Cation exchange chromatography removes FXIa from a 10% intravenous immunoglobulin preparation.

The presence of residual activated coagulation factor XI (FXIa) in some commercial intravenous immunoglobulin (IVIG) products has been identified as the root cause of a small number of thromboembolic events in patients who had received such therapy. Our objectives here were to design and evaluate the manufacturing process of GC5107, a 10% glycine-stabilized IVIG product, for its capacity to remove FXIa. The manufacturing process included a cation exchange chromatography (CEX) step, which employs a resin that binds immunoglobulin G (IgG) with high capacity. Procoagulant activity was assessed using Western blot analysis, enzyme-linked immunosorbent assay, thrombin generation assay, chromogenic FXIa assay, and non-activated partial thromboplastin time (NaPTT) assay. A spiking study in which large quantities of FXIa were added to samples before CEX chromatography was used to examine the robustness of the process to remove FXIa. Western blot and ELISA analyses demonstrated that residual FXIa remained in the intermediate manufacturing products until after CEX chromatography, when it was reduced to undetectable levels. The spiking study demonstrated that CEX chromatography removed >99% of FXI protein and reduced FXI activity to below detection limits, even in samples containing 158-fold greater FXIa levels than that of normal samples. Procoagulant activity in 9 consecutive lots of GC5107 was reduced to below the detection limits of the thrombin generation and chromogenic FXIa assays (<1.56 IU/ml and <0.16 IU/ml, respectively). The NaPTT of >250 s in all 9 lots indicated very low levels of procoagulant activity. We demonstrate that a novel 10% IVIG manufacturing process including CEX chromatography is a robust means of removing FXIa from the final preparation.

Correlation between the results of two analytical methods for measuring measles virus neutralizing antibodies in source plasma and therapeutic immunoglobulin products.

Patients with primary immunodeficiency disorders are vulnerable to infectious diseases. Intravenous immunoglobulin (IVIG) therapeutic products manufactured from human plasma are employed widely to protect patients from pathogens such as measles virus, which causes a potentially fatal and contagious disease. Therefore, health authorities stipulate a minimum titer of measles neutralizing antibodies (mnAbs) in IVIG products to ensure efficient protection. In general, mnAb titers are measured in a cell-based neutralization assay; however, this assay is labor intensive and time consuming, and the results are variable. Here, we compared a cell-based neutralizing assay with several ELISA tests to evaluate whether ELISAs can overcome the limitations of cell-based assays. The mnAb concentrations measured by the ELISAs showed a strong and significant positive correlation with those measured in a cell-based assay. Also, strong positive correlations were identified for measurement of individual source plasmas, which are used as raw materials for manufacturing IVIG products. Measurement by ELISA revealed that about 80% of 198 source plasmas had mnAb concentrations of <500 mIU/mL. These results suggest that quantitative ELISAs based on relevant antigens allow reliable and comprehensive measurement of mnAb concentrations in source plasmas and drug product; these ELISAs are also faster and more accurate than cell-based assay.

Characterisation of the site-specific monoPEGylated rhG-CSF analogue pegteograstim.

We describe the characterisation of a novel monoPEGylated recombinant human granulocyte colony-stimulating factor analogue, pegteograstim (Neulapeg), prepared by site-specific 20 kDa maleimide-PEG conjugation. An additional cysteine was inserted between Gly136 and Ala137 of filgrastim (methionyl human granulocyte colony-stimulating factor) for site-specific PEGylation, and Cys18 of filgrastim was replaced with Ser18 to prevent unwanted PEGylation. Pegteograstim was produced by Escherichia coli and purified by cation exchange chromatography, and its structural, physicochemical, biological and immunological properties were investigated. Male Sprague-Dawley rats were administered pegteograstim (100 µg/kg) and the pharmacokinetics and pharmacodynamics compared with those of filgrastim. The results of long-term stability testing of pegteograstim revealed no significant change in its quality attributes at 2-8 °C for 36 months. In addition, pegteograstim was stable under the accelerated conditions (25 ± 2 °C, RH of 60 ± 5%) for 6 months. The site-specific monoPEGylated pegteograstim is a highly pure, stable and novel drug for long-lasting treatment of chemotherapy-induced neutropenia.