Determining the Initiation of Opiate Misuse Resulting in Opioid Use Disorder in Pregnant Women.

The aim of this study was to explore the "opiate misuse footprint" made by obstetrics and gynecology physicians in prescribing opioid medications for postpartum pain control that led to opioid misuse and opioid use disorder. Data were collected using intake information and anonymous surveys administered to pregnant women at local methadone clinics in Indianapolis, Indiana, in 2016-2017. Results from this study revealed that 40% of the 33 participants stated that the first drug they became addicted to was prescription opioids; 71% stated that the first opiate they became addicted to was a prescription pain medication. Prescription opioids were mainly obtained from emergency medicine physicians and friends. Reported use of opioids within the past four months was high, with the most commonly used drugs being methadone (57.6%) and heroin (42.4%). A majority of participants also endorsed a history of sexual and physical abuse, recent incarceration, and mental health disorders. As a large number of pregnant women with opioid use disorder reported their initial drug of misuse as prescription pain medications, it is important to avoid overprescribing opioids in reproductive-age women.

Effect of temperature, pH, dissolved oxygen, and hydrolysate on the formation of triple light chain antibodies in cell culture.

THIOMABs are recombinant antibodies with reactive cysteine residues used for forming THIOMAB-drug conjugates (TDCs). We recently reported a new impurity associated with THIOMABs: one of the engineered cysteines forms a disulfide bond with an extra light chain (LC) to generate a triple light chain antibody (3LC). In our previous investigations, increased LC expression increased 3LC levels, whereas increased glutathione (GSH) production decreased 3LC levels. In this work, on three stably transfected CHO cell lines, we investigated the effects of temperature, pH, dissolved oxygen (DO), and hydrolysate on 3LC formation during THIOMAB fed-batch cell culture production. Although pH between 6.8 and 7.0 had no significant impact on 3LC formation, temperature at 35°C instead of 33 or 31°C generated the lowest 3LC values for two cell lines. The decreased 3LC level correlated with increased GSH production. We implemented a 35°C temperature process for large-scale (2,000 L) production of a THIOMAB. This process reduced 3LC levels by ∼50% compared with a 33°C temperature process. By contrast, DO and hydrolysate had modest effect on 3LC levels for the model cell line studied. Overall, we did not find significant changes in LC expression under the conditions tested, whereas changes in GSH production were more evident. By investigating the impact of bioreactor process and medium conditions on 3LC levels, we identified strategies that reduced 3LC levels.

Triple light chain antibodies: factors that influence its formation in cell culture.

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species--Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression--evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)--correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production--evaluated as the ratio of the cell-specific production rate of GSH (q(GSH)) to the cell-specific production rate of THIOMAB (q(p))--corresponded to decreased 3LC levels. In time-lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and q(GSH)/q(p) ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high q(GSH)/q(p) ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity.