Lectin binding assays for in-process monitoring of sialylation in protein production.

Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galbeta1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(beta1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galbeta1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galbeta1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.

Genome wide expression profiling of human peripheral blood mononuclear cells stimulated with BAY 50-4798, a novel T cell selective interleukin-2 analog.

BAY 50-4798, a novel, engineered form of interleukin (IL)-2, is a selective agonist for the high-affinity IL-2 receptor and induces the proliferation of activated human T cells with potency similar to recombinant IL-2 (rIL-2), but has reduced proliferative activity on natural killer cells and is associated with a diminished secondary cytokine cascade. In the current study, the transcriptional profiles of human peripheral blood mononuclear cells (PBMCs) stimulated in vitro with BAY 50-4798 and rIL-2 were compared using Affymetrix microarray technology in combination with Ingenuity Pathway Analysis (IPA) to determine whether there are quantitative or qualitative differences in the molecular networks activated by these IL-2 analogs. A total of 299 genes were differentially expressed in response to the two IL-2 analogs, with an increase in the number of differences over time. Consistent with the fact that BAY 50-4798 interacts with fewer forms of the IL-2 receptor than rIL-2 to activate fewer cell types, 169 genes were expressed at lower levels in PBMCs cultured with BAY 50-4798 compared with IL-2. These genes were mainly categorized as cytokines and chemokines, and were used to build multiple molecular interaction networks, the most significant of which centered around a subunit of NF-kappaB, which is known to play a pivotal role in inflammation, and was associated with cell death. Of the genes induced in response to BAY 50-4798, only 25% were expressed at lower levels than those induced by rIL-2. Moreover, despite its more selective receptor targeting compared with rIL-2, BAY 50-4798 caused higher levels of expression of 130 genes, which predominantly fell into categories associated with metabolism and transcription. We interpret these results as consistent with the expected transcriptional profile of a mutein engineered and demonstrated to have diminished inflammatory effects yet fully retain selected features of IL-2 activity. In addition to demonstrating that the responses to BAY 50-4798 are characterized by differential expression of genes known to be induced by IL-2, we report for the first time the induction of a significant number of genes not previously reported in the context of IL-2 biology.