Development of an HSV-1 production process involving serum-reduced culturing and bead-to-bead transfer

Abstract

Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.

Keywords: Bead-to-bead transfer; Herpes simplex virus type 1; Microcarriers; Serum-reduced culturing; Stirred-tank bioreactor; Vero cells.

Fig. 1

Performances of different basal media and supplementations. a Doubling times of Vero cells cultured in 5 types of serum-free media (BM1 - BM5). DMEM + 10% FBS was used as a control (Ctrl) (n ≥ 5). b Normalized volumetric titers of virus production in different serum-free media. c Cell-specific infectivity titers (normalized) of virus production in different serum-free media. All cell-specific infectivity titers were normalized to the average of BM1 (n ≥ 4). Doubling time (d) and viability (e) of Vero cells cultured in BM3 (VP-SFM) supplemented with different concentrations of FBS (n ≥ 10). Normalized volumetric titers (f) and cell-specific infectivity titers (normalized) (g) of virus production in BM3 with different FBS concentrations and DMEM + 10% FBS control. All cell-specific infectivity titers were normalized to the average of 0.2% FBS (n = 2)

Fig. 2

Development and performance of the HSV-1 production process in 1 L bioreactors. a Minimum agitation speed determination of a 1 L bioreactor for homogeneous microcarrier mixing. Vero cell growth curves (b) and viability curves (c) cultured with VP-SFM + 0.2% FBS medium (SRM 1 L) or DMEM + 10% FBS medium (SCM 1 L) in 1 L bioreactors. The times of infection are indicated with dashed circles. d, e Normalized volumetric titers and cell-specific infectivity titers (normalized) of virus production with DMEM + 10% FBS (SCM 1 L) and serum-reduced medium (SRM 1 L) in 1 L bioreactors are illustrated in d and e, respectively. All cell-specific infectivity titers were normalized to the value of SCM 1 L

Fig. 3

Preliminary development of the bead-to-bead transfer process. Detaching percentages (a) and viability drops (b) of the digestion process with no agitation, constant agitation, and combined agitation (n = 3). c Overall recovery proportions of the original, optimized, and optimized processes calibrated with sampling loss (n = 2). The calibration was calculated as described in Table S2. Cell viability curves (d), growth rate comparison (e), and cell growth curves (f) of (1) Vero cells cultured in T-flasks and transferred to microcarriers (seed from TF); (2) cells cultured with microcarriers, treated with a bead-to-bead transfer process and cultured without medium exchange after the attaching period (B2B - no exchange); and (3) cells cultured with microcarriers, treated with a bead-to-bead transfer process and cultured with medium exchange after the attaching period (B2B - exchange) (n = 4). g The normalized volumetric titer of virus production in cells subjected to the baseline process (baseline) or bead-to-bead transfer process (B2B), cultured in DMEM + 10%FBS (SCM) or VP-SFM + 0.2%FBS (SRM) media. (SCM baseline, n = 8; SCM B2B, n = 8; SRM baseline, n = 3; SRM B2B, n = 5)

Fig. 4

Virus production optimization of bead-to-bead and subsequent processes. a, b Correlations between operation and performance parameters and normalized volumetric titers were calculated. Parameters with correlation factors ≤−0.4 and ≥ 0.4 are shown in a and b, respectively. c Proportion of variances of the principal components determined with PCA. d Loadings of principal components 1 and 2. The loadings of the top 10 parameters of the two principal components are plotted

Fig. 5

Performance of the optimized bead-to-bead transfer process with serum-reduced medium cultured in spinner flasks. Detaching percentage and overall recovery proportion (a) and viability drop (b) comparison of the original and optimized bead-to-bead transfer processes. Average growth rates of Day 0–3 (c) and normalized volumetric titers (d) in subcultures of original (B2B - ori), optimized with high inoculation density (B2B - opt high) and optimized with low inoculation density (B2B - opt low) bead-to-bead transfer processes

Fig. 6

Technical transfer of the bead-to-bead transfer process to bioreactors. a Microscopy image of confluent Vero cells cultured with Cytodex-1 immediately before digestion (100×). b Microscopy image of the digested mixture showing Cytodex-1 with detached Vero cells (100×). c The overall recovery proportions of the optimized bead-to-bead transfer process conducted in spinner flasks (B2B SRM SP-opt) and bioreactors (B2B SRM BR) with serum-reduced medium. d Cell growth curve of seeds cultured in a 1 L bioreactor (Day 0–2.8) and sub-cultured and virus production in a 1 L and 3 L bioreactor (Day 3.8–8.8). The dashed circles indicate times of infection. e Viability curves. f Correlation between viable cell density measured by NC-200 and permittivity values measured by the Aber sensor. R²_adjs = 0.8950. g The normalized volumetric titers of the baseline serum-reducing process in a 1 L bioreactor (Baseline 1 L), the virus production process with seeds from optimized bead-to-bead transfer in a spinner flask (B2B SP), and the virus production process with seeds from bead-to-bead transfer in 1 L (B2B 1 L) and 3 L bioreactors (B2B 3 L)