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.

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.

Dissolution rate of griseofulvin in bile salt solutions.

Bile salts increase the apparent solubility of lipophilic poorly water-soluble drugs like griseofulvin. In this study, the dissolution kinetics of griseofulvin in solutions of bile salts (sodium taurocholate and sodium cholate) were investigated. A rotating disk apparatus was chosen to monitor dissolution kinetics; it well-defined hydrodynamic conditions allowed for analysis of the behavior of bile salt micelles under different conditions.

On the structure of immune-stimulating saponin-lipid complexes (iscoms).

Immune-stimulating complexes (iscoms) are stable complexes of cholesterol, phospholipid and Quil A, a triterpene saponin mixture in the size range from 40 to 100 nm. They can be used as antigen carriers in subunit vaccines. In this paper it is demonstrated that iscoms are rigid, negatively charged vesicles in which small water soluble molecules like carboxyfluorescein cannot be retained.

Liposomes and biotherapeutics.

Application of liposomes as delivery system for biotherapeutic peptides and proteins may offer important therapeutic advantages over existing delivery methods. Several approaches towards achieving improved delivery of biotherapeutics with liposomes are outlined. Although the literature on this topic is sporadic and frequently incomplete, enough of a research foundation exists to justify the conclusion that liposomes can play an important role in the formulation and delivery of biotherapeutics.

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.

The parenteral controlled release of liposome encapsulated chloroquine in mice.

Free (0.6 mg), and liposome encapsulated chloroquine (0.6, 3 mg), were injected intraperitoneally, intramuscularly and subcutaneously in mice.

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.

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.

Chloroquine blood levels after administration of the liposome-encapsulated drug in relation to therapy of murine malaria.

In a previous report (P. A. M.

Therapeutic effect of chloroquine(CQ)-containing immunoliposomes in rats infected with Plasmodium berghei parasitized mouse red blood cells: comparison with combinations of antibodies and CQ or liposomal CQ.

The potential therapeutic application of chloroquine-containing immunoliposomes (Fab'-lipCQ) in a Plasmodium berghei malaria model was studied. Extending a previously described in vivo model (Peeters et al. (1988) Biochim.

Chloroquine containing liposomes in the chemotherapy of murine malaria.

In this study, the advantage of the use of chloroquine (CQ) containing liposomes (lipCQ) over free CQ in the chemotherapy of murine malaria (Plasmodium berghei) was demonstrated. The maximum permissible dose per intraperitoneal injection was 0.8 and 10 mg for CQ and lipCQ, respectively.

Intestinal absorption of drugs. I: The influence of taurocholate on the absorption of dantrolene in the small intestine of the rat.

The influence of sodium taurocholate (1) on the intestinal absorption of the lipophilic drug dantrolene (2) was studied in vivo in a chronically isolated internal loop in the rat. Concentrations of 2 were kept below the saturation concentration in saline. Absorption kinetics of 2 were evaluated on the basis of steady-state blood levels, which develop during single-pass perfusions, and on the basis of the rate of disappearance of the drug from the perfusate during recirculating perfusions.

Potential pitfalls in in vitro antitumor activity testing of free and liposome-entrapped doxorubicin.

This paper addresses several potential problems which may play a critical role in the outcome of in vitro studies designed to investigate the antitumor activity of drugs. These problems were demonstrated to exist in in vitro assays developed for the evaluation of antitumor activity of free and liposome-entrapped doxorubicin (DXR). The stability of DXR-containing liposomes against drug leakage into the culture medium, as well as the chemical stability and extent of adsorption to tissue culture plastics of both free and liposomal DXR during the liposome-tumor cell incubation, were investigated.

Immunospecific targeting of immunoliposomes, F(ab')2 and IgG to red blood cells in vivo.

In this report a model to study the fate of target cells in the blood circulation after injection of appropriate immunoliposomes is discussed. The effect of intravenous administration of antimouse RBC immunoliposomes, F(ab')2 or IgG on the fate of intravenously injected 51Cr-labelled mouse RBC (Cr-mRBC) in the mouse and, particularly, in the rat was studied. The immunoliposome was of the Fab'-MPBPE-REV type (Fab'-fragments covalently linked to reverse phase evaporation vesicles by maleimido-4-(p-phenylbutyrate)phosphatidylethanolamine).

Immunospecific targeting of liposomes to erythrocytes.

Immunoliposomes were made by covalently linking Fab' fragments (from rabbit antimouse erythrocyte IgG) to reverse-phase evaporation vesicles (REV) via maleimido-4-(p-phenylbutyrate) phosphatidylethanolamine (MPB-PE) as anchor molecule. These immunoliposomes were characterized in terms of size, charge, stability and antigen binding capacity. The effect of the liposomal Fab' density on the interaction with the target cell was studied.

Immunogenicity of liposomes and iscoms containing the major outer membrane protein of Neisseria gonorrhoeae: influence of protein content and liposomal bilayer composition.

The influences of Neisseria gonorrhoeae protein IB content and bilayer composition of liposomes and protein content of iscoms on immunogenicity were investigated. Changes in the protein content of liposomes did not influence the immunoglobulin G response, whereas the response was lowered when the amount of protein in iscoms was increased. Bilayer composition only influenced the primary immunoglobulin G response; immunological memory was not affected.

Release of doxorubicin from peritoneal macrophages exposed in vivo to doxorubicin-containing liposomes.

Intracellular depot formation may be an important component of the mode of action of doxorubicin (DXR)-containing liposomes. In this paper it was investigated whether it is possible that DXR is released from macrophages which have taken up DXR-containing liposomes in vivo. Macrophages were harvested from the peritoneal cavity of LOU/M rats after i.

Incorporation of the major outer membrane protein of Neisseria gonorrhoeae in saponin-lipid complexes (iscoms): chemical analysis, some structural features, and comparison of their immunogenicity with three other antigen delivery systems.

We incorporated the major outer membrane protein (PI) of Neisseria gonorrhoeae into immunostimulating complexes (iscoms) and examined some analytical, physicochemical, and immunological properties of these structures. The immunogenicity was compared with that of three other PI-containing structures, i.e.

A differential scanning calorimetry study of the thermal behavior of water-dipalmitoylphosphatidylcholine mixtures at subzero temperatures: effects of water content, surface charge, and cholesterol.

The thermal behavior of partly and fully hydrated dipalmitoylphosphatidylcholine (DPPC) alone and mixed with dipalmitoylphosphatidylglycerol (DPPG) or cholesterol (chol) was studied at subzero temperatures with differential scanning calorimetry (DSC). The melting characteristics of the frozen water provided information on the fraction of nonfreezing and freezing (subzero melting) hydration water and excess or bulk water (melting at 0 degrees C). Incorporation of DPPG or cholesterol into the DPPC structure increased the subzero temperature interval compared with pure DPPC.

Dissolution kinetics of griseofulvin in sodium dodecylsulphate solutions.

The rate-limiting step in the absorption of poorly water-soluble drugs is often the dissolution process of these compounds. Surface-active agents influence the dissolution process by wetting and solubilizing. In order to study the effect of solubilization, a rotating-disk apparatus was used with griseofulvin as a model drug substance and sodium dodecylsulphate (SDS) as a model surfactant.

Influence of lipid composition on the antitumor activity exerted by doxorubicin-containing liposomes in a rat solid tumor model.

The effect of changes in lipid composition on the antitumor activity of doxorubicin (DXR)-containing liposomes was studied in immunoglobulin solid immunocytoma-bearing Lou/M Wsl rats. Rats bearing a tumor with a diameter between 20 and 30 mm were treated i.v.

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.

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.

Dissolution of theophylline monohydrate and anhydrous theophylline in buffer solutions.

The dissolution kinetics of theophylline monohydrate and anhydrous theophylline were investigated with a rotating-disk apparatus in buffer solutions at 298 K under sink conditions. The observed dissolution rate of theophylline monohydrate under various conditions agreed well with predictions based on the Extended Simultaneous Chemical Reaction and Dissolution concept. Below 337 K, anhydrous theophylline is converted to theophylline monohydrate in contact with water.