Biosorption of process-equipment-related leachables (PERLs) in biomanufacturing: A quantitative approach to study partitioning of PERLs in a cell culture system.

The assessment and potential risk of process equipment-related leachables (PERLs) in the production of biopharmaceuticals and cell therapeutics using single-use (SU) equipment has been discussed previously. However, potential interactions of cells with PERLs have not yet been considered. Here, we present a quantitative adsorption study of neutral, organic small-molecule leachable compounds - known for extractables & leachables (E&L) analysis of SU equipment - in aqueous suspensions of CHO and T cells. The solid-water partition coefficient Kd was obtained for all compounds that showed adsorption. The findings implied that hydrophobic interactions are dominant; however, there was no unambiguous correlation between the derived adsorption coefficient Kd and the octanol-water partition coefficient Kow. Interestingly, a maximum affinity of both cell types to the leachable bis(2,4-di-tert-butylphenyl)phosphate, which is known to be detrimental to cell development, was observed. A comparison of both cell types revealed that they generally interact with the same compounds in most cases but to different extents. Using partition coefficients enables estimation of the concentrations of leachable compounds associated with the biomass phase and in the aqueous suspensions and could be used for risk assessment of SU systems in biopharmaceutical and cell therapy (CT) manufacturing processes.

Determination of "extractables" on polymer materials by means of HPLC-MS.

In biopharmaceutical processes, in the area of food and medical technology a variety of devices is used. These devices consist of various polymers. The detection and identification of potential extractables from these polymers during application are requested by the regulatory bodies. For risk and toxicity assessment, both identification and quantification of extractables are necessary. This article describes the development of a LS-MS methodology transfered from an established HPLC-UV-VIS method for full extractables analysis of sterile-grade filtration cartridges.

Crossflow filtration for CHO cell separation by microfiltration using crossflow systems.

Chinese Hamster Ovary (CHO) cells are used for fermentation of high value proteins in the pharmaceutical- and biotechnology industry. During the fermentation process the cells are grown under specific conditions in a defined media. The CHO cells produce extra cellular proteins. Separation and purification of these proteins result in the final product of high value. The production steps utilized in any bio-pharmaceutical process must deliver the target protein with high purity and yield. The first step in the production process is the separation of cells from the rest of the fermentation broth. Currently, stacked depth filters are used in this separation. The disadvantages with this method are:- 1) low product yield (high hold-up/wetting volume) and 2) the messy "clean-up" that is inherent with stacked filters. One of the main process requirements is that the CHO cell removal be accomplished with low cell mortality. This eases the subsequent process steps to purify the target protein with high yields free of DNA and other intracellular proteins. Additionally, the CHO cells can be reused.

Heterogeneous expression of c-myb protein in human leukemia detected by simultaneous two color flow cytometric analysis.

The expression of c-myb mRNA and protein was analyzed in fresh leukemic cells by Northern-blot analyses and by immunofluorescent staining using monoclonal c-myb specific antibodies. Staining of the cells was evaluated by flow cytometry. The results demonstrate c-myb mRNA expression predominantly in acute lymphocytic leukemia (ALL, 4/4 cases), acute myeloic leukemia (AML, 17/17) and chronic myeloic leukemia (CML, 12/12) but rarely in chronic lymphocytic leukemia (CLL, 1/17). Immunofluorescent analyses revealed expression of c-myb protein in the nucleus of ALL (5/7) and AML (9/9) with a good correlation of c-myb-positive cells and with the number of proliferating (Ki67-positive) blast cells.

Chromosomal rearrangements caused by the aberrant transposition of double Ds elements are formed by Ds and adjacent non-Ds sequences.

The Ds-induced unstable sh-m6258 allele is caused by the insertion of at least 45 kb of non-sh DNA. Genomic clones spanning the two junctions of sh sequences with insert sequences were isolated and analysed. The long insert in the sh-m6258 allele is bordered by Ds sequences on either side. A 3 kb double Ds structure which consists of one complete Ds element of approximately 2 kb and one half Ds element of approximately 1 kb was found in the sh-m6258 allele at the junction between the 3' region of the sh locus and the insert. At the junction between the 5' region of the sh locus and the insert a half Ds element is present. This truncated Ds element was probably left behind after the excision of a 2 kb Ds element from a 3 kb double Ds structure similar to that found at the 3' junction. Sequence analysis of the insert sequences beyond the Ds elements demonstrated that the long inserts in both the sh-m6258 and the sh-m5933 alleles originated from the same contiguous chromosomal segment in their common progenitor strain. One revertant derivative of the sh-m6258 allele was investigated. In the revertant strain, which displays a normal non-shrunken phenotype, a 2 kb Ds element is present at the site of the 45 kb insert in the mutant allele. This 2 kb insertion, which is located in the last but one intervening sequence of the Sh gene, does not inhibit expression of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Experience with midtrimester abortion.

The midtrimester abortion program at a large community hospital was evaluated. During the 3-year study, 1839 patients aborted in the midtrimester by intraamniotic injection of hypertonic saline, prostaglandin F2alpha or a combination of saline and prostaglandin F2alpha. The method, using a combination of saline and prostaglandin F2alpha together with intracervical laminaria, showed significant reduction in the number of failures (4.3 to 1.0%), reduction in the injection-abortion interval from 33.9 to 14.6 hours, shortening of the hospital stay from 2 1/2 to 1 1/3 days, minimum incidence of live abortions (0.9%), infrequent need for oxytocin to effect delivery (7.7%); and low rates of hemorrhage (1.5%) and fever (2.8%). The main disadvantage was an increased rate of incomplete abortions (32.3%), which could be reduced to 27% by patient selection.

PIP: A retrospective study was performed of all patients undergoing midtrimester abortion at the Baystate Medical Center from 1975-1977 (1839 patients). A comparison was made of the 3 methods employed: intraamniotic hypertonic saline, intraamniotic prostaglandin F2 alpha, and a combination of intraamniotic hypertonic saline and prostaglandin F2 alpha. It was found that use of the combination method resulted in significantly fewer failures and a significantly lower injection-abortion interval (from 33.9-14.6 hours) which resulted in a shorter hospital stay. In addition, the combination method resulted in fewer reinjections (as did use of hypertonic saline alone) than use of prostaglandin alone and less frequent use of oxytocin for delivery. The rate of live abortion was .2% for saline, 9% for prostaglandin alone, and .9% for the combination method. The side effects of hemorrhage and fever occurred in 2.8% and .2% of the patients who underwent the combination procedure, respectively.