Truly continuous low pH viral inactivation for biopharmaceutical process integration

Abstract

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities-which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.

Keywords: continuous processing; low pH viral inactivation; residence time distribution; virus clearance; virus inactivation.

Figure 1

System setup depicting the key components. (a) The continuous VI setup is comprised of two syringe pumps used to drive both streams, an in‐line mixer for homogenization, a sampling port placed before the reactor and the packed bed CVIR. The arrows denote the fluid stream. The probability density function for the RTD of CVIR (full line) and in‐line mixer connected before the CVIR (dashed line) at 19.6 cm/hr. (b) The circles and triangles highlight F _0.5%, E _max and F _99.5% for the CVIR alone and for the in‐line mixer connected before the CVIR, respectively, and their corresponding time is given in the top right insert. F _0.5% is used to define the CVIR volume accordingly with the MRT approach. CVIR, continuous viral inactivation reactor; RTD, residence time distribution; VI, viral inactivation [Color figure can be viewed at https://wileyonlinelibrary.com]

Figure 2

(a) Column characterization by the t[F _0.5]/t[F _0.005] metric, (b) height equivalent to a theoretical plate (HETP) in µm, and (c) asymmetry at 10% peak height

Figure 3

Poly(methyl methacrylate) (PMMA) stability at acidic pH. Exemplary results of particle identification and determination by image processing. (a) The dotted circles denote the particle identified by the image processing tool. The scale bar is 500 µm. PMMA particle diameter probability density function (PDF) upon exposure to different conditions. (b–e) The gray dashed line represents the reference data (dry beads) and the solid black line represents the respective condition. The quartiles of the diameter distribution and the number of successfully identified particles are shown in the top, left inset. (f) PMMA packed bed stability upon exposure to pH 3.0. The initial peak steepness was measured five times per contact time point and the average ± standard deviation is shown

Figure 4

Titration to low pH. The test item spiked with either model virus (X‐MuLV in full line or PRV in dashed line) was titrated with the acid stock (2 M glycine [pH 2.7]) to determine the flow rate ratio of both streams for continuous VI runs. For each virus, the titration was performed in triplicate. The dotted line and arrow show the pH target (pH 3.7) and the respective acid fraction. The gray highlight shows the acceptable pH range (pH‐target ± 0.1). PRV, pseudorabies virus; VI, viral inactivation; X‐MuLV, xenotropic murine leukemia virus

Figure 5

pH‐value measurement throughout the continuous operation for (a) xenotropic murine leukemia virus (X‐MuLV) and (b) pseudorabies virus (PRV) experiments. Samples were drawn for off‐line pH confirmation after the mixer (M) and at the reactor outlet after 1, 2, 3, 4, and 5 V _R of operation for 15, 30, and 60 min continuous incubation (black circles, dark gray squares, and light gray diamonds, respectively). The solid lines link the adjacent points and serve only as visual aids. The dashed lines represent the pH target and range (pH 3.7 ± 0.1)

Figure 6

Control experiments at neutral pH with (a) xenotropic murine leukemia virus (X‐MuLV) and (b) pseudorabies virus (PRV). For each model virus, the extreme flow rates were tested, which correspond to 15 and 60 min incubation (black circles and gray squares, respectively). The spiked test item control (SC), the mixer's outlet (M), the continuous reactor's outlet at 1, 2, 3, 4, and 5 V _R and the hold control (HC) were sampled for virus titration

Figure 7

Continuous low pH viral inactivation. Viral titer for (a) X‐MuLV and (b) PRV throughout the continuous experiments. The SC denotes the starting concentration of the spiked test item. The flow‐through volume from 1 to 5 V _R denotes the operation duration. The incubation inside the reactor was 15, 30 or 60 min (black circle, dark gray diamond, and light gray triangle, respectively). The downward arrow denotes a sample below the TCID₅₀ limit of detection. Viral inactivation kinetics at low pH for (c) X‐MuLV and (d) PRV. The average ± standard deviation LRV registered by continuous operation (black triangles) is compared against equivalent data generated in batch mode (gray circles). Note that, due to the constant titers, the standard deviation is 0, which renders the error bars invisible. The upward arrow denotes minimum LRV due to the respective sample being below the limit of detection. The lines link the adjacent points and serve only as visual aids. LRV, logarithmic reduction value; PRV, pseudorabies virus; SC, spike control; TCID₅₀, median tissue culture infective dose; X‐MuLV, xenotropic murine leukemia virus