Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients
Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-09-01
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| Series: | Frontiers in Immunology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2022.970499/full |
| _version_ | 1818067278203191296 |
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| author | Seth G. Thacker Cheng Her Logan Kelley-Baker Derek D C. Ireland Mohanraj Manangeeswaran Eric S. Pang Daniela Verthelyi |
| author_facet | Seth G. Thacker Cheng Her Logan Kelley-Baker Derek D C. Ireland Mohanraj Manangeeswaran Eric S. Pang Daniela Verthelyi |
| author_sort | Seth G. Thacker |
| collection | DOAJ |
| description | Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays. |
| first_indexed | 2024-12-10T15:21:08Z |
| format | Article |
| id | doaj.art-12c252eade24479d8194d84845fe9d06 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-10T15:21:08Z |
| publishDate | 2022-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-12c252eade24479d8194d84845fe9d062022-12-22T01:43:41ZengFrontiers Media S.A.Frontiers in Immunology1664-32242022-09-011310.3389/fimmu.2022.970499970499Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipientsSeth G. Thacker0Cheng Her1Logan Kelley-Baker2Derek D C. Ireland3Mohanraj Manangeeswaran4Eric S. Pang5Daniela Verthelyi6Laboratory of Immunology, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesLaboratory of Immunology, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesLaboratory of Immunology, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesLaboratory of Immunology, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesLaboratory of Immunology, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesDivision of Therapeutic Performance, Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesLaboratory of Immunology, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United StatesUnintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.https://www.frontiersin.org/articles/10.3389/fimmu.2022.970499/fullimmunogenicityexcipientIIRMIIn vitro modelreporter cell linesmasking |
| spellingShingle | Seth G. Thacker Cheng Her Logan Kelley-Baker Derek D C. Ireland Mohanraj Manangeeswaran Eric S. Pang Daniela Verthelyi Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients Frontiers in Immunology immunogenicity excipient IIRMI In vitro model reporter cell lines masking |
| title | Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients |
| title_full | Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients |
| title_fullStr | Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients |
| title_full_unstemmed | Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients |
| title_short | Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients |
| title_sort | detection of innate immune response modulating impurities iirmi in therapeutic peptides and proteins impact of excipients |
| topic | immunogenicity excipient IIRMI In vitro model reporter cell lines masking |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2022.970499/full |
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