RESEARCH ARTICLE – Pharmaceutical Biotechnology
Characterization of Submicron (0.1–1 m) Particles in Therapeutic Proteins by Nanoparticle Tracking Analysis REKHA VASUDEV,1 SAM MATHEW,1 NATALIYA AFONINA2 1 2
Bristol-Myers Squibb, Molecular and Analytical Development, New Jersey AN Biologics Consulting LLC, Pennsylvania
Received 20 October 2014; revised 15 January 2015; accepted 2 February 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24411 ABSTRACT: The importance of 0.1–1 m submicron particles characterization in therapeutic proteins, which was limited because of a lack of suitable methods, has been recognized recently. An application of nanoparticle tracking analysis (NTA) for characterization of 18 lots of recombinant fusion protein (rP1) drug product presentation along with stressed samples of this material exposed to heat at 50°C, agitation, and UV light was studied. In addition, monodisperse polystyrene standards with nominal sizes of 60–800 nm and rP1 samples spiked with 100–400 nm polystyrene standards were analyzed. The NTA technique was capable of demonstrating good sizing of monodisperse polystyrene standards, detect small particle size population in 800 nm standard, and resolve three size populations in the mixture of four standards (60–400 nm). The NTA was also capable of resolving 400 nm polystyrene standard from the main rP1 peak, but was not able to resolve 100 and 200 um standards because of the particle distribution profiles overlap. A characterization of 0.1–1 m submicron particles in rP1 showed a relatively diverse range of mean particle diameters, D90, and size distributions, which was not linked to the lots storage duration prior to analysis. The size distribution profile of rP1was specific for a single lot and did not show significant variability, which allowed detection of larger particle population in stressed samples compared with a control. Overall, the NTA technique is suitable for characterization of submicron particles in a studied therapeutic protein. However, the NTA can only be used as a semiquantitative C 2015 Wiley Periodicals, Inc. and methodology, because frequent sample dilution is required to achieve optimal particle concentration. the American Pharmacists Association J Pharm Sci Keywords: nanoparticle analysis; therapeutic proteins; aggregation; heat; agitation; UV exposure
INTRODUCTION Protein-based therapeutic drugs manufactured in liquid form or those reconstituted from a lyophilized cake typically are colloidal solutions with a presence of a relatively small fraction of protein aggregates when stored at appropriate conditions.1–4 Many different mechanical and/or physicochemical factors outside these conditions, including a presence of small foreign particles from filling pumps (nucleus), changes in pH, temperature, freeze–thaw cycles, and others may impact an existing quasiequilibrium of the protein solution.2,5–8 This may lead to the formation of new aggregates and subvisible particulates, as proteins are inclined to several chemical and physical degradation pathways.9–12 The presence of aggregates in therapeutic proteins could compromise product purity and may lead to unwanted immunogenicity.13,14 Animal studies demonstrated that the presence of aggregates in injected drug products may break drug tolerance while injection of disaggregated drug product material induced it. However, there are no studies that directly correlate specific impact of aggregate conformations on changes in the human immune system (uncertain risk).1,13–16 It has also been discussed that subvisible particles of 2–10 :m size might have a potential to cause immunogenicity because their concentration is much higher than that of ࣙ10 :m size particles and can dramatically increase under certain stress conditions. This is
Correspondence to: Sam Mathew (Telephone: +609-818-5642; Fax: +609-8183809; E-mail: [email protected]) Journal of Pharmaceutical Sciences
C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
specifically critical in long-term chronic treatment.13,16 Therefore, micron (subvisible) particulates in therapeutic proteins need to be characterized and monitored to ensure drug product quality. The importance of analyzing submicron particulates in the range of 0.1–1 :m in therapeutic proteins has been recognized recently and has been evaluated as a characterization tool in conjunction with other techniques used for 2–10 :m subvisible particle assessment.1 The characterization of aggregates and enumeration of particles in therapeutic proteins present a technical challenge because of their heterogeneous sizes (0.1–ࣙ100 :m), complex properties and morphology, difficulties in segregation, and limitation of suitable methods for measurement.2–4,7,8,11,14,17–21 Most importantly, none of the existing methods alone could cover the entire size range of particulate matter in therapeutic protein solutions. There are few approaches to classification and detection of protein aggregates based on their physical–chemical properties and methods.9–12 On the basis of their size, the aggregates are segregated in five major categories: high-molecular-weight species (dimersoligomers, 1 nm to
Characterization of Submicron (0.1–1 m) Particles in Therapeutic Proteins by Nanoparticle Tracking Analysis REKHA VASUDEV,1 SAM MATHEW,1 NATALIYA AFONINA2 1 2
Bristol-Myers Squibb, Molecular and Analytical Development, New Jersey AN Biologics Consulting LLC, Pennsylvania
Received 20 October 2014; revised 15 January 2015; accepted 2 February 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24411 ABSTRACT: The importance of 0.1–1 m submicron particles characterization in therapeutic proteins, which was limited because of a lack of suitable methods, has been recognized recently. An application of nanoparticle tracking analysis (NTA) for characterization of 18 lots of recombinant fusion protein (rP1) drug product presentation along with stressed samples of this material exposed to heat at 50°C, agitation, and UV light was studied. In addition, monodisperse polystyrene standards with nominal sizes of 60–800 nm and rP1 samples spiked with 100–400 nm polystyrene standards were analyzed. The NTA technique was capable of demonstrating good sizing of monodisperse polystyrene standards, detect small particle size population in 800 nm standard, and resolve three size populations in the mixture of four standards (60–400 nm). The NTA was also capable of resolving 400 nm polystyrene standard from the main rP1 peak, but was not able to resolve 100 and 200 um standards because of the particle distribution profiles overlap. A characterization of 0.1–1 m submicron particles in rP1 showed a relatively diverse range of mean particle diameters, D90, and size distributions, which was not linked to the lots storage duration prior to analysis. The size distribution profile of rP1was specific for a single lot and did not show significant variability, which allowed detection of larger particle population in stressed samples compared with a control. Overall, the NTA technique is suitable for characterization of submicron particles in a studied therapeutic protein. However, the NTA can only be used as a semiquantitative C 2015 Wiley Periodicals, Inc. and methodology, because frequent sample dilution is required to achieve optimal particle concentration. the American Pharmacists Association J Pharm Sci Keywords: nanoparticle analysis; therapeutic proteins; aggregation; heat; agitation; UV exposure
INTRODUCTION Protein-based therapeutic drugs manufactured in liquid form or those reconstituted from a lyophilized cake typically are colloidal solutions with a presence of a relatively small fraction of protein aggregates when stored at appropriate conditions.1–4 Many different mechanical and/or physicochemical factors outside these conditions, including a presence of small foreign particles from filling pumps (nucleus), changes in pH, temperature, freeze–thaw cycles, and others may impact an existing quasiequilibrium of the protein solution.2,5–8 This may lead to the formation of new aggregates and subvisible particulates, as proteins are inclined to several chemical and physical degradation pathways.9–12 The presence of aggregates in therapeutic proteins could compromise product purity and may lead to unwanted immunogenicity.13,14 Animal studies demonstrated that the presence of aggregates in injected drug products may break drug tolerance while injection of disaggregated drug product material induced it. However, there are no studies that directly correlate specific impact of aggregate conformations on changes in the human immune system (uncertain risk).1,13–16 It has also been discussed that subvisible particles of 2–10 :m size might have a potential to cause immunogenicity because their concentration is much higher than that of ࣙ10 :m size particles and can dramatically increase under certain stress conditions. This is
Correspondence to: Sam Mathew (Telephone: +609-818-5642; Fax: +609-8183809; E-mail: [email protected]) Journal of Pharmaceutical Sciences
C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
specifically critical in long-term chronic treatment.13,16 Therefore, micron (subvisible) particulates in therapeutic proteins need to be characterized and monitored to ensure drug product quality. The importance of analyzing submicron particulates in the range of 0.1–1 :m in therapeutic proteins has been recognized recently and has been evaluated as a characterization tool in conjunction with other techniques used for 2–10 :m subvisible particle assessment.1 The characterization of aggregates and enumeration of particles in therapeutic proteins present a technical challenge because of their heterogeneous sizes (0.1–ࣙ100 :m), complex properties and morphology, difficulties in segregation, and limitation of suitable methods for measurement.2–4,7,8,11,14,17–21 Most importantly, none of the existing methods alone could cover the entire size range of particulate matter in therapeutic protein solutions. There are few approaches to classification and detection of protein aggregates based on their physical–chemical properties and methods.9–12 On the basis of their size, the aggregates are segregated in five major categories: high-molecular-weight species (dimersoligomers, 1 nm to
