Bing Guan

A liquid chromatography (LC) method with ultraviolet (UV) and tandem mass spectrometry (MS/MS) detection was developed to quantify dimethoxytrityl alcohol (DMT-OH), a small molecule byproduct generated during the detritylation reaction in oligonucleotide synthesis. The pros and cons of quantification via multiple analytical methods including LC coupled with UV, selected ion monitoring (SIM), and multiple reaction monitoring (MRM) were evaluated. The MRM method was ultimately selected for further qualification and exhibited good linearity (R² = 0.997 from 0.5 to 64 ng mL⁻¹), accuracy (recoveries ranging 75–90% with ≤ 2% RSD), repeatability (−1). The MRM method was further applied to analyze DMT-OH in various oligonucleotide intermediates and drug substances. Similar MRM methods for six other small molecule impurities (aniline, benzamide, isobutyramide, 2-phenylacetamide, succinamide, and uny-CTP) as well as their application are also presented.

Krüppel-like factor 5 (KLF5) is frequently deleted and inactivated in prostate cancer, and exerts tumor-suppressing function in prostate cancer cells. However, the function of KLF5 in the apoptosis of prostate cancer cells remains unclear. In the present study, the effect of KLF5 on phorbol 12-myristate 13-acetate (PMA)-induced apoptosis was investigated in prostate cancer LNCaP cells. It was demonstrated that PMA induced the expression of KLF5 at the mRNA and protein level. To identify whether KLF5 regulates the activity of the downstream pathway, stable KLF5 knockdown or overexpression cell lines were constructed with lentivirus harboring shRNA targeting KLF5 or full-length KLF5 in LNCaP cells. Knockdown of KLF5 significantly decreased PMA-induced apoptosis, while cell apoptosis was significantly increased following KLF5 overexpression compared with the corresponding control groups. Consistently, expression of cleaved poly(ADP-ribose) polymerase and caspase-3 induced by PMA was decreased following KLF5 knockdown and increased following KLF5 overexpression. Using the control medium from cells treated with PMA, it was demonstrated that KLF5 is required for the control medium to induce apoptosis. c-Jun N-terminal kinase (JNK) activity is essential for the apoptosis induced by PMA. It was revealed that knockdown of KLF5 decreased, while overexpression of KLF5 increased the phosphorylation of JNK induced by PMA and control medium treatment. Furthermore, inhibition of tumor necrosis factor α (TNFα) decreased KLF5 expression and significantly decreased cell apoptosis induced by PMA, and control medium. This data indicates that KLF5 is essential for the apoptosis induced by PMA in LNCaP prostate cancer cells. Furthermore, KLF5 is essential for activity of the autocrine factor TNFα, which is secreted by cells treated with PMA and mediates the function of PMA-induced apoptosis through regulating the activity of JNK signaling pathway. These results provide novel insights into the complexity of the signaling pathways regulating apoptosis in prostate cancer cells, which could aid in the development of novel treatments for patients with prostate cancer.

A high-throughput method to quantify sialic acids was developed on a ForteBio Octet QK384 platform to support cell-line development. The high-throughput sialylation analysis method using the ForteBio Octet (HTFBS) is based on the binding rate of Maackia amurensis lectin II (MALII) to (α-2,3)-linked sialic acids of proteins bound to Protein-A sensors. The HTFBS method can specifically quantify the sialylation levels of secreted proteins in crude cell culture samples and does not require purified samples. Using this method, thirty crude cell culture samples can be analyzed for titers and sialylation in 60 minutes. In a comparison study between the HTFBS method and HPLC method, a linear correlation was observed between the MALII binding rate obtained by using the ForteBio Octet QK384 and the total sialic acid content obtained by HPLC for samples in the sialic acid content range of 5–24 mol mol⁻¹. The MALII binding rate was consistent for samples with titers between 6.25 and 100 μg mL⁻¹. The HTFBS method is robust and the effect of protein concentration is negligible on the MALII binding rate for samples between 6.25 and 100 μg mL⁻¹. The HTFBS method was successfully applied to the cell-line development platform for screening highly sialylated clones. The current HTFBS method can quantify sialylation levels in crude samples and can readily be used to accelerate cell line development for producing therapeutic proteins with desired product quality.

We developed a novel reversed-phase liquid chromatography method for monitoring multiple ingredients in poloxamers and evaluating their stability.