Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients

Abstract

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.

Keywords: IIRMI; In vitro model; excipient; immunogenicity; masking; reporter cell lines.

Figure 1

Excipient effect on cell viability. RAW-Blue and THP-1-Blue cells were incubated in triplicate with increasing concentrations of (A) Polysorbate 80, (B) Poloxamer 188, (C) Arginine, (D) Histidine, (E) Mannitol, (F) Sucrose, (G) Trehalose, or (H) Albumin for 24 hours. Top of each graph: Scatter plot depicting concentration of the excipient in licensed biological drugs regulated by the Office of Biotechnology Products at the FDA for reference. Cell metabolism/viability was assessed using the CCK8 assay. Results are shown relative to no-excipient control cells. The graph is representative of three independent experiments. Results are presented as the mean ± SD. RAW- and THP-1-Blue cells are solid black or grey, respectively. *p < 0.05.

Figure 2

Impact of excipients on the sensitivity of RAW-Blue cells to detect IIRMI. RAW-Blue cells were stimulated with a single dose of different model IIRMI in the presence of increasing concentration of (A) Polysorbate 80, (B) Poloxamer 188, (C) Arginine, (D) Histidine, (E) Mannitol, (F) Sucrose, (G) Trehalose, or (H) Albumin for 24 hours. IIRMI activation of RAW-Blue cells was measured as NF-κB activation using Quanti-blue detection media. Graphs show the fold changes relative to the corresponding IIRMI stimulation in the absence of excipients. Cells were treated in triplicate and results are presented as the mean ± SD for 3 independent experiments. *p < 0.05 and **p < 0.01.

Figure 3

Impact of excipients on the sensitivity of THP-1-Blue cells to detect IIRMI. THP-1-Blue cells were stimulated with a single dose of model IIRMI in the presence of increasing concentration of (A) Polysorbate 80, (B) Poloxamer 188, (C) Arginine, (D) Histidine, (E) Mannitol, (F) Sucrose, (G) Trehalose, or (H) Albumin for 24 hours. IIRMI activation of THP-1-Blue cells was measured as NF-κB activation using Quanti-blue detection media. Graphs show fold changes relative to cells stimulated in the absence of excipients. Cells were treated in triplicate and results are presented as the mean ± SD of 3 independent experiments. *p < 0.05.

Figure 4

Polysorbate 80 does not mask detection of LPS in vivo. Mice were injected subcutaneously with increasing concentrations of LPS in the presence of saline (blue bars) or 0.02% w/w polysorbate 80 (light blue bars) (n=3/group). Results are shown as the fold change in IL-1β (A) and S100A8 (B) mRNA in local skin 6 hours post injection relative to mice that received the same volume of saline. Data is presented as the geometric mean and geometric standard deviation.

Figure 5

Impact of drug formulations on IIRMI detection by RAW-Blue and THP1 cells. RAW- and THP-1-Blue cells were stimulated with three model IIRMIs [(LPS (1ng), FSL (1ng), or Zymosan (1μg)] for 24 hours in the presence of drug product formulation (25% v/v). IIRMI activation of NF-κB in RAW- and THP-1-Blue cells was measured using Quanti-blue detection media. (A–B) Impact of 25% v/v drug formulation on baseline NF-κB activation in RAW- and THP-1-Blue cells. (C–E) RAW-Blue cells stimulated with IIRMIs for 24 hours in the presence of 25% v/v formulation. (F–H) THP-1-Blue cells stimulated with IIRMIs for 24 hours in the presence of 25% v/v formulation. (I, J) RAW-Blue cells were incubated for 24 hours with LPS or FSL in presence of 25% v/v formulation A or media alone. Results are shown relative to IIRMI stimulation in media alone (no formulation) treated cells. Results are representative of at least 3 independent experiments. Cells were treated in triplicate and results are presented as the mean ± SD. * or ^#p <0.05 and **p < 0.01. * indicates statistical difference between spiked and unspiked cells in the corresponding formulation; # indicates statistical difference to the level of NF-κB activation by the corresponding stimuli in the absence of formulation (black bar, untreated).

Figure 6

Impact of drug formulations on PBMCs in vitro detection of IIRMI. PBMCs were stimulated for 24h with LPS (10pg), FSL (100pg), or Zymosan (1ng) in media alone, drug product or the corresponding formulation (10% v/v) for 3 licensed or approved products. Fold changes in IL-8 gene expression are relative to the unspiked cells. Fold increase in mRNA for IL-8 (A–E), IL-6 (I), and IL-1β (M) expression by PBMC treated with drug product (B-D, J-P) or drug formulation (F-H) (10% v/v) unspiked or spiked with TLR agonists. N=8-18 PBMC from healthy donors per condition. Donors were considered positive if the level of mRNA expression was ≥ 2-fold increase over the unspiked control; the percentage of positive PBMC in each condition is presented under each graph. * indicated p < 0.05 and is a comparison between the expression in cells stimulated with a model IIRMI in media, drug products, or drug formulation and the corresponding unspiked control.

Figure 7

Product A does not mask detection of LPS in vivo. Mice were injected subcutaneously with 1ng of LPS with saline, product A, the formulation A, or polysorbate 80. The induction of IL-1β (A), IL-6 (B), and S100A8 (C) at the site of injection was measured in skin 6 hours post injection. Results are shown as the fold increase over saline alone. Data is presented as the geometric mean and geometric SD. n = 4-5.