Scientific Publication - Silicone Oil Microdroplets and Protein Aggregates in Repackaged Bevacizumab and Ranibizumab: Effects of Long-term Storage and Product Mishandling.

AUTHORS Lu Liu, David A Ammar, Lindsey Ross, Naresh Mandava, Malik Kahook and John Carpenter

Published Online at IOVS Online Library DOI: 10.1167/iovs.10-6431

Purpose: To quantify levels of subvisible particles and protein aggregates in repackaged bevacizumab obtained from compounding pharmacies as well as in samples of bevacizumab and ranibizumab tested in controlled laboratory experiments.

Methods: Repackaged bevacizumab was purchased from four external compounding pharmacies. For controlled laboratory studies, bevacizumab and placebo were drawn into plastic syringes and incubated at -20°C, 4°C and room temperature (with and without exposure to light) for12 weeks. Additionally, mechanical shock occurring during shipping was mimicked with syringes containing bevacizumab.

Particle counts and size distributions were quantified by Micro-Flow Imaging.

Levels of monomer and soluble aggregates of bevacizumab were determined with size exclusion high performance liquid chromatography (SE-HPLC).

Results: Repackaged bevacizumab from the compounding pharmacies had a wide range of particle counts (89,006±56,406 to 602,062±18,349 /mL). Bevacizumab sampled directly from original glass vial had particle counts of 63,839±349/mL. There was up to 10% monomer loss for repackaged bevacizumab. Laboratory samples of repackaged bevacizumab and placebo had initial particle counts, respectively, of 283,675±60,494/mL and 492,314±389,361/mL. Freeze-thawing of both bevacizumab and placebo samples led to >1.2 million particles/mL. In all repackaged samples, the majority of particles were due to silicone oil. SE-HPLC showed no significant differences for repackaged samples incubated in the laboratory under various conditions compared with bevacizumab directly from vial. However, repeated freeze-thawing caused over 10% monomer loss.

Conclusion: Bevacizumab repackaged in plastic syringes could contain protein aggregates and was contaminated by silicone oil microdroplets. Freeze-thawing or other mishandling can further increase levels of particle contaminants.

The complete article can be found at:
http://www.iovs.org/content/early/2010/10/27/iovs.10-6431.abstract

Scientific Publication - High-Molecular-Weight Aggregates in Repackaged Bevacizumab

Editor's Note: This article was also featured in an earlier newsletter, but is included here as it provides useful background information on why MFI was chosen for use in the study mentioned above.

AUTHORS: Kahook MY, Liu L, Ruzycki P, Mandava N, Carpenter JF, Petrash JM, Ammar DA.

Published in Retina 30(6): 887-892.

IN THIS ARTICLE:

In comparison to SEC and Polyacrylamide Gel Electrophoresis methods...

"To quantify the number of particles between different syringes, the more sensitive MFI method was performed. This approach showed a substantially higher number of >1-μm particles in the material sourced from a CP (compounding pharmacy) when compared with material not prepared by a CP." ". . . Of all the analytical studies shown here, MFI is the only one sensitive enough to detect these larger particles because: 1) although micron-sized particles contain millions of proteins, they represent a trace amount of the total protein and would be nearly invisible by polyacrylamide gel electrophoresis; 2) the size of these particles would preclude them from entering the size exclusion chromatography column; and 3) in addition to protein, some of the particles could be resulting from nonprotein materials such as silicone oil from the syringes."

The complete article can be found at:
Protein Particles and Immunogenicity of Therapeutic Proteins (Nov 12-13, 2009)

Meeting Report - Meeting report on protein particles and immunogenicity of therapeutic proteins: Filling in the gaps in risk evaluation and mitigation.

MEETING HELD: Nov 12-13, 2009

AUTHORS: John Carpenter, Barry Cherney, Anthony Lubinecki, Stacey Ma, Ewa Marszal, Anthony Mire-Sluis, Thomas Nikolai, Jeanne Novak, Jack Ragheb and Jan Simak.

Published in Biologicals 38 (2010): 602-611.

ABSTRACT:
This meeting was successful in achieving its main goals: (1) summarize currently available information on the origin, detection, quantification and characterization of sub-visible particulates in protein products, available information on their clinical importance, and potential strategies for evaluating and mitigating risk to product quality, and (2) foster communication among academic, industry, and regulatory scientists to define the capabilities of current analytical methods, to promote the development of improved methods, and to stimulate investigations into the impact of large protein aggregates on immunogenicity. There was a general consensus that a considerable amount of interesting scientific information was presented and many stimulating conversations were begun. It is clear that this aspect of protein characterization is in its initial stages. As the development of these new methods progress, it is hoped that they will shed light on the role of protein particulates on product quality, safety, and efficacy. A topic which seemed appropriate for short term follow up was to hold further discussions concerning the development and preparation of one or more standard preparations of protein particulates. This would be generally useful to facilitate comparison of results among different studies, methods, and laboratories, and to foster further development of a common understanding among laboratories and health authorities which is essential to making further progress in this emerging field.

The complete article can be found at:
http://www3.interscience.wiley.com/journal/121378778/abstract

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