in batch-suspension cultures in the presence of foetal bovine serum (FBS). To acquire a clinical grade product for the reversal of allograft rejection, the clarified and concentrated cell culture supernatant was purified by a two-step chromatographic procedure, involving protein A affinity chromatography and Q Sepharose anion exchange chromatography. After choosing the appropriate conditions on a small scale, the purification process was scaled up. A BioPilot system was used for automated purification of 1 g WT31 Mab in a closed system. In spite of a relatively high initial ratio of bovine IgG to mouse IgG, the residual level of bovine IgG could be reduced to 1% or less with respect to the Mab content. No other serum proteins nor DNA were detected in the purified product. The efficacy of the purification procedure was demonstrated by a combination of several analytical techniques: ELISA (mouse and bovine IgG contents, protein A content), countercurrent immunoelectrophoresis (bovine serum albumin content), fluorescence activated cell sorter analysis (potency), DNA assay, sodium dodecylsulphate polyacrylamide gel electrophoresis, immunoblotting, isoelectric focusing, and gel permeation chromatography.

Animals↗

PubMed1986

Preparation of liposomes via detergent removal from mixed micelles by dilution. The effect of bilayer composition and process parameters on liposome characteristics.

Liposomes were prepared from mixed micelles by a dilution method. Mixed micellar solutions, containing constant octyl glucoside and egg phosphatidylcholine concentrations and varying amounts of cholesterol and/or a charged compound, were diluted at defined rates. After dilution, the resulting liposome dispersions were sequentially concentrated, washed or dialysed, and filtered. The effect of lipid composition and experimental conditions on physicochemical characteristics was studied. Fairly homogeneous liposome dispersions with mean diameters ranging from 100 to over 200 nm could be obtained. The particle size was dependent on cholesterol content and surface charge, and could be reproducibly controlled by adjustment of the dilution rate. Liposomes with a mean diameter below 100 nm could also be obtained, but were heterodisperse and unstable. The incorporation of charged compounds was monitored by microelectrophoresis. 31P-NMR measurements indicated that the liposomes were unilamellar. Dialysis appeared to be more convenient than washing to remove octyl glucoside.

Chemical Phenomena↗

PubMed1986

Immunogenic activity of gonococcal protein I in mice with three different lipoidal adjuvants delivered in liposomes and in complexes.

For several reasons the major outer membrane protein from Neisseria gonorrhoeae (gonococcal protein [PI]) is an attractive component for a gonococcal vaccine. This paper describes the influence of two different physical forms of PI on its immunogenic activity. To this end PI was delivered in liposomes and in protein-detergent complexes. In both forms PI was present in a multimeric form. The liposomes were composed of phosphatidylcholine and cholesterol. The effect of dicetylphosphate as a negatively charged amphiphile and three lipoidal adjuvants was investigated. Two lipoidal adjuvants (Avridine and dimethyldioctadecylammoniumbromide) were positively charged amphiphiles, whereas the third one (tridecyl N-acetylmuramyl-L-alanyl-D-isoglutaminate) was neutral. The protein-detergent complexes were also tested in the presence of the lipoidal adjuvants and in an AlPO4-adsorbed form. The liposome preparations were characterized for their size, charge, and residual amount of detergent. The immunogenic activity of PI in all forms was tested in mice. The results of the antibody assays showed that PI in the liposomes was more immunogenic than PI in the complexes. A second dose with liposomes induced only a small booster effect, whereas such a dose with the complexes produced pronounced booster effects. The incorporation of the positively charged lipoidal adjuvants in the liposomes resulted in enhanced booster effects. The highest immunogenic activity of PI after two injections, however, was observed in the complexed form adsorbed to AlPO4.

Animals↗