A high-throughput method for quantification of glycoprotein sialylation.

Sialic acid can improve qualities of therapeutic glycoproteins such as circulatory half-life, biological activity, and solubility. In production of therapeutic glycoproteins, a high-throughput method is required for process monitoring and optimization to ensure consistent and optimal sialic acid content. Current methods for quantifying sialic acid, however, require chromatographic separation that is time-consuming and cannot rapidly analyze many samples in parallel. Here we present a novel high-throughput method for quantifying glycoprotein sialylation. Using chemical reduction, enzymatic release of sialic acid, and chemical derivatization of the sialic acid, the method can accurately, rapidly (15 min), and specifically analyze many samples in parallel. It requires only 45 µl of sample and has a quantitation limit of 2 µM sialic acid. It has also been validated for monitoring sialylation of recombinant interferon gamma (IFN-γ) produced in Chinese hamster ovary (CHO) cell culture. This method is useful for various applications in upstream and downstream bioprocesses.

High-throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate.

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high-throughput small-scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two-step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two-step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1.

High-throughput analysis of intraclonal variability of glycoprotein sialylation.

Development of recombinant Chinese Hamster Ovary (CHO) cells producing therapeutic proteins requires analyzing the quality, such as sialic acid content, of proteins produced by many cell clones. In order to perform these analyses, high-throughput methods are required. Conventional methods for quantifying sialic acid, however, require protein purification, which is time consuming and cannot be used for high-throughput analysis. Here we used a high-throughput method (HTM) that we recently developed to analyze the intraclonal variability of 24 CHO cell subclones. The sialic acid content varied significantly from 1 to 70 mg sialic acid/g protein, and the concentration of total proteins secreted by the cells varied from 41 to 214 mg/L. In addition, the sialic acid content was negatively correlated with total protein concentration. This trend agrees with previous theoretical and experimental studies. Overall, the HTM can finish these analyses in 15 minutes, while conventional methods used in previous studies will require at least 24 days. Thus, the HTM can significantly accelerate the analyses of clonal and intraclonal variability in cell line development