Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production.

AIMS: The aim of this study was to develop and evaluate a real-time PCR technology for microbiological control methods to examine individualized cell therapeutics, an emerging class of pharmaceutical formulations. METHODS AND RESULTS: Oligonucleotide primers and hybridization probe for bacterial detection targeting the 16SrRNA gene were adapted based on Nadkarni et al. [Microbiology148 (2002) 257]. For detection of yeast and moulds, primers and probe were designed from conserved sequences of the 18SrRNA gene in this study. The real-time PCR assays were tested on genomic DNA of Escherichia coli and Candida albicans to assess efficiency and linear dynamic range. After successful establishment of robust real-time PCRs, applicability of the assays was evaluated by extracting microbial target DNA from cell-based preparations. Different commercial DNA extraction methods were compared identifying the MagNA Pure DNA Isolation Kit III as the method of choice. Sensitivity was examined for different strains and a detection limit of 102 -103 CFU per ml in a sample containing ~106 mammalian cells per ml was achieved. CONCLUSIONS: This study reports the successful establishment of two qualitative real-time PCR assays, enabling in general the broad-range detection of microbial contaminants in a cell-based sample matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Individualized cell therapeutics tend to have a short shelf life. Due to lengthy incubation periods, compendial testing according to current pharmacopoeial guidelines may not be applicable. We report a suitable alternative method upon which future microbiological quality control methods for such products could be based on. However, to implement valid rapid microbiological testing methods using real-time PCR technology, further challenges need to be addressed.

Validation of dry heat inactivation of bacterial endotoxins.

Bacterial endotoxins (ETs) are lipopolysaccharides from the cell wall of Gram negative bacteria. ETs get into the environment as a result of autolytic desintegration of the bacterial cells. There exist a number of depyrogenation methods, either serving to remove or to inactivate ET. The most common means of ET inactivation is dry heat. Unfortunately no uniform regulation exists describing the conditions for sufficient ET inactivation. While the USP and FDA require an ET reduction of 3 log steps, no European regulation exists regarding depyrogenation of final containers for parenterals. However, the Ph. Eur. specifies the temperature and time conditions for depyrogenation of glassware in the pyrogen test monograph, allowing to choose between the two variants 250 degrees C/30 minutes or 200 degrees C/60 minutes which are not equivalent. In the present study those conditions for depyrogenation of glass containers in production of parenterals were investigated which, on the one hand, are technically feasible and, on the other hand, comply with the requirements of the main Pharmacopeias; furthermore, an ET preparation suitable for validation studies was selected. The preparation of ET indicators, the dry-heat inactivation and the recovery of ET are described in detail. Based on the results obtained, it is recommended to follow a defined treating temperature and period for safe depyrogenation of glass containers for parenterals, which results in a 4 log step reduction in ET without fillers. Thereby the USP/FDA requirement for a 3 log step reduction as well as the 200 degrees C/60 minutes requirement variant given in the Ph. Eur. can be fulfilled.

The Magnuson-Stack procedure for treatment of recurrent glenohumeral dislocations.

The Magnuson-Stack procedure was developed in 1943 as a treatment for recurrent anterior dislocation of the glenohumeral joint. Since that time, very little literature has been published on this procedure. The purpose of our study is to evaluate its efficacy in preventing further dislocations and, also, introduce the Cybex Isokinetic Dynamometer as a quantitative tool in scientifically evaluating total shoulder function. At the Bryn Mawr (Pennsylvania) Hospital from 1971 to 1978, 43 patients underwent a Magnuson-Stack procedure. Twenty-nine returned detailed questionnaires; 26 of these patients returned for clinical and roentgenographic examination by one of the authors, and 18 had their shoulder motion and power quantitatively compared to their contralateral normal arms with the Cybex Isokinetic Dynamometer. Eighty-five percent of shoulders did not dislocate postoperatively; 90% considered their results satisfactory. By gross physical examination, no shoulder atrophy was noted. Strength seemed to equal the uninjured shoulder. Range of motion showed a 10 degrees loss of external rotation as measured with a hand-held goniometer. However, by testing range of motion with the Cybex system, a 25 degrees lack of external rotation was noted. Force values measured at 60 degrees of arc/sec and 180 degrees of arc/sec showed an 18 and 10% deficit, respectively, in external rotation. The authors believe that the Magnuson-Stack procedure has a place in the treatment of recurrent anterior dislocations of the shoulder. Also, the Cybex II isolated joint testing system provides an accurate reproducible method of comparing the postoperative results of all patients treated for dislocation of the shoulder.(ABSTRACT TRUNCATED AT 250 WORDS)