John E. More

Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C‐reactive protein, for clinical use

The human pentraxin proteins, serum amyloid P component (SAP) and C‐reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non‐specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL‐6 or IL‐8, nor does SAP cause release of IL‐1β or IL‐10. Furthermore neither of our preparations was pro‐inflammatory in mice in vivo.

A direct comparison of the antigen‐specific antibody profiles of intravenous immunoglobulins derived from US and UK donor plasma

Background and Objectives Intravenous immunoglobulin (IVIG) is used in a range of immunodeficiency states that require a broad spectrum of protective antibodies to a range of common pathogens. A comparison of the antigen‐specific antibody profile of preparations of an IVIG (Vigam®) derived from US and UK sourced plasma was performed, and these preparations were also compared with three other IVIG products from different manufacturers.

Clinical Experience with a New Solvent Detergent–Treated Intravenous Immunoglobulin Free of Hypotensive Effects

Objective: To see if modifications to the processing of intravenous immunoglobulin to include a virus inactivation stage alter immunoglobulin G (IgG) resulting in hypotension in patients. Methods: Clinical trials were done involving extensive patient monitoring during infusion: in vitro – testing for markers of hypotension, and in vivo – an animal model which closely simulates clinical use. Results: No hypotensive response was seen in the animal model or clinical trial. Conclusions: The production process used does not damage IgG or create vaso–active kinins as the preparation was free of hypotensive effects.

Virus inactivation in albumin by a combination of alkali conditions and high temperature

Non-enveloped viruses such as HAV and B19 are of potential concern in plasma products. In the case of albumin, pasteurisation at 60 °C for 10 h is generally used for virus inactivation. However this procedure is only partially effective against some non-enveloped viruses. Using a range of non-enveloped viruses i.e. HAV, SV40, CPV, treatment at a high pH of about 9.5 and a temperature of 60 °C for 10 h was found to be effective for virus inactivation. These extreme conditions caused no increase in aggregate composition of the albumin. In addition the albumin composition was stable over a period of at least 6 months. The ligand binding properties of the albumin, as determined using the dye phenol red, were also not affected by this treatment. This procedure has the potential for increasing the spectrum of viruses inactivated by the 60 °C pasteurisation step.

Development of an intravenous immunoglobulin with improved safety and functional activity.

The development and properties of a liquid intravenous immunoglobulin (Gammaplex(®)), of high purity, stability and functional activity, is described. Virus and TSE reduction by specific steps in the process were evaluated by spiking studies using small-scale models. The removal of procoagulant activity was determined using immunochemical and functional activity assays. Neutralisation and opsonic activity were used to demonstrate the functional activity of the IgG. The final low pH formulated product was stable at room temperature and was of high purity and functional activity. Three dedicated virus inactivation steps, i.e. solvent detergent, low pH and virus filtration, were shown to be effective. When combined with the B + I ethanol precipitation step, this gave a total reduction of >21 to >24 log for the enveloped and >10 to >13 log for the non-enveloped viruses tested. Several steps in the process were shown to contribute to TSE removal using scrapie. Potential procoagulant activity including Factor XI/XIa, was reduced to very low/undetectable levels in the final product. A new high purity liquid IVIG product has been developed, of high purity and good functional activity and stability. The process includes various steps for the removal of pathogens and procoagulant activity.

Concerning the Letter to the Editor by Rubinstein, A.I. et al. [1]: Dry Heat Treatment of Intravenous Immunoglobulin —Some Practical Considerations

We read with interest the Letter to the Editor by Rubinstein et al. [I] concerning the value of dry heat treatment of intravenous immunoglobulin for the assurance of its safe therapeutic use. While the Letter format is restricted in the amount of information given, we feel that the conclusions drawn by the authors were wholly unsupported by the evidence presented. We should like to share our own experience of dry heat treatment of similar intravenous immunoglobulin preparations in support of our comments. Rubinstein et al. [ I ] have correctly quoted clinical and laboratory evidence that the inactivationiclearance provided by Cohn fractionation alone is insufficient to eliminate the transmission of hepatitis C virus. However, there has been no clinical evidence of HIV and HBV transmission by intravenous immunoglobulin, and model studies support the view that adequate clearance of these agents is achieved during Cohn fractionation [2, 31. The authors cite hepatitis A transmission by coagulation factor concentrates treated with the solvent-detergent process to illustrate another potential threat posed by intravenous immunoglobulin, but fail to point out that unlike the coagulation factors, normal immunoglobulin and most specific hyperimmune immunoglobulins contain considerable titres of anti-hepatitis A antibodies which would serve to reduce the risk of hepatitis A transmission from an intravenous immunoglobulin product. The authors also fail to mention that all fractionators since early 1993 have been obliged to use plasma sourced only from those donors shown to be anti-HCV negative, adding a further assurance of non-transmission of this virus by the products manufactured from this plasma. Concerning the Letter to the Editor by Rubinstein, A.I. et al. 111

Automated Multi-Stage Chromatographic Systems for Production of Biotherapeutics

Systems for multi-stage chromatographic purification of biotechnologysourced therapeutic proteins have been designed and built using carmercially available components, modified where necessary. The systems operate as sterile self-contained units totally under computer control. The chemistries of the individual stages are integrated so that the product peak from one stage is loaded directly on to the next stage. Automatic CIP can be added and programmed as part of the process cycle. Two such systems are in operation: the first is for the purification of human growth hormone from a recombinant mammalian cell line; the second is for the purification of therapeutic monoclonal antibodies. Both systems have been used for the production of material for clinical trials.