Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process.

An increasing number of non-mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log10 reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.

Measurement of spinal kyphosis: implications for the management of Scheuermann's kyphosis.

STUDY DESIGN: Repeat clinical radiograph measurement of kyphosis was performed. OBJECTIVES: To evaluate precision in kyphosis measurement, the presence of intra- and interobserver error, and the effect of endplate selection and curve magnitude on error. SUMMARY OF BACKGROUND DATA: One study thus far has shown no interobserver difference in readings and an error interval of +/-11 degrees. METHODS: Four experienced examiners measured 30 radiographs of varying angles of kyphosis without preselected end vertebrae twice using the Cobb method. RESULTS: The mean intraobserver variance was 4.3 degrees (95% confidence interval, +/-9.6 degrees). One examiner had significantly better precision (P= 0.02) than the other examiners, who had no significant difference among them (P = 0.41). This examiner's mean intraobserver difference was 2.3 degrees (95% confidence limit, +/-6.2 degrees ). The 95% confidence limit for the interobserver difference was +/-8.7 degrees. The vertebral error index did not have a rank correlation with precision between readings. Magnitude of curve did not correlate with variance in measurement. CONCLUSIONS: The broad range in intra- and interobserver differences in the measurement of kyphosis should be taken into account in making management decisions or evaluating the success or failure of a treatment program. Careful technique in measurement may allow for improvement in individual precision.