Water loss from silicone tubing and effect on protein concentration during drug product manufacturing.

Silicone tubing is used in various unit operations during drug product (DP) manufacturing. Hold of protein formulations in silicone tubing over time may have an impact on product quality, particularly protein concentration. This study evaluated the change in protein concentration of a test monoclonal antibody (mAb) formulation over various hold times in silicone tubing as a function of tubing internal diameter (ID) and wall thickness. It was hypothesized that the rate of water diffusion through the semi-permeable membrane is a function of the tubing ID and wall thickness. The weight and protein concentration of various formulation-filled tubings over time was measured. The weight of water lost varied linearly with the change in protein concentration. It was observed to be independent of mAb type, formulation composition, and initial protein concentration for a given tubing ID and wall thickness. The effect of formulation water activity on the water loss rate was investigated. A mechanistic diffusion-based model was developed that predicts the change in tubing weight and therefore protein concentration over various hold times for a given formulation and tubing. Overall, this study suggests that water loss from silicone tubing affects protein concentration and should be monitored during DP process development and manufacturing.

Lyophilizer Leak Rate Testing - An Industry Survey and Best Practice Recommendation.

The vacuum integrity of freeze dryers is critical for attaining adequate process control and maintaining confidence in sterility assurance which is key for the manufacture of sterile pharmaceutical products. Although discussions on the topic have been published, there is no industry standard established that is based on empirical data or that has a justifiable scientific rationale. This article provides a review of the scientific literature in the public domain and most importantly, a perspective from 14 Pharmaceutical companies on the leak rate specifications commonly used in industry. Using this information we recommend a best practice for the lyophilizer leak rate test which includes detailing necessary preparation activities following Steam-In-Place (SIP) sterilization, defining a period of stabilization to eliminate pressure and temperature fluctuations and details of the test conditions and the test period. We conclude that for routine manufacturing practice the operational leak rate should not exceed 20 µbar L/s and we provide additional guidance for large volume and older lyophilisation equipment.

Transcription factor ABI3 auto-activates its own expression during dehydration stress response.

The transcription factor abscisic acid insensitive 3 (ABI3) has recently been shown to mediate the dehydration stress response in nonseed and seed plants by regulation of several downstream genes. Here, we show how ABI3 autoregulates its transcription in response to dehydration stress signalling. Autoactivation is primarily through the Sph/RY element CATGCA present at the promoter region of ABI3. Along with other known cis-elements found at the ABI3 promoter, CATGCA remains occluded by nucleosomes during transcription repression. The nucleosomes tend to reposit during active transcription and are associated with several histone modifications, such as H3K9 and K27 acetylations and H3K4 trimethylation. This work thus, reveals the genetic and epigenetic essentials required for expression of the ABI3 gene, a crucial factor regulating dehydration stress signalling in Arabidopsis thaliana.

ABI3 mediates dehydration stress recovery response in Arabidopsis thaliana by regulating expression of downstream genes.

ABI3, originally discovered as a seed-specific transcription factor is now implicated to act beyond seed physiology, especially during abiotic stress. In non-seed plants, ABI3 is known to act in desiccation stress signaling. Here we show that ABI3 plays a role in dehydration stress response in Arabidopsis. ABI3 gene was upregulated during dehydration stress and its expression was maintained during subsequent stress recovery phases. Comparative gene expression studies in response to dehydration stress and stress recovery were done with genes which had potential ABI3 binding sites in their upstream regulatory regions. Such studies showed that several genes including known seed-specific factors like CRUCIFERIN1, CRUCIFERIN3 and LEA-group of genes like LEA76, LEA6, DEHYDRIN LEA and LEA-LIKE got upregulated in an ABI3-dependent manner, especially during the stress recovery phase. ABI3 got recruited to regions upstream to the transcription start site of these genes during dehydration stress response through direct or indirect DNA binding. Interestingly, ABI3 also binds to its own promoter region during such stress signaling. Nucleosomes covering potential ABI3 binding sites in the upstream sequences of the above-mentioned genes alter positions, and show increased H3 K9 acetylation during stress-induced transcription. ABI3 thus mediates dehydration stress signaling in Arabidopsis through regulation of a group of genes that play a role primarily during stress recovery phase.