(M1030-02-14) Case Study: Viral Inactivation in Bioprocessing Using Hydroxypropyl Beta-Cyclodextrins, a Compendial Excipient

Purpose: Virus inactivation is an essential step in the purification of biologics (mAbs, viral vectors, blood products, etc.) derived from cell expression systems, plasma fractionation, and other sources. Conventionally, S/D (solvent/detergent) is widely applied to inactive enveloped viruses, among which Triton X-100 is the most used detergent. However, due to its environmental risk, Triton X-100 has been banned in Europe.¹ The industry has been searching for suitable replacements for Triton X-100.² This study aims to validate the virus inactivation efficiency of hydroxypropyl beta-cyclodextrin (HPBCD) when applied to the harvested cell culture fluid (HCCF). The compatibility of HPBCD in subsequent downstream processes will also be investigated.

Methods: Hydroxypropyl beta-cyclodextrin (KLEPTOSE® HP Biopharma) was obtained from Roquette Frères (Lestrem, France). Tri-n-butyl phosphate (TNBP) and Triton X-100 were sourced from Merck KGaA. The HCCF, protein A eluate of mAb X, and model viruses - Xenotropic Murine Leukemia Virus (X-MuLV) and Herpes Simplex Virus type 1 (HSV-1) - were provided by Syngene International. Ipilimumab was produced in-house using the stable CHO cell line. To evaluate virus inactivation efficiency, the HCCF of mAb X was spiked with 5% X-MuLV or HSV-1. Subsequently, the virus inactivation reagents, including different concentrations of HPBCD, 1% polysorbate, 1% Triton X-100, and combinations thereof with 0.3% TNBP, were added and incubated for a defined period of time. These samples were tested along with load and processing controls (i.e., media spiked with the virus but without virus inactivation reagents) using a cell-based infectivity assay. The virus titers were quantified, and log₁₀ reduction values (LRV) were calculated statistically. The compatibility of HPBCD with downstream processes was evaluated by assessing protein stability and impurity removal efficiency. HCCF of ipilimumab, spiked with 5% or 10% HPBCD, was subjected to protein A chromatography, followed by anion exchange chromatography and cation exchange chromatography. The size and charge profiles of ipilimumab after each chromatography step were assessed using size exclusion chromatography (SEC-HPLC) and cation exchange chromatography (CIEX-HPLC). The host cell protein (HCP), host cell DNA (HCDNA), and leached protein A in the same samples were measured using an ELISA kit and Q-PCR assay, respectively.

Results: KLEPTOSE® HP is capable of extracting cholesterol and other lipids from the viral envelope, thereby disrupting the viral structure and leading to virus inactivation. The virus inactivation effect of KLEPTOSE® HP is concentration-dependent. In the X-MuLV inactivation study (Figure 1), a 5% HPBCD solution resulted in a relatively modest virus inactivation, achieving approximately a 1.7 log₁₀ reduction at both 15 and 60 minutes, comparable to the reduction observed with 1% polysorbate. In contrast, a 10% KLEPTOSE® HP solution, as well as 5% KLEPTOSE® HP in combination with 0.3% TNBP achieved greater than 4 log₁₀ reduction, comparable to the reductions observed with 1% Triton X-100, either alone or in combination with 0.3% TNBP. In the HSV-1 inactivation study (Figure 2), a 15% KLEPTOSE® HP solution achieved a greater than 4 log₁₀ reduction within 30 minutes. Furthermore, similar to the results observed with X-MuLV, the combination of 5% KLEPTOSE® HP with 0.3% TNBP, as well as 1% Triton X-100 alone or in combination with 0.3% TNBP, resulted in a rapid reduction exceeding 4 log₁₀. Additionally, HPBCD is fully compatible with downstream processes. The data indicate that the size and charge profiles of purified ipilimumab remain consistent across all samples, regardless of whether 5% or 10% KLEPTOSE® HP was added to the HCCF. The removal efficiency for impurities, including HCP, HCDNA, and leaked protein A is also comparable among the test samples (Figure 3).

Conclusion: HPBCD is an effective virus-inactivation agent that ensures the viral safety of biologics. It is compatible with downstream processes, maintaining product stability and purity. Given its established safety for parenteral administration - demonstrated by its inclusion in numerous approved drugs - HPBCD can potentially remain in the final drug formulation as a stabilizer. Moreover, multiple studies have shown that HPBCD protects biologics from the stresses encountered during various manufacturing steps, further supporting its role as a stabilizing agent.

References: 1. Regulation (EC) No. 1907/2006 of the European Parliament and of the Council of 18 December 2006. EUR-Lex 1 May 2022; https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02006R1907-20220501.
2. Luo Wen et al. “Identification and characterization of a triton X-100 replacement for virus inactivation.” Biotechnol Prog. vol.36,6 (2020): e3036

The inactivation of X-MuLV by different reagents in harvested cell culture fluid (HCCF).

The inactivation of HSV-1 by different reagents in harvested cell culture fluid (HCCF).

Impurities removal of (A) host cell protein, (B) host cell DNA, and (C) leached protein A after each chromatography step for the starting HCCF load with different concentrations of HPBCD (KLEPTOSE® HP Biopharma).