Wei Z.,Process Purification science |
Bilbulian S.,Novavax |
Li J.,Process Purification science |
Pandey R.,Novavax |
And 3 more authors.
Journal of Separation Science | Year: 2015
A new analytical method has been developed for the quantitative determination of ethylene glycol-containing nonionic surfactants, such as polyethylene glycol 8000, polysorbate 80, and Pluronic F-68. These surfactants are commonly used in pharmaceutical protein preparations, thus, testing in the presence of protein is required. This method is based on the capillary gas chromatographic analysis of ethylene glycol diacetate formed by hydrolysis and acetylation of surfactants that contain ethylene glycol. Protein samples containing free surfactants were hydrolyzed and acetylated with acetic anhydride in the presence of p-toluene sulfonic acid. Acetylated ethylene glycol was extracted with dichloromethane and analyzed by gas chromatography using a flame ionization detector. The amount of nonionic surfactant in the sample was determined by comparing the released ethylene glycol diacetate signal to that measured from calibration standards. The limits of quantitation of the method were 5.0 μg/mL for polyethylene glycol 8000 and Pluronic F-68, and 50 μg/mL for polysorbate 80. This method can be applied to determine the polyethylene glycol content in PEGylated proteins or the final concentration of polysorbate 80 in a protein drug in a quality control environment. © 2015 MedImmune.
Grillo A.O.,Biopharmaceutical Development |
Grillo A.O.,University of the Incarnate Word |
Perkins M.,Biopharmaceutical Development
BioPharm International | Year: 2010
Design of experiments (DOE) is a valuable tool for identifying aspects of a formulation that are critical to product quality. The formulation design space can be characterized by performing excipient robustness studies that use DOE. This paper presents considerations for performing robustness studies as well as two case studies in which DOE was used to determine the robustness of protein formulations to changes in protein, excipient, and pH levels. The results from the DOE studies identified formulation components that must be tightly controlled and showed that variations had a minimal impact to product in formulation component levels within the formulation design space.
Singh S.K.,Pfizer |
Afonina N.,Bristol Myers Squibb |
Awwad M.,Pfizer |
Bechtold-Peters K.,Boehringer Ingelheim |
And 9 more authors.
Journal of Pharmaceutical Sciences | Year: 2010
Concern around the lack of monitoring of proteinaceous subvisible particulates in the 0.1-10 μm range has been heightened (Carpenter et al., 2009, J Pharm Sci 98: 1202-1205), primarily due to uncertainty around the potential immunogenicity risk from these particles. This article, representing the opinions of a number of industry scientists, aims to further the discussion by developing a common understanding around the technical capabilities, limitations, as well as utility of monitoring this size range; reiterating that the link between aggregation and clinical immunogenicity has not been unequivocally established; and emphasizing that such particles are present in marketed products which remain safe and efficacious despite the lack of monitoring. Measurement of subvisible particulates in the < 10 μm size range has value as an aid in product development and characterization. Limitations in measurement technologies, variability from container/closure, concentration, viscosity, history, and inherent batch heterogeneity, make these measurements unsuitable as specification for release and stability or for comparability, at the present time. Such particles constitute microgram levels of protein with currently monitored sizes ≥10 μm representing the largest fraction. These levels are well below what is detected or reported for other product quality attributes. Subvisible particles remain a product quality attribute that is also qualified in clinical trials. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association.
Bailey L.A.,University of Manchester |
Hatton D.,Biopharmaceutical Development |
Field R.,Biopharmaceutical Development |
Dickson A.J.,University of Manchester
Biotechnology and Bioengineering | Year: 2012
Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies, due to their ability to perform correct post-translational modifications. A potential issue when utilizing CHO cells for therapeutic protein production is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval for the desired therapeutic. In this study, we investigated a model unstable GS-CHO cell line over a continuous period of approximately 100 generations to determine markers of mechanisms that underlie instability. In this cell line, stability of expression was retained for 40-50 generations after which time a 40% loss in antibody production was detected. The instability observed within the cell line was not due to a loss in recombinant gene copy number or decreased expression of mRNA encoding for recombinant antibody H or L chain, but was associated with lower cumulative cell time values and an apparent increased sensitivity to cellular stress (exemplified by increased mRNA expression of the stress-inducible gene GADD153). Changes were also noted in cellular metabolism during LTC (alterations to extracellular alanine accumulation, and enhanced rates of glucose and lactate utilization, during the exponential and decline phase of batch culture, respectively). Our data indicates the breadth of changes that may occur to recombinant CHO cells during LTC ranging from instability of recombinant target production at a post-mRNA level to metabolic events. Definition of the mechanisms, regulatory events, and linkages underpinning cellular phenotype changes require further detailed analysis at a molecular level. © 2012 Wiley Periodicals, Inc.
Veneziale R.W.,Pfizer |
Kishnani N.S.,Merck And Co. |
Nelson J.,Charles River Preclinical Services Nevada |
Resendez J.C.,MPI Research |
And 12 more authors.
Gene Therapy | Year: 2012
The safety and toxicokinetics of SCH 721015, an adenovirus encoding the human interferon alpha-2b gene, and Syn3 (SCH 209702), a novel excipient, were assessed in cynomolgus monkeys administered intravesical doses of 2.5 × 10E11 or 1.25 × 10E13 particles SCH 721015 in 25 mg Syn3 or 25 mg Syn3 alone on study days 1 and 91. There was no systemic toxicity. Monkeys dosed with SCH 721015 in Syn3 were positive for SCH 721015-specific DNA in the urine for 2 to 3 days following each dose and had interferon alpha-2b protein in the urine for 1-3 days after a single dose and in fewer animals after a second dose. Intracystic administration was associated with inflammation and focal/multifocal ulceration in the urinary bladder and irritation in the ureters and urethra at necropsy. The physical trauma from catheterization and filling/emptying of the bladder was likely a contributing factor and Syn3 exacerbated the trauma. There was nearly complete resolution of these findings 2 months after the last dose. The trauma to the bladder likely contributed to low, transient systemic exposure to Syn3, SCH 721015 and human interferon protein. The results of this study support the clinical investigation of SCH 721015 in Syn3. © 2012 Macmillan Publishers Limited All rights reserved.
PubMed | Biopharmaceutical Development
Type: Journal Article | Journal: Journal of pharmaceutical sciences | Year: 2016
This study highlights the significance of the freezing step and the critical role it can play in modulating process performance and product quality during freeze-drying. For the model protein formulation evaluated, the mechanism of freezing had a significant impact on cake appearance, a potential critical product quality attribute for a lyophilized drug product. Contrary to common knowledge, a freezing step with annealing resulted in 20% increase in primary drying time compared to without annealing. In addition, annealing resulted in poor cake appearance with shrinkage, cracks, and formation of a distinct skin at the top surface of the cake. Finally, higher product resistance (7.5 cm(2).Torr.hr/g) was observed in the case of annealing compared to when annealing was not included (5 cm(2).Torr.hr/g), which explains the longer primary drying time due to reduced sublimation rates. An alternative freezing option using controlled ice nucleation resulted in reduced primary drying time (i.e., 30% reduction compared to annealing) and a more homogenous batch with elegant uniform (i.e., significantly improved) cake appearance. Here, a mechanistic understanding of the distinct differences in cake appearance as a function of freezing mechanism is proposed within the context of ice nucleation temperature, ice crystal growth, and presumed solute distribution within the frozen matrix.