Evaluation of single-use optical and electrochemical pH sensors in upstream bioprocessing

Culture pH is a critical process parameter during CHO cell bioreactor operations that is key for proper cell growth, protein production, and maintaining the critical quality attributes of a monoclonal antibody drug substance. The traditional means of measuring pH in bioreactors is with an electrochemical probe that can withstand and maintain accuracy through repeated sterilization cycles. An alternative technique for measuring pH is an optical sensor composed of a fluorescent dye that is sensitive to the hydrogen ion concentration. In this work we explore single-use electrochemical and single-use optical pH sensors in stirred-tank and rocking bioreactors, respectively, to understand how their overall performance compares to traditional electrochemical probes in benchtop glass stirred tank bioreactors. We found that the single-use optical pH sensors were generally less accurate than the electrochemical probes, especially in detecting large pH drifts from the setpoint. The single-use electrochemical probes were increasingly accurate as pH was increased from <7.0 to 7.5 but tended to decrease in accuracy as the batch age increased. In conclusion, single-use pH sensors offer a convenient means to measure pH during an upstream bioprocess, but the limitations of these sensors should be built into process control such that deviations in process pH, and consequently potential fluctuations in product quality, can be avoided

Isoniazid Inhibits Human Erythroid 5-aminolevulinate Synthase: Molecular Mechanism and Tolerance Study with Four X-linked Protoporphyria Patients

Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5′-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Ki of 1.2 μM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5 mg·kg− 1·day− 1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias