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Hassan A, Diederichs T, Garidel P, Heerklotz H. Anomalous Clouding Behavior of Polysorbate 80─Deciphering the Role of Nonesterified Components. Mol Pharm 2025. [PMID: 40365937 DOI: 10.1021/acs.molpharmaceut.4c01268] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/15/2025]
Abstract
Polysorbates (PSs) are key excipients for the colloidal stability of biopharmaceuticals with unique properties. A comprehensive understanding of the physicochemical properties of these multicomponent products is essential to address potential stability issues without compromising their functionality. Here, we demonstrate that polysorbate 80 HP (PS80) shows an anomalous clouding, i.e., a thermotropic liquid-liquid phase separation behavior, which cannot adequately be explained by the conventional interpretation assuming a pseudobinary system. In a binary two-phase system of surfactant and buffer, an increase in the total surfactant concentration increases the fraction of the surfactant-rich phase in the respective proportion (lever rule). PS80 within about 7 K of the lower critical solution temperature fails to comply with this; concentrations and compositions of the coexisting phases change with the total concentration. This renders the phases more alike and, at some point, eliminates phase separation. This significant deviation from the pseudobinary phase behavior can be resolved by conceptually dividing the numerous chemical species in PS80 into two independent pseudocomponents, PS80-I and -II. Ternary phase diagrams derived from this approach successfully explain the observed anomalous behavior. RP-UPLC-MS analysis indicated a concentration-dependent redistribution of the nonesterified components (NECs), suggesting, along with other evidence, that NECs are key constituents of component II. Specifically, free polyethylene glycol (PEG) and/or PEG-sorbitans seem to function as intrinsic cosurfactant(s) within PS80, modulating its wetting and clouding properties. The latter is important for interaction, association, and phase separation properties in biologics.
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Affiliation(s)
- Alaa Hassan
- Institute of Pharmaceutical Sciences, Department of Pharmaceutics, University of Freiburg, Hermann-Herder-Str. 9, Freiburg 79104, Germany
- Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt
| | - Tim Diederichs
- PDB-TIP, Innovation Unit, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer St. 65, Biberach an der Riss 88397, Germany
| | - Patrick Garidel
- PDB-TIP, Innovation Unit, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer St. 65, Biberach an der Riss 88397, Germany
- Martin Luther University Halle-Wittenberg | MLU · Institute of Chemistry, Physical Chemistry, von-Danckelmann-Platz 4, Halle D-06120, Germany
| | - Heiko Heerklotz
- Institute of Pharmaceutical Sciences, Department of Pharmaceutics, University of Freiburg, Hermann-Herder-Str. 9, Freiburg 79104, Germany
- Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto M5S 3M2, Ontario, Canada
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2
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Geyer FA, Domján J, Szalai TV, Rapi Z, Varga Z, Marosi G, Nagy ZK, Hirsch E. Stabilizing effect of HP-β-CD on infliximab in liquid formulations and a solid formulation produced by electrospinning. Eur J Pharm Sci 2025; 206:107014. [PMID: 39818362 DOI: 10.1016/j.ejps.2025.107014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2024] [Revised: 12/30/2024] [Accepted: 01/13/2025] [Indexed: 01/18/2025]
Abstract
The development of stable biopharmaceutical formulations, such as monoclonal antibodies, poses a great challenge in the pharmaceutical industry. This study investigated the stabilizing effect of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in liquid and solid formulations of infliximab during processing and storage. The solid formulation was produced by a scaled-up high-speed electrospinning method, resulting in a product suitable for reconstitution with excellent dissolution properties. Liquid formulations contained exclusively HP-β-CD as a stabilizing excipient at different concentrations. Both types of formulations were stored under various conditions for up to 6 months, and infliximab stability was compared based on monomer recovery and the analysis of fragments and aggregates. The solid formulation stored at low (-18 °C, 4 °C) temperatures and lower humidity conditions show the most promising results. Moreover, the monomer loss was less than 10 % even at room temperature storage after 2 months, indicating a potentially good temperature tolerance. Increasing HP-β-CD content in liquid formulations significantly improved the long-term stability of infliximab while enhancing protection against mechanical stress. Furthermore, to better understand the stabilizing mechanism of HP-β-CD on infliximab, the molecular interactions were investigated by bio-layer interferometry and isothermal titration calorimetry methods. The experiments proved that the presence of HP-β-CD decreased infliximab self-interaction, correlating with the aggregation-suppressing effect of HP-β-CD observed during the stress- and long-term stability studies. The results demonstrate the potential of HP-β-CD as a stabilizing excipient in liquid and solid formulations of infliximab.
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Affiliation(s)
- Fanni Angéla Geyer
- Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111, Budapest, Hungary
| | - Júlia Domján
- Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111, Budapest, Hungary
| | - Tibor Viktor Szalai
- Department of Inorganic and Analytical Chemistry, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Műegyetem rkp. 3., H-1111, Budapest, Hungary; Medicinal Chemistry Research Group, HUN-REN Research Centre for Natural Sciences, Magyar tudósok krt. 2, 1117, Budapest, Hungary
| | - Zsolt Rapi
- Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111, Budapest, Hungary
| | - Zoltán Varga
- Biological Nanochemistry Research Group, Institute of Materials and Environmental Chemistry, HUN-REN Research Center for Natural Sciences, Magyar tudósok körútja 2, H-1117, Budapest, Hungary; Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, Műegyetem rkp. 3, Budapest, 1111, Hungary
| | - György Marosi
- Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111, Budapest, Hungary
| | - Zsombor K Nagy
- Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111, Budapest, Hungary
| | - Edit Hirsch
- Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, H-1111, Budapest, Hungary.
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Arte KS, Chen M, Patil CD, Huang Y, Qu L, Zhou Q. Recent advances in drying and development of solid formulations for stable mRNA and siRNA lipid nanoparticles. J Pharm Sci 2025; 114:805-815. [PMID: 39694272 DOI: 10.1016/j.xphs.2024.12.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2024] [Revised: 12/03/2024] [Accepted: 12/04/2024] [Indexed: 12/20/2024]
Abstract
Current RNA lipid nanoparticle (LNP) based products are typically liquid formulations that require ultra-cold storage temperatures for stability. To address this limitation, recent efforts have focused on enhancing stability and enabling room temperature storage by converting these formulations into solid forms through drying processes such as lyophilization, spray drying, and spray-freeze drying. Nevertheless, the drying process itself can influence the stability of RNA/LNP formulations. Therefore, understanding the factors that contribute to instability during drying is essential. The choice of drying technique for LNPs depends on factors such as the mode of delivery, lipid components, and desired final product characteristics. Additionally, the drying mechanism and associated stresses must also be carefully considered. Drying methods involve a range of process parameters related to formulation, process settings, and the manufacturing environment. It is essential to understand how these parameters influence the final solid-state products' attributes, including appearance, moisture content, flow properties, and reconstitution time, as these can significantly affect the physical and chemical stability of the formulation. This review focuses on various drying techniques and their impact on the stability of RNA/LNP-based systems.
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Affiliation(s)
- Kinnari Santosh Arte
- Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA
| | - Manlin Chen
- Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA
| | - Chanakya D Patil
- Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA
| | - Yijing Huang
- Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA
| | - Li Qu
- Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
| | - Qi Zhou
- Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
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4
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Ling J, Du Y, Wuelfing WP, Buist N, Krishnamachari Y, Xi H, Templeton AC, Su Y. Molecular mechanisms for stabilizing biologics in the solid state. J Pharm Sci 2025; 114:736-765. [PMID: 39617053 DOI: 10.1016/j.xphs.2024.11.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2024] [Revised: 11/09/2024] [Accepted: 11/15/2024] [Indexed: 12/19/2024]
Abstract
Protein drugs exhibit challenges of biophysical and biochemical instability due to their structural complexity and rich dynamics. Solid-state biologics aim to enhance stability by increasing molecular rigidity within the formulation matrix, representing a primary category of drug products alongside sterile liquid formulations. Understanding the molecular mechanisms behind the stabilization and destabilization of protein drugs, influenced by formulation composition and drying processes, provides scientific rationale for drug product design. This review aims to elaborate on the two primary models of water-to-sugar substitution and matrix vitrification, respectively, via thermodynamic and kinetic stabilization. It offers an up-to-date review of experimental investigations into these hypotheses, specifically elucidating protein structure and protein-excipient interactions at the molecular level, molecular dynamics across a broad range of motion regimes, and microscopic attributes such as protein-sugar and protein-salt miscibility and microenvironmental acidity, in relevant liquid, frozen, and solid states, using advanced biophysical techniques for solid-state analysis. Moreover, we discuss how these mechanistic understandings facilitate the investigation and prediction of critical stability behaviors and enables the design of solid biological drug products.
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Affiliation(s)
- Jing Ling
- Pharmaceutical Sciences and Clinical Supply, Merck & Co., Inc., Rahway, NJ 07065, USA
| | - Yong Du
- Analytical Research and Development, Merck & Co., Inc., Rahway, NJ 07065, USA
| | - W Peter Wuelfing
- Pharmaceutical Sciences and Clinical Supply, Merck & Co., Inc., Rahway, NJ 07065, USA
| | - Nicole Buist
- Pharmaceutical Sciences and Clinical Supply, Merck & Co., Inc., Rahway, NJ 07065, USA
| | - Yogita Krishnamachari
- Pharmaceutical Sciences and Clinical Supply, Merck & Co., Inc., Rahway, NJ 07065, USA
| | - Hanmi Xi
- Analytical Research and Development, Merck & Co., Inc., Rahway, NJ 07065, USA
| | - Allen C Templeton
- Pharmaceutical Sciences and Clinical Supply, Merck & Co., Inc., Rahway, NJ 07065, USA.
| | - Yongchao Su
- Pharmaceutical Sciences and Clinical Supply, Merck & Co., Inc., Rahway, NJ 07065, USA; Analytical Research and Development, Merck & Co., Inc., Rahway, NJ 07065, USA.
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5
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Felix MN, Waerner T, Lakatos D, Reisinger B, Fischer S, Garidel P. Polysorbates degrading enzymes in biotherapeutics - a current status and future perspectives. Front Bioeng Biotechnol 2025; 12:1490276. [PMID: 39867473 PMCID: PMC11760601 DOI: 10.3389/fbioe.2024.1490276] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Accepted: 12/02/2024] [Indexed: 01/28/2025] Open
Abstract
Polysorbates, in particular polysorbate (PS) 20 and 80, are the most commonly used surfactants for stabilising biotherapeutics produced by biotechnological processes. PSs are derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids of varying chain length and degree of saturation. In the past, these surfactants have been reported to have specific liabilities. Chemical (oxidations and hydrolyses) and enzymatic degradations have been reported to affect the stability of PS in drug products. Specifically, the presence of trace amounts (sub-ppm) of certain host cell proteins (HCPs) can induce enzymatic PS degradation, which can lead to the release of free fatty acids during storage over time. Enzymatic polysorbate degradation may impair the functionality of the surfactant in stabilising therapeutic proteins, leading to the formation of visible and/or sub-visible particles in biopharmaceutical drug products. This review summarises the enzymes currently known to be involved in the degradation of polysorbate in mammalian biotechnological processes for therapeutic proteins. In recent years, advanced analytical methods have been developed to qualify and quantify the PS-degrading enzymes. Most of these assays are based on mass spectrometry with a preceding HCP enrichment approach. Efforts were made to measure the enzyme activity and correlate it with observed PS degradation. The impact on drug product quality attributes, including fatty acid solubility and phase separation, up to the formation of visible particles, and the potential induction of protein and protein/fatty acid mixed particles as well as the sensitivity of specific PS quality towards enzymatic degradation, was considered. Various drug substance (DS) mitigation strategies related to the occurrence of PS degrading enzymes are discussed as amongst them the generation of stable HCP knockout cell lines, which are also carefully analysed. The underlying opinion article reflects the undergoing discussions related to PS degrading enzymes and focusses on (i) impact on drug product, (ii) analytics for identification/quantification (characterisation) of the PS degrading enzymes, (iii) enzyme activity (iv) currently identified enzymes, and (v) potential mitigation strategies to avoid enzymatic PS degradation during DS manufacturing.
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Affiliation(s)
- Marius Nicolaus Felix
- Analytical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co., KG, Innovation Unit, Biberach an der Riss, Germany
| | - Thomas Waerner
- Analytical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co., KG, Innovation Unit, Biberach an der Riss, Germany
| | - Daniel Lakatos
- Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co., KG, Innovation Unit, Biberach an der Riss, Germany
| | - Bernd Reisinger
- Analytical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co., KG, Innovation Unit, Biberach an der Riss, Germany
| | - Simon Fischer
- Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co., KG, Innovation Unit, Biberach an der Riss, Germany
| | - Patrick Garidel
- Pharmaceutical Development Biologicals, TIP, Boehringer Ingelheim Pharma GmbH & Co., KG, Innovation Unit, Biberach an der Riss, Germany
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6
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Lay-Fortenbery A, Tower CW, Ezeajughi E, Calahan J, Duru C, Matejtschuk P, Munson EJ. Predicting the Stability of Lyophilized Human Serum Albumin Formulations Containing Sucrose and Trehalose Using Solid-State NMR Spectroscopy: Effect of Storage Temperature on 1H T 1 Relaxation Times. AAPS J 2024; 26:40. [PMID: 38570383 DOI: 10.1208/s12248-024-00900-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2023] [Accepted: 02/14/2024] [Indexed: 04/05/2024] Open
Abstract
In a lyophilized protein/disaccharide system, the ability of the disaccharide to form a homogeneous mixture with the protein and to slow the protein mobility dictates the stabilization potential of the formulation. Human serum albumin was lyophilized with sucrose or trehalose in histidine, phosphate, or citrate buffer. 1H T1 relaxation times were measured by solid-state NMR spectroscopy and were used to assess the homogeneity and mobility of the samples after zero, six, and twelve months at different temperatures. The mobility of the samples decreased after 6 and 12 months storage at elevated temperatures, consistent with structural relaxation of the amorphous disaccharide matrix. Formulations with sucrose had lower mobility and greater stability than formulations with trehalose.
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Affiliation(s)
- Ashley Lay-Fortenbery
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky, 40536, USA
| | - Cole W Tower
- Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University, West Lafayette, IN, 47907, USA
| | - Ernest Ezeajughi
- Analytical and Biological Sciences, Medicines & Healthcare Products Regulatory Agency, South Mimms, Hertfordshire, EN6 3QG, UK
| | - Julie Calahan
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky, 40536, USA
| | - Chinwe Duru
- Analytical and Biological Sciences, Medicines & Healthcare Products Regulatory Agency, South Mimms, Hertfordshire, EN6 3QG, UK
| | - Paul Matejtschuk
- Analytical and Biological Sciences, Medicines & Healthcare Products Regulatory Agency, South Mimms, Hertfordshire, EN6 3QG, UK.
| | - Eric J Munson
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky, 40536, USA.
- Department of Industrial and Molecular Pharmaceutics, College of Pharmacy, Purdue University, West Lafayette, IN, 47907, USA.
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7
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Tristan Osanlóo D, Mahlin D, Bjerregaard S, Bergenståhl B, Millqvist-Fureby A. Formulation factors affecting foam properties during vacuum foam-drying. Int J Pharm 2024; 652:123803. [PMID: 38218506 DOI: 10.1016/j.ijpharm.2024.123803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2023] [Revised: 12/28/2023] [Accepted: 01/09/2024] [Indexed: 01/15/2024]
Abstract
This paper explores how vacuum foam-drying of a protein is influenced by formulation parameters by investigating the foam structure, physical properties of the foam, and the stability of the protein. Recombinant human bile salt-stimulated lipase was used as a model of a protein drug. The stability of the lipase was evaluated through activity measurements. Two disaccharides (sucrose and trehalose), strongly tending to an amorphous form, were used as matrix formers, and the physical properties were assessed through residual water content, glass transition temperature, and crystalline state. Moreover, some formulations included surfactants with different sizes and structures of the head group. The alkyl chain length was kept constant to only investigate the impact of the surfactant head group, in the presence of the lipase, on the foamability and surface coverage of the lipase. The study demonstrated that the lipase allowed for a dry, solid foam with a foam overrun of up to 2600 %. The wall thickness of the dry, solid foam was estimated to be 20-50 µm. Clear differences between sucrose and trehalose as matrix former were identified. The lipase showed no tendency to lose activity because of the drying and rehydration, despite a proportion of the lipase covering the surfaces of the dry material.
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Affiliation(s)
- Daniel Tristan Osanlóo
- RISE Research Institutes of Sweden, Box 5604, Stockholm SE-114 86, Sweden; University of Lund, Department of Food Technology, Engineering, and Nutrition, Box 117, Lund SE-221 00, Sweden.
| | - Denny Mahlin
- RISE Research Institutes of Sweden, Box 5604, Stockholm SE-114 86, Sweden.
| | - Simon Bjerregaard
- Ferring Pharmaceuticals A/S, Amager Strandvej 405, Kastrup 2770, Denmark.
| | - Björn Bergenståhl
- University of Lund, Department of Food Technology, Engineering, and Nutrition, Box 117, Lund SE-221 00, Sweden.
| | - Anna Millqvist-Fureby
- RISE Research Institutes of Sweden, Box 5604, Stockholm SE-114 86, Sweden; University of Lund, Department of Food Technology, Engineering, and Nutrition, Box 117, Lund SE-221 00, Sweden.
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8
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Sonje J, Thakral S, Krueger S, Suryanarayanan R. Enabling Efficient Design of Biological Formulations Through Advanced Characterization. Pharm Res 2023; 40:1459-1477. [PMID: 36959413 DOI: 10.1007/s11095-023-03495-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Accepted: 03/01/2023] [Indexed: 03/25/2023]
Abstract
The present review summarizes the use of differential scanning calorimetry (DSC) and scattering techniques in the context of protein formulation design and characterization. The scattering techniques include wide angle X-ray diffractometry (XRD), small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). While DSC is valuable for understanding thermal behavior of the excipients, XRD provides critical information about physical state of solutes during freezing, annealing and in the final lyophile. However, as these techniques lack the sensitivity to detect biomolecule-related transitions, complementary characterization techniques such as small-angle scattering can provide valuable insights.
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Affiliation(s)
- Jayesh Sonje
- Department of Pharmaceutics, College of Pharmacy, University of Minnesota, 308 Harvard St. SE, Minneapolis, MN, 55455, USA
- BioTherapeutics, Pharmaceutical Sciences, Pfizer Inc., 1 Burtt Road, Andover, USA
| | - Seema Thakral
- Boehringer Ingelheim Pharmaceuticals, Inc, 900 Ridgebury Road, Ridgefield, CT, 06877, USA
| | - Susan Krueger
- Center for Neutron Research, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD, 20899, USA
- Department of Materials Science and Engineering, University of Maryland, College Park, MD, 20742, USA
| | - Raj Suryanarayanan
- Department of Pharmaceutics, College of Pharmacy, University of Minnesota, 308 Harvard St. SE, Minneapolis, MN, 55455, USA.
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9
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Mutukuri TT, Ling J, Du Y, Su Y, Zhou QT. Effect of Buffer Salts on Physical Stability of Lyophilized and Spray-Dried Protein Formulations Containing Bovine Serum Albumin and Trehalose. Pharm Res 2023; 40:1355-1371. [PMID: 35764755 PMCID: PMC9794634 DOI: 10.1007/s11095-022-03318-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Accepted: 06/14/2022] [Indexed: 12/30/2022]
Abstract
This study examined the effect of buffer salts on the physical stability of spray-dried and lyophilized formulations of a model protein, bovine serum albumin (BSA). BSA formulations with various buffers were dried by either lyophilization or spray drying. The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Particle characterizations such as Brunauer-Emmett-Teller (BET) surface area, particle size distribution, and particle morphology were also performed. Results from conventional techniques such as ssFTIR did not exhibit correlations with the physical stability of studied formulations. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a satisfactory correlation with the loss of the monomeric peak area measured by SEC (R2 of 0.8722 for spray-dried formulations and 0.8428 for lyophilized formulations) in the 90-day accelerated stability study conducted at 40°C. mDSC and PXRD was unable to measure phase separation in the samples right after drying. In contrast, ssNMR successfully detected the occurrence of phase separation between the succinic buffer component and protein in the lyophilized formulation, which results in a distribution of microenvironmental acidity and the subsequent loss of long-term stability. Moreover, our results suggested that buffer salts have less impact on physical stability for the spray-dried formulations than the lyophilized solids.
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Affiliation(s)
- Tarun Tejasvi Mutukuri
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana, 47907, USA
| | - Jing Ling
- Discovery Pharmaceutical Sciences, Merck & Co., Inc., South San Francisco, California, 94080, USA
| | - Yong Du
- Analytical Research and Development, Merck & Co., Inc., Rahway, New Jersey, 07065, USA
| | - Yongchao Su
- Analytical Research and Development, Merck & Co., Inc., Rahway, New Jersey, 07065, USA.
| | - Qi Tony Zhou
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana, 47907, USA.
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10
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Prediction of Unwanted Crystallization of Freeze-Dried Protein Formulations Using α-Relaxation Measurements. Pharmaceutics 2023; 15:pharmaceutics15020703. [PMID: 36840026 PMCID: PMC9963158 DOI: 10.3390/pharmaceutics15020703] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2023] [Revised: 02/10/2023] [Accepted: 02/18/2023] [Indexed: 02/22/2023] Open
Abstract
There is a lack of methods to predict the isothermal crystallization behavior of amorphous freeze-dried formulations stored below the glass transition temperature. This study applies isothermal microcalorimetry to predict long-term crystallization during product storage time. The relaxation curve of a fresh sample recorded within 12 h after lyophilization is correlated with the long-term crystallization time at the same temperature. Storage conditions of 25 °C and 40 °C are examined and five model formulations containing either sucrose or trehalose with different concentrations of an IgG1 antibody are investigated. The amorphous formulations were created by different freeze-drying processes only differing in their freezing step (random nucleation; additional annealing step of 1.5 h and 3 h, controlled nucleation; quench cooling). Samples that crystallized during the study time of 12 months showed a promising correlation between their relaxation time and crystallization behavior upon storage. Furthermore, the study shows that polysorbate 20 strongly accelerates crystallization of sucrose and that the freezing step itself has a strong impact on the relaxation phenomena that is not levelled out by primary and secondary drying.
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11
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Ghosh I, Gutka H, Krause ME, Clemens R, Kashi RS. A systematic review of commercial high concentration antibody drug products approved in the US: formulation composition, dosage form design and primary packaging considerations. MAbs 2023; 15:2205540. [PMID: 37243580 DOI: 10.1080/19420862.2023.2205540] [Citation(s) in RCA: 30] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2022] [Revised: 04/15/2023] [Accepted: 04/18/2023] [Indexed: 05/29/2023] Open
Abstract
Three critical aspects that define high concentration antibody products (HCAPs) are as follows: 1) formulation composition, 2) dosage form, and 3) primary packaging configuration. HCAPs have become successful in the therapeutic sector due to their unique advantage of allowing subcutaneous self-administration. Technical challenges, such as physical and chemical instability, viscosity, delivery volume limitations, and product immunogenicity, can hinder successful development and commercialization of HCAPs. Such challenges can be overcome by robust formulation and process development strategies, as well as rational selection of excipients and packaging components. We compiled and analyzed data from US Food and Drug Administration-approved and marketed HCAPs that are ≥100 mg/mL to identify trends in formulation composition and quality target product profile. This review presents our findings and discusses novel formulation and processing technologies that enable the development of improved HCAPs at ≥200 mg/mL. The observed trends can be used as a guide for further advancements in the development of HCAPs as more complex antibody-based modalities enter biologics product development.
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Affiliation(s)
- Indrajit Ghosh
- Sterile Product Development, Bristol Myers Squibb, New Brunswick, NJ, USA
| | - Hiten Gutka
- Sterile Product Development, Bristol Myers Squibb, New Brunswick, NJ, USA
| | - Mary E Krause
- Sterile Product Development, Bristol Myers Squibb, New Brunswick, NJ, USA
| | - Ryan Clemens
- College of Pharmacy, University of Illinois at Chicago, Chicago, USA
| | - Ramesh S Kashi
- Sterile Product Development, Bristol Myers Squibb, Summit, NJ, USA
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12
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Malkawi WI, Laird NZ, Phruttiwanichakun P, Mohamed E, Elangovan S, Salem AK. Application of Lyophilized Gene-Delivery Formulations to Dental Implant Surfaces: Non-Cariogenic Lyoprotectant Preserves Transfection Activity of Polyplexes Long-Term. J Pharm Sci 2023; 112:83-90. [PMID: 36372226 PMCID: PMC9772140 DOI: 10.1016/j.xphs.2022.11.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Revised: 11/04/2022] [Accepted: 11/04/2022] [Indexed: 11/13/2022]
Abstract
Titanium is the metal of choice for dental implants because of its biocompatibility and ability to merge with human bone tissue. Despite the great success rate of dental implants, early and late complications occur. Coating titanium dental implant surfaces with polyethyleneimine (PEI)-plasmid DNA (pDNA) polyplexes improve osseointegration by generating therapeutic protein expression at the implantation site. Lyophilization is an approach for stabilizing polyplexes and extending their shelf life; however, most lyoprotectants are sugars that can aid bacterial growth in the peri-implant environment. In our research, we coated titanium surfaces with polyplex solutions containing varying amounts of lyoprotectants. We used two common lyoprotectants (sucrose and polyvinylpyrrolidone K30) and showed for the first time that sucralose (a sucrose derivative used as an artificial sweetener) might act as a lyoprotectant for polyplex solutions. Human embryonic kidney (HEK) 293T cells were used to quantify the transfection efficiency and cytotoxicity of the polyplex/lyoprotectant formulations coating titanium surfaces. Polyplexes that were lyophilized in the presence of a lyoprotectant displayed both preserved particle size and high transfection efficiencies. Polyplexes lyophilized in 2% sucralose have maintained transfection efficacy for three years. These findings suggest that modifying dental implants with lyophilized polyplexes might improve their success rate in the clinic.
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Affiliation(s)
- Walla I Malkawi
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States
| | - Noah Z Laird
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States
| | - Pornpoj Phruttiwanichakun
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States
| | - Esraa Mohamed
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States
| | - Satheesh Elangovan
- Department of Periodontics, College of Dentistry and Dental Clinics, The University of Iowa, Iowa City, IA, 52242, United States
| | - Aliasger K Salem
- Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, 52242, United States.
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13
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Ukidve A, Rembert KB, Vanipenta R, Dorion P, Lafarguette P, McCoy T, Saluja A, Suryanarayanan R, Patke S. Succinate Buffer in Biologics Products: Real-world Formulation Considerations, Processing Risks and Mitigation Strategies. J Pharm Sci 2023; 112:138-147. [PMID: 35667631 DOI: 10.1016/j.xphs.2022.05.026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2022] [Revised: 05/28/2022] [Accepted: 05/29/2022] [Indexed: 10/18/2022]
Abstract
The succinic acid/succinate system has an excellent buffering capacity at acidic pH values (4.5-6.0), promising to be a buffer of choice for biologics having slightly acidic to basic isoelectric points (pI 6 - 9). However, its prevalence in drug products is limited due to the propensity (risk) of its components to crystallize during freezing and the consequent shift in the pH which might affect the product stability. Most of these previous assessments have been performed under operational conditions that do not simulate typical drug product processing conditions. In this work, we have characterized the physicochemical behavior of succinate formulations under representative pharmaceutical conditions. Our results indicate that the pH increases by ∼ 1.2 units in 25 mM and 250 mM succinate buffers at pharmaceutically relevant freezing conditions. X-ray diffractometry studies revealed selective crystallization of monosodium succinate, which is posed as the causative mechanism. This salt crystallization was not observed in the presence of 2% w/v sucrose, suggesting that this pH shift can be mitigated by including sucrose in the formulation. Additionally, three monoclonal antibodies (mAbs) that represent different IgG subtypes and span a range of pIs (5.9 - 8.8) were formulated with succinate and sucrose and subjected to freeze-thaw, frozen storage and lyophilization. No detrimental impact on quality attributes (QA) such as high molecular weight (HMW) species, turbidity, alteration in protein concentration and sub-visible particles, was observed of any of the mAbs tested. Lastly, drug formulations lyophilized in succinate buffer with sucrose demonstrated acceptable QA profiles upon accelerated kinetic storage stability, supporting the use of succinate buffers in mAb drug products.
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Affiliation(s)
- Anvay Ukidve
- Biologics Drug Product Development, Sanofi, One Mountain Road, Framingham, MA, 01701, USA
| | - Kelvin B Rembert
- Biologics Drug Product Development, Sanofi, One Mountain Road, Framingham, MA, 01701, USA
| | - Ragaleena Vanipenta
- Biologics Drug Product Development, Sanofi, One Mountain Road, Framingham, MA, 01701, USA
| | - Patrick Dorion
- Biologics Drug Product Development, Sanofi, One Mountain Road, Framingham, MA, 01701, USA
| | - Pierre Lafarguette
- Physical Characterization, Analytics, Sanofi, 94400, Vitry-Sur-Seine, France
| | - Timothy McCoy
- Biologics Drug Product Development, Sanofi, One Mountain Road, Framingham, MA, 01701, USA
| | - Atul Saluja
- Biologics Drug Product Development, Sanofi, One Mountain Road, Framingham, MA, 01701, USA
| | - Raj Suryanarayanan
- Department of Pharmaceutics, University of Minnesota, Minneapolis, MN, 55455, USA
| | - Sanket Patke
- Biologics Drug Product Development, Sanofi, One Mountain Road, Framingham, MA, 01701, USA.
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14
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Castañeda Ruiz AJ, Shetab Boushehri MA, Phan T, Carle S, Garidel P, Buske J, Lamprecht A. Alternative Excipients for Protein Stabilization in Protein Therapeutics: Overcoming the Limitations of Polysorbates. Pharmaceutics 2022; 14:2575. [PMID: 36559072 PMCID: PMC9781097 DOI: 10.3390/pharmaceutics14122575] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Revised: 11/11/2022] [Accepted: 11/15/2022] [Indexed: 11/25/2022] Open
Abstract
Given their safety and efficiency in protecting protein integrity, polysorbates (PSs) have been the most widely used excipients for the stabilization of protein therapeutics for years. In recent decades, however, there have been numerous reports about visible or sub-visible particles in PS-containing biotherapeutic products, which is a major quality concern for parenteral drugs. Alternative excipients that are safe for parenteral administration, efficient in protecting different protein drugs against various stress conditions, effective in protein stabilization in high-concentrated liquid formulations, stable under the storage conditions for the duration of the product's shelf-life, and compatible with other formulation components and the primary packaging are highly sought after. The aim of this paper is to review potential alternative excipients from different families, including surfactants, carbohydrate- and amino acid-based excipients, synthetic amphiphilic polymers, and ionic liquids that enable protein stabilization. For each category, important characteristics such as the ability to stabilize proteins against thermal and mechanical stresses, current knowledge related to the safety profile for parenteral administration, potential interactions with other formulation components, and primary packaging are debated. Based on the provided information and the detailed discussion thereof, this paper may pave the way for the identification or development of efficient excipients for biotherapeutic protein stabilization.
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Affiliation(s)
- Angel J. Castañeda Ruiz
- Department of Pharmaceutical Technology and Biopharmaceutics, University of Bonn, 53121 Bonn, Germany
| | | | - Tamara Phan
- Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Birkendorfer Straße 65, 88397 Biberach an der Riss, Germany
| | - Stefan Carle
- Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Birkendorfer Straße 65, 88397 Biberach an der Riss, Germany
| | - Patrick Garidel
- Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Birkendorfer Straße 65, 88397 Biberach an der Riss, Germany
| | - Julia Buske
- Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Birkendorfer Straße 65, 88397 Biberach an der Riss, Germany
| | - Alf Lamprecht
- Department of Pharmaceutical Technology and Biopharmaceutics, University of Bonn, 53121 Bonn, Germany
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15
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Mehanna MM, Abla KK. Recent Advances in Freeze-Drying: Variables, Cycle Optimization, and Innovative Techniques. Pharm Dev Technol 2022; 27:904-923. [PMID: 36174214 DOI: 10.1080/10837450.2022.2129385] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
Freeze-drying (FD) is the most substantial drying technique utilized in the pharmaceutical and biopharmaceutical industries. It is a drying process where the solvent is crystallized at low temperatures and then sublimed from the solid-state directly into the vapor phase. Although FD possesses several merits as its suitability for thermolabile materials and its ability to produce dry products with high-quality attributes, it is a complex and prolonged process that requires optimization of both; process and formulation variables. This review attains to disassemble freeze-drying complications through a detailed explanation of the lyophilization concept, stages, the factors influencing the process including controlled ice nucleation, and the modified and innovative freeze-drying technologies proposed in recent years to overcome the shortage of traditional freeze-drying. In addition, this work points out the quality by design (QbD), critical quality of attributes (CQAs), limitations, and drawbacks of lyophilization.HighlightsLyophilization is a propitious drying technique for thermolabile materials.Optimizing the lyophilization cycle requires controlling the process parameters.The formulation excipients and the dispersion medium play crucial roles in designing a successful process.Numerous approaches were developed to ameliorate the lyophilization performance.
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Affiliation(s)
- Mohammed M Mehanna
- Department of Industrial Pharmacy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
| | - Kawthar K Abla
- Pharmaceutical Nanotechnology Research Lab, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon
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16
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Matejtschuk P, Bird C, Ezeajughi E, MacLellan-Gibson K, Wadhwa M. Impact of Formulation Choices on the Freeze-Drying of an Interleukin-6 Reference Material. Front Mol Biosci 2022; 9:868460. [PMID: 35860358 PMCID: PMC9289548 DOI: 10.3389/fmolb.2022.868460] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Accepted: 05/11/2022] [Indexed: 11/20/2022] Open
Abstract
Formulation is critical to successful delivery of lyophilized biologics. We have compared the impact of buffer choice and the addition of sodium chloride (a formulant often viewed as unfavorable for freeze-drying applications) on the outcome of trial lyophilization of an interleukin-6 reference material. While phosphate buffer was a preferred choice and yielded well-formed cakes associated with fair recovery of biological activity, the resultant residual moisture content was high (2–4% w/w). By inclusion of isotonic levels of NaCl, the freeze-dried appearance and process were not impaired, but the residual moisture delivered was considerably reduced to levels <1% w/w. We postulate that this is due to the presence of a more open-cake structure and support this with evidence from thermal analysis and scanning electron microscopy. This work illustrates the importance of wide ranging empirical investigation of formulation options in order to optimize freeze-drying outcomes for biologics.
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Affiliation(s)
- Paul Matejtschuk
- Analytical and Biological Sciences, NIBSC, Medicines and Healthcare Products Regulatory Agency, Potters Bar, United Kingdom
- *Correspondence: Paul Matejtschuk,
| | - Christopher Bird
- Biotherapeutics, NIBSC, Medicines and Healthcare products Regulatory Agency, Potters Bar, United Kingdom
| | - Ernest Ezeajughi
- Analytical and Biological Sciences, NIBSC, Medicines and Healthcare Products Regulatory Agency, Potters Bar, United Kingdom
| | - Kirsty MacLellan-Gibson
- Analytical and Biological Sciences, NIBSC, Medicines and Healthcare Products Regulatory Agency, Potters Bar, United Kingdom
| | - Meenu Wadhwa
- Biotherapeutics, NIBSC, Medicines and Healthcare products Regulatory Agency, Potters Bar, United Kingdom
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17
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Sonje J, Thakral S, Mayhugh B, Sacha G, Nail S, Srinivasan J, Suryanarayanan R. Mannitol Hemihydrate in Lyophilized Protein Formulations:Impact of its Dehydration During Storage on Sucrose Crystallinity and Protein Stability. Int J Pharm 2022; 624:121974. [PMID: 35787458 DOI: 10.1016/j.ijpharm.2022.121974] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 06/18/2022] [Accepted: 06/29/2022] [Indexed: 10/17/2022]
Abstract
The high propensity of mannitol to crystallize in frozen solutions along with its high eutectic temperature enabling higher primary drying temperatures makes it a good bulking agent. In protein formulations, addition of a sugar (sucrose) that has the ability to remain amorphous throughout processing as well as storage, it is imperative to retain the protein in its native state. It is well known that in the presence of amorphous excipients and protein, mannitol can crystallize as a mixture of anhydrous polymorphs - α-, β- and δ-forms and a hemihydrate form [mannitol hemihydrate (MHH); C6H14O6·0.5H2O]. The conditions of formation of MHH due to processing and formulation variables are well established in the literature. However, MHH's dehydration kinetics on storage and its impact on the stability of a protein has not been systematically evaluated. The overall objective was to identify conditions (temperature and humidity) at which MHH can dehydrate on storage and the consequences of the release of associated water on sucrose phase behavior and protein stability. In a mannitol-sucrose-protein lyophile, the purpose of this study was (i) to investigate the dehydration behavior of MHH (ii) to determine the influence of dehydration on sucrose crystallization and (iii) the effect of moisture released due to MHH dehydration on model protein (Bovine serum albumin, BSA or Human serum albumin, HSA) aggregation. MHH dehydration and sucrose crystallization was observed in cases where the relative humidity was ≥ 55% (open vials). A relative humidity of ≤ 33% RH prevented MHH dehydration while retaining sucrose amorphous. No protein aggregation was observed irrespective of presence of MHH or its dehydration.
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Affiliation(s)
- Jayesh Sonje
- Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, United States
| | - Seema Thakral
- Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, United States
| | - Brendan Mayhugh
- Baxter Global Science and Technology, 927 S. Curry Pike, Bloomington, IN 47403
| | - Gregory Sacha
- Baxter Global Science and Technology, 927 S. Curry Pike, Bloomington, IN 47403
| | - Steve Nail
- Baxter Global Science and Technology, 927 S. Curry Pike, Bloomington, IN 47403
| | - Jayasree Srinivasan
- Baxter Global Science and Technology, 927 S. Curry Pike, Bloomington, IN 47403.
| | - Raj Suryanarayanan
- Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, United States.
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18
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Davagnino J, Gabrielson JP. Developing Lyophilized Formulations for Protein Biopharmaceuticals Containing Salt that Produce Placebos of Corresponding Appearance. J Pharm Sci 2022; 111:2930-2937. [DOI: 10.1016/j.xphs.2022.06.020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Revised: 06/22/2022] [Accepted: 06/24/2022] [Indexed: 11/15/2022]
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19
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Kharatyan T, Gopireddy SR, Ogawa T, Kodama T, Nishimoto N, Osada S, Scherließ R, Urbanetz NA. Quantitative Analysis of Glassy State Relaxation and Ostwald Ripening during Annealing Using Freeze-Drying Microscopy. Pharmaceutics 2022; 14:pharmaceutics14061176. [PMID: 35745749 PMCID: PMC9231378 DOI: 10.3390/pharmaceutics14061176] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2022] [Revised: 05/27/2022] [Accepted: 05/27/2022] [Indexed: 02/06/2023] Open
Abstract
Supercooling during the freezing of pharmaceutical solutions often leads to suboptimal freeze-drying results, such as long primary drying times or a collapse in the cake structure. Thermal treatment of the frozen solution, known as annealing, can improve those issues by influencing properties such as the pore size and collapse temperature of the lyophilisate. In this study we aimed to show that annealing causes a rearrangement of water molecules between ice crystals, as well as between the freeze-concentrated amorphous matrix and the crystalline ice phase in a frozen binary aqueous solution. Ice crystal sizes, as well as volume fractions of the crystalline and amorphous phases of 10% (w/w) sucrose and trehalose solutions, were quantified after annealing using freeze-drying microscopy and image labelling. Depending on the annealing time and temperature, the amorphous phase was shown to decrease its volume due to the crystallisation of vitreous water (i.e., glassy state relaxation) while the crystalline phase was undergoing coarsening (i.e., Ostwald ripening). These results allow, for the first time, a quantitative comparison of the two phenomena. It was demonstrated that glassy state relaxation and Ostwald ripening, although occurring simultaneously, are distinct processes that follow different kinetics.
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Affiliation(s)
- Tigran Kharatyan
- Department of Pharmaceutics and Biopharmaceutics, Kiel University, 24118 Kiel, Germany; (T.K.); (R.S.)
- Pharmaceutical Development, Daiichi-Sankyo Europe GmbH, 85276 Pfaffenhofen an der Ilm, Germany;
| | - Srikanth R. Gopireddy
- Pharmaceutical Development, Daiichi-Sankyo Europe GmbH, 85276 Pfaffenhofen an der Ilm, Germany;
- Correspondence:
| | - Toru Ogawa
- Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd., Hiratsuka 254-0014, Japan; (T.O.); (T.K.); (N.N.); (S.O.)
| | - Tatsuhiro Kodama
- Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd., Hiratsuka 254-0014, Japan; (T.O.); (T.K.); (N.N.); (S.O.)
| | - Norihiro Nishimoto
- Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd., Hiratsuka 254-0014, Japan; (T.O.); (T.K.); (N.N.); (S.O.)
| | - Sayaka Osada
- Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd., Hiratsuka 254-0014, Japan; (T.O.); (T.K.); (N.N.); (S.O.)
| | - Regina Scherließ
- Department of Pharmaceutics and Biopharmaceutics, Kiel University, 24118 Kiel, Germany; (T.K.); (R.S.)
| | - Nora A. Urbanetz
- Pharmaceutical Development, Daiichi-Sankyo Europe GmbH, 85276 Pfaffenhofen an der Ilm, Germany;
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20
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Huang C, Chen L, Franzen L, Anderski J, Qian F. Spray-Dried Monoclonal Antibody Suspension for High-Concentration and Low-Viscosity Subcutaneous Injection. Mol Pharm 2022; 19:1505-1514. [PMID: 35417176 DOI: 10.1021/acs.molpharmaceut.2c00039] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Administration of highly concentrated monoclonal antibodies (mAbs) through injection is often not possible as the viscosity can be readily above 50 mPa·s when the concentration exceeds 150 mg/mL. Besides, highly concentrated mAb solutions always exhibit increased aggregation propensity and lower stability, which raise the difficulty for the successful development of highly concentrated mAb formulations. We hereby explored the possibility of suspension as another formulation form for high-concentration proteins to reduce viscosity and maintain stability. Specifically, we demonstrated that spray drying can serve as a process to prepare particles for suspension. Particles prepared from formulations with different mAb/trehalose mass ratios displayed good physical stability and antibody binding affinity, as indicated by circular dichroism, fluorescence spectroscopy, and surface plasmon resonance (SPR)-based bioassay analyses. During spray drying, a surface tension-dominated enrichment of mAb on the particle surface was observed, but this did not show a significant negative impact on mAb stability. Spray-dried particles were subsequently suspended into benzyl benzoate, and the resulting suspension showed good stability and a lower viscosity when compared to its counterpart solution. Furthermore, mAbs recovered from the suspension maintained their conformational structure. Our study demonstrated that the suspension displayed low viscosity and good physical stability, so it may offer novel opportunities for the preparation of highly concentrated protein formulations.
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Affiliation(s)
- Chengnan Huang
- School of Pharmaceutical Sciences, Beijing Advanced Innovation Center for Structural Biology, and Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University, Beijing, 100084, P. R. China
| | - Linc Chen
- Bayer Healthcare Co. Ltd., Beijing, 100020, P. R. China
| | - Lutz Franzen
- Research & Development, Pharmaceuticals, Bayer AG, Wuppertal, 42096, Germany
| | - Juliane Anderski
- Research & Development, Pharmaceuticals, Bayer AG, Wuppertal, 42096, Germany
| | - Feng Qian
- School of Pharmaceutical Sciences, Beijing Advanced Innovation Center for Structural Biology, and Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University, Beijing, 100084, P. R. China
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21
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Systematic studies on stabilization of AAV vector formulations by lyophilization. J Pharm Sci 2022; 111:2288-2298. [DOI: 10.1016/j.xphs.2022.03.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2022] [Revised: 03/02/2022] [Accepted: 03/02/2022] [Indexed: 01/31/2023]
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22
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Zhang Y, Soto M, Ghosh D, Williams RO. Manufacturing Stable Bacteriophage Powders by Including Buffer System in Formulations and Using Thin Film Freeze-drying Technology. Pharm Res 2021; 38:1793-1804. [PMID: 34697726 DOI: 10.1007/s11095-021-03111-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2021] [Accepted: 09/14/2021] [Indexed: 11/27/2022]
Abstract
PURPOSE Bacteriophage (phage) therapy has re-gained attention lately given the ever-increasing prevalence of multi-drug resistance 'super-bugs'. To develop therapeutic phage into clinically usable drug products, the strategy of solidifying phage formulations has been implemented to diversify the dosage forms and to overcome the storage condition limitations for liquid phage formulations. METHOD In our work, we hypothesize and tested that an advanced technology, thin film freeze-drying (TFFD), can be used to produce phage containing dry powders without significantly losing phage viability. Here we selected T7 phage as our model phage in a preliminary screening study. RESULTS We found that a binary excipient matrix of sucrose and leucine at ratios of 90:10 or 75:25 by weight, protected phage from the stresses encountered during the TFFD process. In addition, we confirmed that incorporating a buffer system in the formulation significantly improved the survival of phage during the initial freezing step and subsequent sublimation step in the solidifying processes. The titer loss of phage in SM buffer (Tris/NaCl/MgSO4) containing formulation was as low as 0.19 log plaque forming units, which indicated that phage function was well preserved after the TFFD process. The presence of buffers markedly reduced the geometric particle sizes as determined by a dry dispersion method using laser diffraction, which indicated that the TFFD phage powder formulations were easily sheared into smaller powder aggregates, an ideal property for facilitating a variety of topical drug delivery routes including pulmonary delivery through dry powder inhalers, nebulization after reconstitution, and intranasal or wound therapy, etc. CONCLUSION: From these findings, we show that introducing buffer system can stabilize phage during dehydration processes, and TFFD, as a novel particle engineering method, can successfully produce phage containing powders that possess the desired properties for bioactivity and potentially for inhalation therapy.
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Affiliation(s)
- Yajie Zhang
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA.,Formulation Development Department, Regeneron Pharmaceuticals, Tarrytown, NY, USA
| | - Melissa Soto
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA
| | - Debadyuti Ghosh
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA
| | - Robert O Williams
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA.
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23
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Park SK, Noh GY, Yu HW, Lee EC, Jeong J, Park YM, Han HK, Jeong SH, Kim NA. Lessons Learned in Protein Precipitation Using a Membrane Emulsification Technique to Produce Reversible and Uniform Microbeads. Pharmaceutics 2021; 13:pharmaceutics13101738. [PMID: 34684031 PMCID: PMC8540039 DOI: 10.3390/pharmaceutics13101738] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2021] [Revised: 10/08/2021] [Accepted: 10/16/2021] [Indexed: 11/16/2022] Open
Abstract
The effects of the manufacturing process and the regeneration of Shirasu porous glass (SPG) membranes were investigated on the reproducibility of protein precipitants, termed protein microbeads. Intravenous immunoglobulin (IVIG) was selected as a model protein to produce its microbeads in seven different cases. The results showed that the hydrophobically modified SPG membrane produced finer microbeads than the hydrophilic SPG membrane, but this was inconsistent when using the general regeneration method. Its reproducibility was determined to be mostly dependent on rinsing the SPG membrane prior to the modification and on the protein concentration used for emulsification. The higher concentration could foul and plug the membrane during protein release and thus the membrane must be washed thoroughly before hydrophobic modification. Moreover, the membrane regenerated by silicone resin dissolved in ethanol had better reproducibility than silicone resin dissolved in water. On the other hand, rinsing the protein precipitant with cold ethanol after the emulsification was not favorable and induced protein aggregation. With the addition of trehalose, the purity of the IVIG microbeads was almost the same as before microbeadification. Therefore, the regeneration method, protein concentration, and its stabilizer are key to the success of protein emulsification and precipitation using the SPG membrane.
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Affiliation(s)
- Sang-Koo Park
- BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 10326, Korea; (S.-K.P.); (G.Y.N.); (H.W.Y.); (E.C.L.); (J.J.); (H.-K.H.)
| | - Ga Yeon Noh
- BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 10326, Korea; (S.-K.P.); (G.Y.N.); (H.W.Y.); (E.C.L.); (J.J.); (H.-K.H.)
| | - Hyun Woo Yu
- BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 10326, Korea; (S.-K.P.); (G.Y.N.); (H.W.Y.); (E.C.L.); (J.J.); (H.-K.H.)
| | - Eun Chae Lee
- BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 10326, Korea; (S.-K.P.); (G.Y.N.); (H.W.Y.); (E.C.L.); (J.J.); (H.-K.H.)
| | - Junoh Jeong
- BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 10326, Korea; (S.-K.P.); (G.Y.N.); (H.W.Y.); (E.C.L.); (J.J.); (H.-K.H.)
| | - Young-Min Park
- Division of Health and Kinesiology, Incheon National University, Incheon 22012, Korea;
| | - Hyo-Kyung Han
- BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 10326, Korea; (S.-K.P.); (G.Y.N.); (H.W.Y.); (E.C.L.); (J.J.); (H.-K.H.)
| | - Seong Hoon Jeong
- BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 10326, Korea; (S.-K.P.); (G.Y.N.); (H.W.Y.); (E.C.L.); (J.J.); (H.-K.H.)
- Correspondence: (S.H.J.); (N.A.K.); Tel.: +82-10-5679-0621 (S.H.J.); +82-10-5590-1018 (N.A.K.)
| | - Nam Ah Kim
- BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 10326, Korea; (S.-K.P.); (G.Y.N.); (H.W.Y.); (E.C.L.); (J.J.); (H.-K.H.)
- Correspondence: (S.H.J.); (N.A.K.); Tel.: +82-10-5679-0621 (S.H.J.); +82-10-5590-1018 (N.A.K.)
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Das TK, Sreedhara A, Colandene JD, Chou DK, Filipe V, Grapentin C, Searles J, Christian TR, Narhi LO, Jiskoot W. Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality. J Pharm Sci 2021; 111:868-886. [PMID: 34563537 DOI: 10.1016/j.xphs.2021.09.030] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2021] [Revised: 09/21/2021] [Accepted: 09/21/2021] [Indexed: 01/22/2023]
Abstract
Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.
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Affiliation(s)
- Tapan K Das
- Bristol Myers Squibb, Biologics Development, New Brunswick, New Jersey 08903, USA.
| | | | - James D Colandene
- GlaxoSmithKline, Biopharmaceutical Product Sciences, 1250 S Collegeville Road, Collegeville, PA 19425, USA
| | - Danny K Chou
- Compassion BioSolution, LLC, Lomita, CA 90717, USA
| | | | - Christoph Grapentin
- Lonza AG, Drug Product Services, Hochbergerstrasse 60G, 4057 Basel, Switzerland
| | - Jim Searles
- Pfizer Inc., Biotherapeutics Pharmaceutical Sciences Research and Development, 875 Chesterfield Pkwy W, Chesterfield, MO 63017 USA
| | | | | | - Wim Jiskoot
- Leiden University, Division of BioTherapeutics, Leiden Academic Centre for Drug Research, Leiden, the Netherlands; Coriolis Pharma, Martinsried, Germany
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25
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Dong M, Meinerz NM, Walker KD, Garcea RL, Randolph TW. Thermostability of a trivalent, capsomere-based vaccine for human papillomavirus infection. Eur J Pharm Biopharm 2021; 168:131-138. [PMID: 34438020 DOI: 10.1016/j.ejpb.2021.08.008] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2021] [Revised: 08/09/2021] [Accepted: 08/19/2021] [Indexed: 12/17/2022]
Abstract
Currently licensed vaccines require a cold-chain to maintain efficacy. This cold-chain requirement reduces the availability of vaccines in resource-poor areas of the world. Commercially available human papillomavirus (HPV) vaccines protect against the most common HPV types related to cervical cancer; however, their impact is limited in many regions due to cold-chain requirements. The goal of this study was to test the thermostability of an adjuvanted, trivalent HPV L1 capsomere-based vaccine (containing HPV types 16, 18, and 31) that was formulated by using lyophilization to embed the antigens within a solid, glassy matrix. Thermal stabilities were determined by storing the vaccine formulations for 3 months at 50 °C, followed by immunization of BALB/c mice and measurement of antibody responses. Antibody responses to capsomere vaccines formulated with alum were unchanged after storage for 3 months at 50 °C. Neutralizing responses to these vaccines were unchanged by high-temperature storage, and were equivalent to those generated after administration of the commercially available liquid HPV vaccine Gardasil®9.
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Affiliation(s)
- Miao Dong
- Center for Pharmaceutical Biotechnology, Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA
| | - Natalie M Meinerz
- The BioFrontiers Program, University of Colorado, Boulder, CO, USA; The Department of Molecular, Cellular, Developmental Biology, University of Colorado, Boulder, CO, USA
| | - Kathryne D Walker
- The BioFrontiers Program, University of Colorado, Boulder, CO, USA; The Department of Molecular, Cellular, Developmental Biology, University of Colorado, Boulder, CO, USA
| | - Robert L Garcea
- The BioFrontiers Program, University of Colorado, Boulder, CO, USA; The Department of Molecular, Cellular, Developmental Biology, University of Colorado, Boulder, CO, USA
| | - Theodore W Randolph
- Center for Pharmaceutical Biotechnology, Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA.
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26
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Mutukuri TT, Maa YF, Gikanga B, Sakhnovsky R, Zhou QT. Electrostatic spray drying for monoclonal antibody formulation. Int J Pharm 2021; 607:120942. [PMID: 34324986 DOI: 10.1016/j.ijpharm.2021.120942] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Revised: 07/20/2021] [Accepted: 07/22/2021] [Indexed: 11/25/2022]
Abstract
This study explored the feasibility of electrostatic spray drying for producing a monoclonal antibody (mAb) powder formulation at lower drying temperatures than conventional spray drying and its effect on protein stability. A mAb formulation was dried by either conventional spray drying or electrostatic spray drying with charge (ESD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Conventional spray drying of the mAb formulation at the inlet temperature of 70 °C failed to generate dry powders due to poor drying efficiency; electrostatic spray drying at the same temperature and 5 kV charge enabled the formation of powder formulation with satisfactory moisture contents. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a good correlation with the loss of the monomeric peak area measured by size exclusion chromatography in the 90-day accelerated stability study conducted at 40 °C. Low-temperature (70 °C inlet temperature) drying with an electrostatic charge (5 kV) led to better protein physical stability as compared with the samples spray-dried at the high temperature (130 °C inlet temperature) without charge. This study shows that electrostatic spray drying can produce solid monoclonal antibody formulation at lower inlet temperature than traditional spray drying with better physical stability.
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Affiliation(s)
- Tarun Tejasvi Mutukuri
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA
| | - Yuh-Fun Maa
- Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA
| | - Benson Gikanga
- Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA
| | | | - Qi Tony Zhou
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA.
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27
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Mastrocco A, Cazzolli D, Prittie J. The effect of storage at 4°C on canine-specific albumin on product sterility and albumin concentration. J Vet Emerg Crit Care (San Antonio) 2021; 31:574-577. [PMID: 34297893 DOI: 10.1111/vec.13090] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Revised: 12/27/2019] [Accepted: 01/10/2020] [Indexed: 12/01/2022]
Abstract
OBJECTIVES To determine if lyophilized canine-specific albumin (CSA) can be stored beyond the manufacturer recommended 6 hours post-reconstitution without significant concern for bacterial growth. DESIGN Laboratory research. SETTING Large private teaching hospital. INTERVENTIONS Six bottles of lyophilized CSA were retrieved from a medical grade (4°C) refrigerator and were reconstituted with 0.9% NaCl to a 5% solution. Time to complete dissolution of all bottles at room temperature without agitation was recorded in minutes. A 1.5-mL sample was collected from each bottle at the following time points: time 0 (at the time of complete dissolution), and then 6, 12, and 24 hours after removal from 4°C. Each aliquot was inoculated into a blood culture tube and was immediately submitted for aerobic and anaerobic bacterial culture. A positive and negative control were cultured at the 24-hour time point. At each time point, an additional 0.5-mL aliquot from each albumin bottle was collected, and these were combined to determine the average albumin concentration (g/L, g/dL) as measured by an in-house analyzer. The albumin was stored at 4°C between sample collection. MEASUREMENTS AND MAIN RESULTS The reconstitution of CSA and storage at 4°C for up to 24 hours did not result in anaerobic or aerobic bacterial growth in any of the 24 cultured samples. Storage did not have a clinically significant effect on the albumin concentration of the solution. The average albumin concentration of all samples was 42 g/L (4.2 g/dL). No growth was noted in the negative control, and the positive control grew 4 strains of bacteria. CONCLUSIONS It appears safe to use lyophilized CSA up to 24 hours post-reconstitution without significant risk of bacterial growth or change in albumin concentration.
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Affiliation(s)
- Alicia Mastrocco
- Section of Emergency and Critical Care, The Animal Medical Center, New York, New York, USA
| | - Dava Cazzolli
- Section of Emergency and Critical Care, The Animal Medical Center, New York, New York, USA
| | - Jennifer Prittie
- Section of Emergency and Critical Care, The Animal Medical Center, New York, New York, USA
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28
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Development of a stable lyophilized adeno-associated virus gene therapy formulation. Int J Pharm 2021; 606:120912. [PMID: 34298099 DOI: 10.1016/j.ijpharm.2021.120912] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Revised: 07/13/2021] [Accepted: 07/18/2021] [Indexed: 11/21/2022]
Abstract
Adeno-associated viruses (AAV) are among the most actively investigated vectors for gene therapy. Supply of early clinical studies with frozen drug product (DP) can accelerate timelines and minimize degradation risks. In the long-term, logistical challenges of frozen DP may limit patient access. In this work, we developed a lyophilized (freeze-dried) formulation of AAV. The mass concentration of AAV is typically low, and AAV also requires a minimum ionic strength to inhibit aggregation. These factors result in a low collapse temperature, which is limiting to lyophilization. Mannitol crystallization was found to cause extensive degradation and potency loss of AAV during the freezing step. With further development, we determined that AAV could be lyophilized in a sucrose and citrate formulation with a more desirable high glass transition temperature of the dried cake. An optimal residual moisture range (1-3%) was found to be critical to maintaining AAV8 stability. Glycerol was found to protect AAV8 from over-drying by preventing capsid damage and genome DNA release. A lyophilized formulation was identified that maintained potency for 24 months at 2-8 °C, indicating the feasibility of a dried formulation for AAV gene therapy.
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29
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Kim NA, Yu HW, Noh GY, Park SK, Kang W, Jeong SH. Protein microbeadification to achieve highly concentrated protein formulation with reversible properties and in vivo pharmacokinetics after reconstitution. Int J Biol Macromol 2021; 185:935-948. [PMID: 34237365 DOI: 10.1016/j.ijbiomac.2021.07.012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Revised: 07/01/2021] [Accepted: 07/02/2021] [Indexed: 10/20/2022]
Abstract
A protein precipitation technique was optimized to produce biophysically stable 'protein microbeads', applicable to highly concentrated protein formulation. Initially, production of BSA microbeads was performed using rapid dehydration by vortexing in organic solvents followed by cold ethanol treatment and a vacuum drying. Out of four solvents, n-octanol produced the most reversible microbeads upon reconstitution. A Shirasu porous glass (SPG) membrane emulsification technique was utilized to enhance the size distribution and manufacturing process of the protein microbeads with a marketized human IgG solution. Process variants such as dehydration time, temperature, excipients, drying conditions, and initial protein concentration were evaluated in terms of the quality of IgG microbeads and their reversibility. The hydrophobized SPG membrane produced a narrow size distribution of the microbeads, which were further enhanced by shorter dehydration time, low temperature, minimized the residual solvents, lower initial protein concentration, and addition of trehalose to the IgG solution. Final reversibility of the IgG microbeads with trehalose was over 99% at both low and high protein concentrations. Moreover, the formulation was highly stable under repeated mechanical shocks and at an elevated temperature compared to its liquid state. Its in vivo pharmacokinetic profiles in rats were consistent before and after the 'microbeadification'.
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Affiliation(s)
- Nam Ah Kim
- College of Pharmacy, Dongguk University-Seoul, Gyeonggi 13026, Republic of Korea.
| | - Hyun Woo Yu
- College of Pharmacy, Dongguk University-Seoul, Gyeonggi 13026, Republic of Korea
| | - Ga Yeon Noh
- College of Pharmacy, Dongguk University-Seoul, Gyeonggi 13026, Republic of Korea
| | - Sang-Koo Park
- College of Pharmacy, Dongguk University-Seoul, Gyeonggi 13026, Republic of Korea
| | - Wonku Kang
- College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea
| | - Seong Hoon Jeong
- College of Pharmacy, Dongguk University-Seoul, Gyeonggi 13026, Republic of Korea.
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30
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Baudhuin H, Van Bockstal PJ, De Beer T, Vaneycken I, Bridoux J, Raes G, Caveliers V, Keyaerts M, Devoogdt N, Lahoutte T, Xavier C. Lyophilization of NOTA-sdAbs: First step towards a cold diagnostic kit for 68Ga-labeling. Eur J Pharm Biopharm 2021; 166:194-204. [PMID: 34186190 DOI: 10.1016/j.ejpb.2021.06.012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 05/27/2021] [Accepted: 06/22/2021] [Indexed: 12/30/2022]
Abstract
Lyophilization is commonly used in the production of pharmaceutical compounds to increase the stability of the Active Pharmaceutical Ingredient (API) by removing solvents. This study investigates the possibility to lyophilize an anti-HER2 and an anti-MMR single-domain antibody fragment (sdAb)-based precursor as a first step in the development of a diagnostic kit for PET imaging. METHODS NOTA-sdAb precursors have been lyophilized with the following formulation: 100 µg NOTA-sdAb in 0.1 M NaOAc (NaOAc), 5% (w/v%) mannitol-sucrose mix at a 2:1 ratio and 0.1 mg/mL polysorbate 80. During development of the formulation and drying cycle, factors such as cake appearance, glass transition temperature and residual moisture were analyzed to ensure qualitative and stable lyophilized samples. Stability studies of lyophilized precursor were conducted up to 18 months after storage at 2-8 °C by evaluating the precursor integrity, aggregation, functionality and 68Ga-labeling efficiency. A comparative biodistribution study (lyophilized vs non-lyophilized precursor) was conducted in wild type mice (n = 3) and in tumor bearing mice (n = 6). RESULTS The lyophilized NOTA-anti-HER2 precursor shows consistent stability data in vitro for up to 12 months at 2-8 °C in three separate batches, with results indicating stability even for up to T18m. No aggregation, degradation or activity loss was observed. Radiochemical purity after 68Ga-labeling is consistent over a period of 12 months (RCP ≥ 95% at T12m). In vivo biodistribution analyses show a typical [68Ga]Ga-NOTA-anti-HER2 sdAb distribution profile and a comparable tumor uptake for the lyophilized compound vs non-lyophilized (5.5% vs 5.7 %IA/g, respectively). In vitro results of lyophilized NOTA-anti-MMR precursor indicates stability for up to 18 months, while in vivo data show a comparable tumor uptake (2.5% vs 2.8 %IA/g, respectively) and no significant difference in kidney retention (49.4% vs 47.5 %IA/g, respectively). CONCLUSION A formulation and specific freeze-drying cycle were successfully developed to lyophilize NOTA-sdAb precursors for long-term storage at 2-8 °C. In vivo data show no negative impact of the lyophilization process on the in vivo behavior or functionality of the lyophilized precursor. These results highlight the potential to develop a kit for the preparation of 68Ga-sdAb-based radiopharmaceuticals.
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Affiliation(s)
- Henri Baudhuin
- Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Brussels, Belgium.
| | - Pieter-Jan Van Bockstal
- Laboratory of Pharmaceutical Process Analytical Technology (LPPAT), Universiteit Gent, Ghent, Belgium.
| | - Thomas De Beer
- Laboratory of Pharmaceutical Process Analytical Technology (LPPAT), Universiteit Gent, Ghent, Belgium.
| | - Ilse Vaneycken
- Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Brussels, Belgium; Nuclear Medicine Department (NUCG), Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium.
| | - Jessica Bridoux
- Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Brussels, Belgium.
| | - Geert Raes
- Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium; Myeloid Cell Immunology Laboratory, VIB Center for Inflammation Research, Brussels, Belgium.
| | - Vicky Caveliers
- Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Brussels, Belgium; Nuclear Medicine Department (NUCG), Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium.
| | - Marleen Keyaerts
- Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Brussels, Belgium; Nuclear Medicine Department (NUCG), Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium.
| | - Nick Devoogdt
- Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Brussels, Belgium.
| | - Tony Lahoutte
- Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Brussels, Belgium; Nuclear Medicine Department (NUCG), Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium.
| | - Catarina Xavier
- Department of Medical Imaging (MIMA), Vrije Universiteit Brussel, Brussels, Belgium.
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31
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Kim BS, Kim JU, So KH, Hwang NS. Supercritical Fluid-Based Decellularization Technologies for Regenerative Medicine Applications. Macromol Biosci 2021; 21:e2100160. [PMID: 34121330 DOI: 10.1002/mabi.202100160] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2021] [Revised: 05/24/2021] [Indexed: 12/14/2022]
Abstract
Supercritical fluid-based extraction technologies are currently being increasingly utilized in high purity extract products for food industries. In recent years, supercritical fluid-based extraction technology is transformed in biomaterials process fields to be further utilized for tissue engineering and other biomedical applications. In particular, supercritical fluid-based decellularization protocols have great advantage over the conventional decellularization as it may allow preservation of extracellular matrix components and structures. In this review, the latest technological development utilizing the supercritical fluid-based decellularization for regenerative medicine is introduced.
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Affiliation(s)
- Beom-Seok Kim
- Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea
| | - Jeong-Uk Kim
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea
| | - Kyoung-Ha So
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea
| | - Nathaniel S Hwang
- Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea.,School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea.,Bio-MAX Institute, Institute of Bio-Engineering, Seoul National University, Seoul, 08826, Republic of Korea
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32
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Thakral S, Sonje J, Munjal B, Suryanarayanan R. Stabilizers and their interaction with formulation components in frozen and freeze-dried protein formulations. Adv Drug Deliv Rev 2021; 173:1-19. [PMID: 33741437 DOI: 10.1016/j.addr.2021.03.003] [Citation(s) in RCA: 57] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Revised: 02/06/2021] [Accepted: 03/03/2021] [Indexed: 02/06/2023]
Abstract
This review aims to provide an overview of the current knowledge on protein stabilization during freezing and freeze-drying in relation to stress conditions commonly encountered during these processes. The traditional as well as refined mechanisms by which excipients may stabilize proteins are presented. These stabilizers encompass a wide variety of compounds including sugars, sugar alcohols, amino acids, surfactants, buffers and polymers. The rational selection of excipients for use in frozen and freeze-dried protein formulations is presented. Lyophilized protein formulations are generally multicomponent systems, providing numerous possibilities of excipient-excipient and protein-excipient interactions. The interplay of different formulation components on the protein stability and excipient functionality in the frozen and freeze-dried systems are reviewed, with discussion of representative examples of such interactions.
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33
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Chen CA, Yuan H, Chen CW, Chien YS, Sheng WH, Chen CF. An electricity- and instrument-free infectious disease sensor based on a 3D origami paper-based analytical device. LAB ON A CHIP 2021; 21:1908-1915. [PMID: 34008628 DOI: 10.1039/d1lc00079a] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
Infectious diseases cause millions of deaths annually in the developing world. Recently, microfluidic paper-based analytical devices (μPADs) have been developed to diagnose such diseases, as these tests are low cost, biocompatible, and simple to fabricate. However, current μPADs are difficult to use in resource-limited areas due to their reliance on external instrumentation to measure and analyze the test results. In this work, we propose an electricity and external instrumentation-free μPAD sensor based on the colorimetric enzyme-linked immunosorbent assay (ELISA) for the diagnosis of infectious disease (3D-tPADs). Designed based on the principle of origami, the proposed μPAD enables the sequential steps of the colorimetric ELISA test to be completed in just ∼10 min. In addition, in order to obtain an accurate ELISA result without using any instrument, we have integrated an electricity-free "timer" within the μPAD that can be controlled by the buffer viscosity and fluid path volume to indicate the appropriate times for washing and color development steps, which can avoid false positive or false negative results caused by an extended or shortened amount of washing and development times. Due to the low background noise and high positive signal intensity of the μPAD, positive and negative detection results can be distinguished by just the naked eye. Furthermore, the ELISA result can be semi-quantified by comparing the results shown on the μPAD with a color chart diagram with a detection limit of HIV type 1(HIV-1) p24 antigen as low as 0.03 ng mL-1. These results demonstrate the proposed sensor can perform infectious disease diagnosis without external instrumentation or electricity, extending the application of the μPAD test for on-site detection and use in resource-limited settings.
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Affiliation(s)
- Chung-An Chen
- Institute of Applied Mechanics, National Taiwan University, Taipei 106, Taiwan.
| | - Hao Yuan
- Institute of Applied Mechanics, National Taiwan University, Taipei 106, Taiwan.
| | - Chiao-Wen Chen
- Institute of Applied Mechanics, National Taiwan University, Taipei 106, Taiwan.
| | - Yuh-Shiuan Chien
- Institute of Applied Mechanics, National Taiwan University, Taipei 106, Taiwan.
| | - Wang-Huei Sheng
- Division of Infectious Diseases, Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan
| | - Chien-Fu Chen
- Institute of Applied Mechanics, National Taiwan University, Taipei 106, Taiwan.
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34
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Chen Y, Mutukuri TT, Wilson NE, Zhou QT. Pharmaceutical protein solids: Drying technology, solid-state characterization and stability. Adv Drug Deliv Rev 2021; 172:211-233. [PMID: 33705880 PMCID: PMC8107147 DOI: 10.1016/j.addr.2021.02.016] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 01/18/2021] [Accepted: 02/22/2021] [Indexed: 01/30/2023]
Abstract
Despite the boom in biologics over the past decade, the intrinsic instability of these large molecules poses significant challenges to formulation development. Almost half of all pharmaceutical protein products are formulated in the solid form to preserve protein native structure and extend product shelf-life. In this review, both traditional and emerging drying techniques for producing protein solids will be discussed. During the drying process, various stresses can impact the stability of protein solids. However, understanding the impact of stress on protein product quality can be challenging due to the lack of reliable characterization techniques for biological solids. Both conventional and advanced characterization techniques are discussed including differential scanning calorimetry (DSC), solid-state Fourier transform infrared spectrometry (ssFTIR), solid-state fluorescence spectrometry, solid-state hydrogen deuterium exchange (ssHDX), solid-state nuclear magnetic resonance (ssNMR) and solid-state photolytic labeling (ssPL). Advanced characterization tools may offer mechanistic investigations into local structural changes and interactions at higher resolutions. The continuous exploration of new drying techniques, as well as a better understanding of the effects caused by different drying techniques in solid state, would advance the formulation development of biological products with superior quality.
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Affiliation(s)
- Yuan Chen
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA
| | - Tarun Tejasvi Mutukuri
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA
| | - Nathan E Wilson
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA
| | - Qi Tony Zhou
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA.
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35
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Hall MP, Kincaid VA, Jost EA, Smith TP, Hurst R, Forsyth SK, Fitzgerald C, Ressler VT, Zimmermann K, Lazar D, Wood MG, Wood KV, Kirkland TA, Encell LP, Machleidt T, Dart ML. Toward a Point-of-Need Bioluminescence-Based Immunoassay Utilizing a Complete Shelf-Stable Reagent. Anal Chem 2021; 93:5177-5184. [PMID: 33730483 DOI: 10.1021/acs.analchem.0c05074] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Enzyme-linked immunosorbent assays (ELISAs) are used extensively for the detection and quantification of biomolecules in clinical diagnostics as well as in basic research. Although broadly used, the inherent complexities of ELISAs preclude their utility for straightforward point-of-need testing, where speed and simplicity are essential. With this in mind, we developed a bioluminescence-based immunoassay format that provides a sensitive and simple method for detecting biomolecules in clinical samples. We utilized a ternary, split-NanoLuc luciferase complementation reporter consisting of two small peptides (11mer, 13mer) and a 17 kDa polypeptide combined with a luminogenic substrate to create a complete, shelf-stable add-and-read assay detection reagent. Directed evolution was used to optimize reporter constituent sequences to impart chemical and thermal stability, as well as solubility, while formulation optimization was applied to stabilize an all-in-one reagent that can be reconstituted in aqueous buffers or sample matrices. The result of these efforts is a robust, first-generation bioluminescence-based homogenous immunoassay reporter platform where all assay components can be configured into a stable lyophilized cake, supporting homogeneous, rapid, and sensitive one-step biomolecule quantification in complex human samples. This technology represents a promising alternative immunoassay format with significant potential to bring critical diagnostic molecular detection testing closer to the point-of-need.
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Affiliation(s)
- Mary P Hall
- Promega Corporation, Madison, Wisconsin 53711, United States
| | | | - Emily A Jost
- Promega Corporation, Madison, Wisconsin 53711, United States
| | - Thomas P Smith
- Promega Biosciences LLC, San Luis Obispo, California 93401, United States
| | - Robin Hurst
- Promega Corporation, Madison, Wisconsin 53711, United States
| | | | - Connor Fitzgerald
- Promega Biosciences LLC, San Luis Obispo, California 93401, United States
| | | | - Kris Zimmermann
- Promega Corporation, Madison, Wisconsin 53711, United States
| | - Dan Lazar
- Promega Corporation, Madison, Wisconsin 53711, United States
| | - Monika G Wood
- Promega Corporation, Madison, Wisconsin 53711, United States
| | - Keith V Wood
- Promega Corporation, Madison, Wisconsin 53711, United States
| | - Thomas A Kirkland
- Promega Biosciences LLC, San Luis Obispo, California 93401, United States
| | - Lance P Encell
- Promega Corporation, Madison, Wisconsin 53711, United States
| | | | - Melanie L Dart
- Promega Corporation, Madison, Wisconsin 53711, United States
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36
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Li Z, Han X, Hong X, Li X, Gao J, Zhang H, Zheng A. Lyophilization Serves as an Effective Strategy for Drug Development of the α9α10 Nicotinic Acetylcholine Receptor Antagonist α-Conotoxin GeXIVA[1,2]. Mar Drugs 2021; 19:md19030121. [PMID: 33668830 PMCID: PMC7996264 DOI: 10.3390/md19030121] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2021] [Revised: 02/18/2021] [Accepted: 02/22/2021] [Indexed: 12/22/2022] Open
Abstract
α-Conotoxin GeXIVA[1,2] is a highly potent and selective antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype. It has the advantages of strong efficacy, no tolerance, and no effect on motor function, which has been expected help patients with neuropathic pain. However, drug development for clinical use is severely limited owing to its instability. Lyophilization is applied as the most preferred method to solve this problem. The prepared lyophilized powder is characterized by differential scanning calorimetry (DSC), powder X-ray diffractometry (PXRD), and Fourier transform infrared spectroscopy (FTIR). Molecular simulation is also used to explore the internal distribution and forces formed in the system. The analgesic effect on paclitaxel-induced neuropathic pain following single and 14-day repeated administrations are evaluated by the von Frey test and the tail-flick test. Trehalose combined with mannitol in a ratio of 1:1 is employed as the excipients in the determined formulation, where trehalose acts as the stabilizer and mannitol acts as the bulking agent, according to the results of DSC, PXRD, and FTIR. Both GeXIVA[1,2] (API) and GeXIVA[1,2] lyophilized powder (formulation) could produce stable analgesic effect. These results indicated that GeXIVA[1,2] lyophilized powder could improve the stability and provide an effective strategy to push it into clinical use as a new analgesic drug.
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Affiliation(s)
| | | | | | | | | | | | - Aiping Zheng
- Correspondence: ; Tel.: +86-010-66931694 or +86-13520467936
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37
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Gupta D, Tiwari P, Haque MA, Sachdeva E, Hassan MI, Ethayathulla AS, Kaur P. Structural insights into the transient closed conformation and pH dependent ATPase activity of S.Typhi GyraseB N- terminal domain. Arch Biochem Biophys 2021; 701:108786. [PMID: 33548211 DOI: 10.1016/j.abb.2021.108786] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Revised: 01/06/2021] [Accepted: 01/29/2021] [Indexed: 11/28/2022]
Abstract
DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.
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Affiliation(s)
- Deepali Gupta
- Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India
| | - Pragya Tiwari
- Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India
| | - Md Anzarul Haque
- Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India
| | - Ekta Sachdeva
- Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India
| | - Md Imtaiyaz Hassan
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 10025, India
| | - Abdul S Ethayathulla
- Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India
| | - Punit Kaur
- Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India.
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38
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Mutukuri TT, Wilson NE, Taylor LS, Topp EM, Zhou QT. Effects of drying method and excipient on the structure and physical stability of protein solids: Freeze drying vs. spray freeze drying. Int J Pharm 2020; 594:120169. [PMID: 33333176 DOI: 10.1016/j.ijpharm.2020.120169] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Revised: 12/04/2020] [Accepted: 12/07/2020] [Indexed: 11/25/2022]
Abstract
This study aims to determine the impacts of drying method and excipient on changes in protein structure and physical stability of model protein solids. Protein solids containing one of two model proteins (lysozyme or myoglobin) were produced with or without excipients (sucrose or mannitol) using freeze drying or spray freeze drying (SFD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), circular dichroism spectrometry (CD), size exclusion chromatography (SEC), BET surface area measurements and solid-state hydrogen deuterium exchange with mass spectrometry (ssHDX-MS). ssFTIR and CD could identify little to no difference in structure of the proteins in the formulation. ssHDX-MS was able to identify the population heterogeneity, which was undetectable by conventional characterization techniques of ssFTIR and CD. ssHDX-MS metrics such as Dmax and peak area showed a good correlation with the protein physical instability (loss of the monomeric peak area by size exclusion chromatography) in 90-day stability studies conducted at 40 °C for lysozyme. Higher specific surface area was associated with greater loss in monomer content for myoglobin-mannitol formulations as compared to myoglobin-only formulations. Spray freeze drying seems a viable manufacturing technique for protein solids with appropriate optimization of formulations. The differences observed within the formulations and between the processes using ssHDX-MS, BET surface area measurements and SEC in this study provide an insight into the influence of drying methods and excipients on protein physical stability.
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Affiliation(s)
- Tarun Tejasvi Mutukuri
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA
| | - Nathan E Wilson
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA
| | - Lynne S Taylor
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA
| | - Elizabeth M Topp
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA; National Institute for Bioprocessing Research and Training, Belfield, Blackrock, Co. Dublin A94 X099, Ireland
| | - Qi Tony Zhou
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA.
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Butreddy A, Janga KY, Ajjarapu S, Sarabu S, Dudhipala N. Instability of therapeutic proteins - An overview of stresses, stabilization mechanisms and analytical techniques involved in lyophilized proteins. Int J Biol Macromol 2020; 167:309-325. [PMID: 33275971 DOI: 10.1016/j.ijbiomac.2020.11.188] [Citation(s) in RCA: 77] [Impact Index Per Article: 15.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Revised: 11/23/2020] [Accepted: 11/26/2020] [Indexed: 01/06/2023]
Abstract
Solid-state is the preferred choice for storage of protein therapeutics to improve stability and preserve the biological activity by decreasing the physical and chemical degradation associated with liquid protein formulations. Lyophilization or freeze-drying is an effective drying method to overcome the instability problems of proteins. However, the processing steps (freezing, primary drying and secondary drying) involved in the lyophilization process can expose the proteins to various stress and harsh conditions, leading to denaturation, aggregation often a loss in activity of protein therapeutics. Stabilizers such as sugars and surfactants are often added to protect the proteins against physical stress associated with lyophilization process and storage conditions. Another way to curtail the degradation of proteins due to process related stress is by modification of the lyophilization process. Slow freezing, high nucleation temperature, decreasing the extent of supercooling, and annealing can minimize the formation of the interface (ice-water) by producing large ice crystals with less surface area, thereby preserving the native structure and stability of the proteins. Hence, a thorough understanding of formulation composition, lyophilization process parameters and the choice of analytical methods to characterize and monitor the protein instability is crucial for development of stable therapeutic protein products. This review provides an overview of various stress conditions that proteins might encounter during lyophilization process, mechanisms to improve the stability and analytical techniques to tackle the proteins instability during both freeze-drying and storage.
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Affiliation(s)
- Arun Butreddy
- Formulation R&D, Biological E. Limited, IKP Knowledge Park, Shameerpet, Hyderabad, Telangana State 500078, India; Laboratory of Nanotechnology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana State 506009, India
| | - Karthik Yadav Janga
- Laboratory of Nanotechnology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana State 506009, India
| | - Srinivas Ajjarapu
- Industrial Research Laboratory, Department of Pharmacy, Birla Institute of Technology and Science, Pilani 333031, India
| | - Sandeep Sarabu
- Laboratory of Nanotechnology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana State 506009, India
| | - Narendar Dudhipala
- Laboratory of Nanotechnology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana State 506009, India; Department of Pharmaceutics, Vaagdevi College of Pharmacy, Warangal, Telangana State 506 005, India..
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40
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Seifert I, Friess W. Improvement of arginine hydrochloride based antibody lyophilisates. Int J Pharm 2020; 589:119859. [DOI: 10.1016/j.ijpharm.2020.119859] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2020] [Revised: 09/02/2020] [Accepted: 09/03/2020] [Indexed: 01/06/2023]
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41
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Wood VE, Groves K, Cryar A, Quaglia M, Matejtschuk P, Dalby PA. HDX and In Silico Docking Reveal that Excipients Stabilize G-CSF via a Combination of Preferential Exclusion and Specific Hotspot Interactions. Mol Pharm 2020; 17:4637-4651. [PMID: 33112626 DOI: 10.1021/acs.molpharmaceut.0c00877] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Assuring the stability of therapeutic proteins is a major challenge in the biopharmaceutical industry, and a better molecular understanding of the mechanisms through which formulations influence their stability is an ongoing priority. While the preferential exclusion effects of excipients are well known, the additional presence and impact of specific protein-excipient interactions have proven to be more elusive to identify and characterize. We have taken a combined approach of in silico molecular docking and hydrogen deuterium exchange-mass spectrometry (HDX-MS) to characterize the interactions between granulocyte colony-stimulating factor (G-CSF), and some common excipients. These interactions were related to their influence on the thermal-melting temperatures (Tm) for the nonreversible unfolding of G-CSF in liquid formulations. The residue-level interaction sites predicted in silico correlated well with those identified experimentally and highlighted the potential impact of specific excipient interactions on the Tm of G-CSF.
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Affiliation(s)
- Victoria E Wood
- Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, United Kingdom
| | - Kate Groves
- National Measurement Laboratory at LGC Ltd., Queens Road, Teddington TW11 0LY, United Kingdom
| | - Adam Cryar
- National Measurement Laboratory at LGC Ltd., Queens Road, Teddington TW11 0LY, United Kingdom
| | - Milena Quaglia
- National Measurement Laboratory at LGC Ltd., Queens Road, Teddington TW11 0LY, United Kingdom
| | - Paul Matejtschuk
- National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom
| | - Paul A Dalby
- Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, United Kingdom
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42
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Zhang Y, Zhang H, Ghosh D, Williams RO. Just how prevalent are peptide therapeutic products? A critical review. Int J Pharm 2020; 587:119491. [PMID: 32622810 PMCID: PMC10655677 DOI: 10.1016/j.ijpharm.2020.119491] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2020] [Revised: 05/28/2020] [Accepted: 05/29/2020] [Indexed: 02/08/2023]
Abstract
How prevalent are peptide therapeutic products? How innovative are the formulations used to deliver peptides? This review provides a critical analysis of therapeutic peptide products and the formulations approved by the United States Food and Drug administration (FDA), the European Medicines Agency (EMA), and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). This review also provides an in-depth analysis of dosage forms and administration routes for delivering peptide therapeutics, including injectables, oral dosage forms, and other routes of administration. We discuss the function of excipients in parenteral formulations in detail, since most peptide therapeutics are parenterally administered. We provide case studies of alternate delivery routes and dosage forms. Based on our analysis, therapeutic peptides administered as injectables remain the most commonly used dosage forms, particularly in the form of subcutaneous, intravenous, or intramuscular injections. In addition, therapeutic peptides are formulated to achieve prolonged release, often through the use of polymer carriers. The limited number of oral therapeutic peptide products and their poor absorption and subsequent low bioavailability indicate a need for new technologies to broaden the formulation design space. Therapeutic peptide products may also be delivered through other administration routes, including intranasal, implant, and sublingual routes. Therefore, an in-depth understanding of how therapeutic peptides are now formulated and administered is essential to improve peptide delivery, improve patient compliance, and reduce the healthcare burden for these crucial therapeutic agents.
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Affiliation(s)
- Yajie Zhang
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA
| | - Hairui Zhang
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA
| | - Debadyuti Ghosh
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA
| | - Robert O Williams
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA.
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43
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Zhang Y, Williams III RO, Tucker HO. Formulation strategies in immunotherapeutic pharmaceutical products. World J Clin Oncol 2020; 11:275-282. [PMID: 32728530 PMCID: PMC7360519 DOI: 10.5306/wjco.v11.i5.275] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/19/2020] [Revised: 05/05/2020] [Accepted: 05/13/2020] [Indexed: 02/06/2023] Open
Abstract
Development of immunologic-based biopharmaceutical products have strikingly increased in recent years and have made evident contributions to human health. Antibodies are the leading entity in immunotherapy, while chimeric antigen receptor T cells therapies are the advent of a novel strategy in this area. In order to enable antibody candidates or cells available as products, formulation is critical in terms of stabilize molecules or cells to achieve practical shelf life, storage and handling conditions. Here we provide a concise and contemporary review of ongoing formulation strategies and excipients used in approved antibodies and cellular therapeutic products. Excipients are categorized, and their function in formulations are discussed.
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Affiliation(s)
- Yajie Zhang
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, United States
| | - Robert O Williams III
- Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, United States
| | - Haley Oana Tucker
- Departments of Bioengineering and Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, United States
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44
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Excipients in freeze-dried biopharmaceuticals: Contributions toward formulation stability and lyophilisation cycle optimisation. Int J Pharm 2020; 576:119029. [DOI: 10.1016/j.ijpharm.2020.119029] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Revised: 01/08/2020] [Accepted: 01/09/2020] [Indexed: 02/08/2023]
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45
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Wilson NE, Mutukuri TT, Zemlyanov DY, Taylor LS, Topp EM, Zhou QT. Surface Composition and Formulation Heterogeneity of Protein Solids Produced by Spray Drying. Pharm Res 2019; 37:14. [DOI: 10.1007/s11095-019-2738-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Accepted: 11/22/2019] [Indexed: 11/29/2022]
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46
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Madani F, Hsein H, Busignies V, Tchoreloff P. An overview on dosage forms and formulation strategies for vaccines and antibodies oral delivery. Pharm Dev Technol 2019; 25:133-148. [DOI: 10.1080/10837450.2019.1689402] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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47
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Geraldes DC, Beraldo-de-Araújo VL, Pardo BOP, Pessoa Junior A, Stephano MA, de Oliveira-Nascimento L. Protein drug delivery: current dosage form profile and formulation strategies. J Drug Target 2019; 28:339-355. [DOI: 10.1080/1061186x.2019.1669043] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Affiliation(s)
- Danilo Costa Geraldes
- Faculty of Pharmaceutical Sciences, State University of Campinas, Campinas, SP, Brazil
- Biochemistry and Tissue Biology Department, Biology Institute, State University of Campinas, Campinas, SP, Brazil
| | - Viviane Lucia Beraldo-de-Araújo
- Faculty of Pharmaceutical Sciences, State University of Campinas, Campinas, SP, Brazil
- Biochemistry and Tissue Biology Department, Biology Institute, State University of Campinas, Campinas, SP, Brazil
| | | | | | | | - Laura de Oliveira-Nascimento
- Faculty of Pharmaceutical Sciences, State University of Campinas, Campinas, SP, Brazil
- Biochemistry and Tissue Biology Department, Biology Institute, State University of Campinas, Campinas, SP, Brazil
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48
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Formulation Composition and Process Affect Counterion for CSP7 Peptide. Pharmaceutics 2019; 11:pharmaceutics11100498. [PMID: 31569515 PMCID: PMC6835953 DOI: 10.3390/pharmaceutics11100498] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2019] [Revised: 09/19/2019] [Accepted: 09/25/2019] [Indexed: 12/26/2022] Open
Abstract
Counterions commonly remain with peptides in salt form after peptide purification. In animal and human studies, acetate counterions are a safer and more acceptable choice for peptides than others (e.g., trifluoroacetate counterions). Various salt forms of caveolin-1 scaffolding domain (CSP7) affect counterion volatilization. The development of lyophilized formulations containing volatile compounds is a challenge because these compounds sublimate away during the process. This work aims to investigate the effect of excipients and lyophilization parameters on the preservation of volatile compounds after lyophilization. The peak areas obtained from 1H and 19F NMR spectra were used to calculate the molar ratio of counterions to CSP7. We found that the pH modifier excipient had the greatest impact on the loss of counterions. By optimizing the molar ratio of bulking agent to CSP7, volatile compounds can be preserved after lyophilization. Higher chamber pressure during lyophilization can lower the sublimation rate of volatile compounds. Moreover, the loss of volatile compounds affects the stability of CSP7 due to the pH shift of reconstituted solutions, thereby causing peptide aggregation. The optimization of the formulation and processing helps preserve volatile compounds, thus minimizing the pH change of reconstituted solutions and maintaining the stability of peptide.
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49
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Jain D, Mahammad SS, Singh PP, Kodipyaka R. A review on parenteral delivery of peptides and proteins. Drug Dev Ind Pharm 2019; 45:1403-1420. [DOI: 10.1080/03639045.2019.1628770] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Affiliation(s)
- Divisha Jain
- Custom Pharma Services (CPS), Dr. Reddy’s Laboratories Ltd, Hyderabad, India
| | - S. Shahe Mahammad
- Custom Pharma Services (CPS), Dr. Reddy’s Laboratories Ltd, Hyderabad, India
| | - Pirthi Pal Singh
- Custom Pharma Services (CPS), Dr. Reddy’s Laboratories Ltd, Hyderabad, India
| | - Ravinder Kodipyaka
- Custom Pharma Services (CPS), Dr. Reddy’s Laboratories Ltd, Hyderabad, India
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50
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Wilson NE, Topp EM, Zhou QT. Effects of drying method and excipient on structure and stability of protein solids using solid-state hydrogen/deuterium exchange mass spectrometry (ssHDX-MS). Int J Pharm 2019; 567:118470. [PMID: 31252148 DOI: 10.1016/j.ijpharm.2019.118470] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2019] [Revised: 06/23/2019] [Accepted: 06/24/2019] [Indexed: 12/01/2022]
Abstract
Powders containing one of four model proteins (myoglobin, bovine serum albumin, lysozyme, β-lactoglobulin) were formulated with either sucrose, trehalose, or mannitol and dried using lyophilization or spray-drying. The powders were characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), solid-state fluorescence spectroscopy, differential scanning calorimetry (DSC) and solid-state hydrogen/deuterium exchange mass spectrometry (ssHDX-MS). ssFTIR and fluorescence spectroscopy identified minor structural differences among powders with different excipients and drying methods for some proteins. Using ssHDX-MS, differences in protein structure were observed among protein formulations containing sucrose or trehalose and mannitol, and/or with varying processing conditions, including proteins like β-lactoglobulin, for which standard characterization techniques showed no differences. Proteins processed by spray-drying typically showed greater heterogeneity by ssHDX-MS than those lyophilized; these differences were not detected by ssFTIR or solid-state fluorescence spectroscopy. The ssHDX-MS metrics were better correlated with protein physical instability measured by size-exclusion chromatography in 90-day stability studies (40 °C, 33% RH) than with the results of DSC, ssFTIR, or fluorescence spectroscopy. Thus, ssHDX-MS detected subtle changes in conformation and/or matrix interactions for these proteins that were correlated with storage stability, suggesting that the method can be used to design robust solid-state pharmaceutical protein products more rapidly.
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Affiliation(s)
- Nathan E Wilson
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA
| | - Elizabeth M Topp
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA.
| | - Qi Tony Zhou
- Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA.
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