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Flow Imaging: Moving Toward Best Practices for Subvisible Particle Quantitation in Protein Products
LESSONS LEARNED Flow Imaging: Moving Toward Best Practices for Subvisible Particle Quantitation in Protein Products GLENN A. WILSON,1 MARK CORNELL MANNING2,3 1
West Coast BioDesign, Santa Barbara, California
2
Legacy BioDesign, Johnstown, Colorado
3
Department of Chemistry, Colorado State University, Fort Collins, Colorado
Received 29 November 2012; accepted 17 December 2012 Published online 9 January 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23445 ABSTRACT: The potential impact of subvisible particles (SVPs) in protein therapeutic products has received a great deal of attention recently. As a result, new analytical methods have emerged to characterize and quantify SVPs. Among these, flow imaging (also called flow R and Micro-Flow microscopy) has been widely employed. As we have used both FlowCAM TM Imaging in a variety of development projects, we wished to share our experiences and observations, with the intention of fostering a discussion that will lead to best practices for the use of flow imaging to quantify SVP levels in biopharmaceuticals. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1133–1134, 2013 Keywords: flow imaging; subvisible particles; image analysis; imaging methods; protein formulation; particle size; analytical biochemistry
INTRODUCTION In the development of biopharmaceutical drug products, a great deal of attention has turned to subvisible particles (SVPs) and their potential to impact drug safety.1,2 As a result, a number of analytical methods have been devised over the last few years to detect, characterize, and quantify particles in the 1–50 :m range. Among these, flow imaging (also called flow microscopy) has become the most prominent. Although a number of articles have appeared using these methods, virtually nothing has been presented on the importance of sample handing, data collection procedures, and postprocessing of data when using these techniques. Our goal for this article is to move the industry rationally toward best practices for flow imaging techniques. The two most commonly used instruments for flow R imaging of protein aggregates are the FlowCAM (Fluid Imaging Technologies, Yarmouth, ME) and Micro-Flow ImagingTM (MFI, Protein Simple, Santa Clara, CA) systems. This article is not meant as an endorsement or an indictment of either instrument, Correspondence to: +970-231-9744; Fax: LegacyBioDesign.com)
Mark Cornell Manning +970-663-6006; E-mail:
(Telephone: Manning@
Journal of Pharmaceutical Sciences, Vol. 102, 1133–1134 (2013) © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association
nor of any competing technologies or vendors. Its purpose is solely to raise awareness that attention needs to be paid to the factors that can impact the quality of flow imaging data. As we share our experiences R , across many projects using both MFI and FlowCAM we hope to engender a discussion among all users of flow imaging systems. There are some differences between these two flow imaging systems that are worth noting. The MFI equipment uses a 5× objective, a fixed 100 :m flow cell, and two software packages, one for data collection R can and one for data analysis. In contrast, FlowCAM employ either a 4× or a 10× objective, can accommodate both disposable and permanent flow cells, and uses a single software package for data collection and analysis. Our experience has shown that neither system provides truly usable data for particles of less than 2 :m in size because of insufficient pixel resolution, despite both instruments allowing the capture of information on particles from 1 :m and larger. Thus, in our opinion, only data for particles of 2 :m or greater in size are worth considering.
LESSONS LEARNED REGARDING FLOW IMAGING In particular, there are five topics we wish to emphasize. Although other aspects of data collection and
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013
1133
1134
WILSON AND MANNING
processing are important, these should receive particular attention. Definition of Particle Size These two manufacturers define the “size” of a particle differently. In the case of MFI, size is defined as equivalent circular diameter (ECD). The ECD of an object is expressed in micrometers and represents the diameter of a sphere that occupies the same twodimensional surface area as the particle. The MFI product platform converts the area of an object into an ECD value using proprietary conversion techniques to avoid the error inherent with performing direct calculations based on field of view dimensions. In contrast, individual particle size using the Fluid Imaging Technologies software employs a measurement technique known as equivalent spherical diameter (ESD). ESD is the mean Feret measurement of the particle based on 36 sample measurements (conducted every 5◦ ). A Feret measurement is the perpendicular distance between parallel tangents touching opposite sides of the particle. While these algorithms provide similar sizes for stable particles, they are not identical definitions. Calibration and Sample Preparation As it is critical not to introduce particles to the samples, high quality water (e.g., 18 M, 0.2 :m filtered) must be used. All sample handling and preparation should be performed in a clean hood to minimize the accumulation of dust and nonproteinaceous particles. Running blanks throughout the course of sample analysis is recommended to ensure system suitability, as is performed with other analytical methods, such as high performance liquid chromatography (HPLC). Care and Use of Flow Cells First, it should be noted that flow cells are not interchangeable between the two platforms. The flow cell from MFI, which is permanent (i.e., not disposable), is coated with silane and is sensitive to cleaning agents and chemicals that will degrade the coating, for example, ethanol or isopropyl alcohol. Although a nonsilane-coated cell is available, it is not recommended by Protein Simple for use with proteins. A flow cell integrity check feature is available, but must be monitored closely to ensure that the integrity of the cell illumination are acceptable. Furthermore, the flow cell cannot be reoriented to ensure the optimum view R offers several window. On the contrary, FlowCAM types of flow cells (both permanent, field of view cells
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013
and disposable cells), all of which are uncoated and generally insensitive to cleaning and chemical agents. These cells must be focused to ensure high quality imR , this is performed prior to the ages. With FlowCAM start of each new method and when the cell is reversed or flipped for cleaning, which is a fairly quick process. On the MFI instrument, focusing is carried out manually when a new flow cell is installed and can take up to three quarters of an hour. Cleaning procedures are important, but vary widely between the two platforms. A discussion of the details is beyond the scope of this article, but the importance for obtaining precise and accurate results cannot be overemphasized. Software Interface With all analytical instrumentation, there is a balance between ease of use and allowing the scientist a greater degree of control over the operating parameters. With MFI, data collection is easy to set up with few parameters to adjust. On the contrary, R has a number of parameters one must FlowCAM define. This allows for greater customization of the method, but does result in a steeper initial learning R is curve. If a method needs to be refined, FlowCAM easier to modify, whereas MFI requires resetting up the entire method. Data Analysis There are significant differences between MFI and R FlowCAM in terms of data analysis, postprocessing capabilities, and particle visualization. The MFI instrument uses a second software package, reports are limited to.pdf format, and easy access to all but a small selection of the particle images is time consumR uses a single software package for ing. FlowCAM data collection and processing, allows export of data reports in a variety of formats, and access to images is much easier.
REFERENCES 1. Carpenter JF, Randoph TW, Jiskoot W, Crommelin DJA, Middaugh CR, Winter G, Fan Y-X, Kirschner S, Verthelyi D, Kozlowski S, Clouse KA, Swann PG, Rosenberg A, Cherney B. 2009. Overlooking subvisible particles in therapeutic protein products: Gaps that may compromise product quality. J Pharm Sci 98(4):1201–1205. 2. Singh SK, Afonina N, Awwad M, Bechtold-Peters K, Blue JT, Chou D, Cromwell M, Krause H-J, Mahler H-C, Meyer BK, Nahri L, Nesta DP, Spitznagel T. 2010. An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics. J Pharm Sci 99(8):3302–3321.
DOI 10.1002/jps
West Coast BioDesign, Santa Barbara, California
2
Legacy BioDesign, Johnstown, Colorado
3
Department of Chemistry, Colorado State University, Fort Collins, Colorado
Received 29 November 2012; accepted 17 December 2012 Published online 9 January 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23445 ABSTRACT: The potential impact of subvisible particles (SVPs) in protein therapeutic products has received a great deal of attention recently. As a result, new analytical methods have emerged to characterize and quantify SVPs. Among these, flow imaging (also called flow R and Micro-Flow microscopy) has been widely employed. As we have used both FlowCAM TM Imaging in a variety of development projects, we wished to share our experiences and observations, with the intention of fostering a discussion that will lead to best practices for the use of flow imaging to quantify SVP levels in biopharmaceuticals. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1133–1134, 2013 Keywords: flow imaging; subvisible particles; image analysis; imaging methods; protein formulation; particle size; analytical biochemistry
INTRODUCTION In the development of biopharmaceutical drug products, a great deal of attention has turned to subvisible particles (SVPs) and their potential to impact drug safety.1,2 As a result, a number of analytical methods have been devised over the last few years to detect, characterize, and quantify particles in the 1–50 :m range. Among these, flow imaging (also called flow microscopy) has become the most prominent. Although a number of articles have appeared using these methods, virtually nothing has been presented on the importance of sample handing, data collection procedures, and postprocessing of data when using these techniques. Our goal for this article is to move the industry rationally toward best practices for flow imaging techniques. The two most commonly used instruments for flow R imaging of protein aggregates are the FlowCAM (Fluid Imaging Technologies, Yarmouth, ME) and Micro-Flow ImagingTM (MFI, Protein Simple, Santa Clara, CA) systems. This article is not meant as an endorsement or an indictment of either instrument, Correspondence to: +970-231-9744; Fax: LegacyBioDesign.com)
Mark Cornell Manning +970-663-6006; E-mail:
(Telephone: Manning@
Journal of Pharmaceutical Sciences, Vol. 102, 1133–1134 (2013) © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association
nor of any competing technologies or vendors. Its purpose is solely to raise awareness that attention needs to be paid to the factors that can impact the quality of flow imaging data. As we share our experiences R , across many projects using both MFI and FlowCAM we hope to engender a discussion among all users of flow imaging systems. There are some differences between these two flow imaging systems that are worth noting. The MFI equipment uses a 5× objective, a fixed 100 :m flow cell, and two software packages, one for data collection R can and one for data analysis. In contrast, FlowCAM employ either a 4× or a 10× objective, can accommodate both disposable and permanent flow cells, and uses a single software package for data collection and analysis. Our experience has shown that neither system provides truly usable data for particles of less than 2 :m in size because of insufficient pixel resolution, despite both instruments allowing the capture of information on particles from 1 :m and larger. Thus, in our opinion, only data for particles of 2 :m or greater in size are worth considering.
LESSONS LEARNED REGARDING FLOW IMAGING In particular, there are five topics we wish to emphasize. Although other aspects of data collection and
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013
1133
1134
WILSON AND MANNING
processing are important, these should receive particular attention. Definition of Particle Size These two manufacturers define the “size” of a particle differently. In the case of MFI, size is defined as equivalent circular diameter (ECD). The ECD of an object is expressed in micrometers and represents the diameter of a sphere that occupies the same twodimensional surface area as the particle. The MFI product platform converts the area of an object into an ECD value using proprietary conversion techniques to avoid the error inherent with performing direct calculations based on field of view dimensions. In contrast, individual particle size using the Fluid Imaging Technologies software employs a measurement technique known as equivalent spherical diameter (ESD). ESD is the mean Feret measurement of the particle based on 36 sample measurements (conducted every 5◦ ). A Feret measurement is the perpendicular distance between parallel tangents touching opposite sides of the particle. While these algorithms provide similar sizes for stable particles, they are not identical definitions. Calibration and Sample Preparation As it is critical not to introduce particles to the samples, high quality water (e.g., 18 M, 0.2 :m filtered) must be used. All sample handling and preparation should be performed in a clean hood to minimize the accumulation of dust and nonproteinaceous particles. Running blanks throughout the course of sample analysis is recommended to ensure system suitability, as is performed with other analytical methods, such as high performance liquid chromatography (HPLC). Care and Use of Flow Cells First, it should be noted that flow cells are not interchangeable between the two platforms. The flow cell from MFI, which is permanent (i.e., not disposable), is coated with silane and is sensitive to cleaning agents and chemicals that will degrade the coating, for example, ethanol or isopropyl alcohol. Although a nonsilane-coated cell is available, it is not recommended by Protein Simple for use with proteins. A flow cell integrity check feature is available, but must be monitored closely to ensure that the integrity of the cell illumination are acceptable. Furthermore, the flow cell cannot be reoriented to ensure the optimum view R offers several window. On the contrary, FlowCAM types of flow cells (both permanent, field of view cells
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 3, MARCH 2013
and disposable cells), all of which are uncoated and generally insensitive to cleaning and chemical agents. These cells must be focused to ensure high quality imR , this is performed prior to the ages. With FlowCAM start of each new method and when the cell is reversed or flipped for cleaning, which is a fairly quick process. On the MFI instrument, focusing is carried out manually when a new flow cell is installed and can take up to three quarters of an hour. Cleaning procedures are important, but vary widely between the two platforms. A discussion of the details is beyond the scope of this article, but the importance for obtaining precise and accurate results cannot be overemphasized. Software Interface With all analytical instrumentation, there is a balance between ease of use and allowing the scientist a greater degree of control over the operating parameters. With MFI, data collection is easy to set up with few parameters to adjust. On the contrary, R has a number of parameters one must FlowCAM define. This allows for greater customization of the method, but does result in a steeper initial learning R is curve. If a method needs to be refined, FlowCAM easier to modify, whereas MFI requires resetting up the entire method. Data Analysis There are significant differences between MFI and R FlowCAM in terms of data analysis, postprocessing capabilities, and particle visualization. The MFI instrument uses a second software package, reports are limited to.pdf format, and easy access to all but a small selection of the particle images is time consumR uses a single software package for ing. FlowCAM data collection and processing, allows export of data reports in a variety of formats, and access to images is much easier.
REFERENCES 1. Carpenter JF, Randoph TW, Jiskoot W, Crommelin DJA, Middaugh CR, Winter G, Fan Y-X, Kirschner S, Verthelyi D, Kozlowski S, Clouse KA, Swann PG, Rosenberg A, Cherney B. 2009. Overlooking subvisible particles in therapeutic protein products: Gaps that may compromise product quality. J Pharm Sci 98(4):1201–1205. 2. Singh SK, Afonina N, Awwad M, Bechtold-Peters K, Blue JT, Chou D, Cromwell M, Krause H-J, Mahler H-C, Meyer BK, Nahri L, Nesta DP, Spitznagel T. 2010. An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics. J Pharm Sci 99(8):3302–3321.
DOI 10.1002/jps