Use of Monocyte-derived macrophages and dendritic cells as a platform to detect Innate Immune Response Modulating Impurities (IIRMIs) in Therapeutic Proteins

EDUARDO MUFARREGE; ETCHEVERRIGARAY, MARINA; DANIELA VERTHELYI

Leesburg

Workshop; NIH Immunology Interest Group Workshop; 2017

National Institute of Health (NIH)

Biotherapeutics are agents produced through complex multi-step manufacturing methods. However, some contaminants derived from different sources (host cell or manufacturing process) may escape the purification strategy and are found in the final product. These contaminants, previously defined as innate immune response modulating impurities (IIRMIs), have the ability to elicit an immune response even when present at trace levels. This response may lead to the development of neutralizing antibodies (NAbs) against the product and an exacerbated production of pro-inflammatory cytokines and chemokines. The use of PBMC has been proposed to assess the potential of impurities that activate the immune cells. However, macrophages and dendritic cells, which are rich in pattern recognition receptors (PRR) and are known to initiate immune responses, are present at very low frequency or absent in peripheral blood (PB). To address this, we evaluated the use of monocyte-derived macrophages (mo-MØ) and immature dendritic cells (mo-iDC) as screening tools to detect IIRMIs. Using purified PRRAgonists (PRRAgs) as model IIRMI we show that both primary cultures are more sensitive than PBMC in detecting IIRMI. Interestingly, mo-MØ and mo-iDC showed different limits of detection (LLOD) for individual PRRAgs. In addition PRRAgs induced increased expression of a set of pro-inflammatory genes in mo-iDC and the profile of genes induced varied with the TLR agonist and concentration. Finally, we tested the capability of human PBMC, mo-MØ and mo-iDC for detecting impurities in a commercially available product for the treatment of autoimmune inflammatory diseases. While PBMC samples were not activated by impurities present in the product, mo-MØ and mo-iDC were activated even when low amount of product where added to the cell culture.