Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes

<div><p>The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween^®, either Triton^™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.</p></div

Representative electron microscope pictures of influenza virus particles, before and after split.

<p>Enlargement pictures to the left 300.000x, pictures to the right 400.000x.Top row whole virus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>, fraction 3.0), bottom row left panel ether split formaldehyde inactivated virus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>, fraction 5.1FE), bottom right panel BPL inactivated Triton split virus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>, fraction 5.1BT). Pictures at top: HA and NA spikes are clearly visible on the outside of the particles. The pictures at the bottom show disrupted, heterologous structures.</p

The main characteristics of the products from the six different downstream processes.

<p>If applicable the requirements are listed. All products comply with the requirements, except product 5.1FE that has low antigenic HA content</p

SDS PAGE of reduced and reduced plus de-glycosylated samples as a fingerprint of principle proteins present.

<p>Lanes M were loaded with marker proteins, with the corresponding molecular weight presented to the left. The fraction sample identity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>) is noted above the lane. Left gel: 1.2 before ZUC, 2.1F after ZUC in phosphate, 2.1B after ZUC in citrate, followed by the six bulks (5.1F, 5.1FE, 5.1FT, 5.1B, 5.1BE and 5.1BT); the migration distance of heavily glycosylated HA proteins varies, causing diffuse bands. In such a case the HA1 band range (~64–79 kD) may be difficult to discriminate from the Nucleoprotein band (~55–66 kD) and the HA2 band range (~23–25 kD) may cover the location of M1 band (~26 kD) as reported by Harvey [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.ref022" target="_blank">22</a>]. After de-glycosylation the HA bands are more distinct and migration distance has increased (right gel, bulks 5.1F, 5.1FE, 5.1FT, 5.1B, 5.1BE and 5.1BT). NP and M1 protein bands have not changed position due to the applied de-glycosylation. In the lanes to the right of the right gel, for comparison products prepared at Intravacc site were applied: 5.1 is WIV BPL inactivated bulk, 5.1S is BPL inactivated Triton split bulk and 3.1 is BPL inactivated influenza before splitting with Triton</p

Overview of the process flows, starting with inoculation of 25.000 eggs and resulting in 6 vaccine products.

<p>In the boxes the unit operations are presented. Fraction identification number is written below the unit operation box. Fraction 1.2 (clarified allantoic fluid) was equally divided over the six process streams. The processes from left to right, with the end product, given in the bottom boxes below the unit operation ‘Sterile Filtration’: 5.1FE standard Cantacuzino Institute process for H3N2 strain, 5.1F Whole Inactivated Virus (WIV) inactivated by formaldehyde, 5.1FT formaldehyde inactivated, Triton split virus product, 5.1BE beta-propiolactone (BPL) inactivated, ether split virus product, 5.1B WIV inactivated by BPL, 5.1BT standard Intravacc process.</p

Parameters of the bulk material quantified/determined, including specification and reference to method used.

<p>Parameters of the bulk material quantified/determined, including specification and reference to method used.</p

SDS PAGE of samples (reduced) taken during removal of Triton of BPL inactivated virus (3.3BT).

<p>Samples were taken approximately every half hour (start at t = 0, last sample at t = 7). Lane 3.3BT t₀ fraction before removal of Triton, lane 3.3BT t₇ fraction after removal of Triton. Lane M presents molecular weight (MW) markers, with right of lane M the MW indicated in kD. M1 matrix protein (~26 kD) band is present in both lanes, as are all other clearly visible bands, indicating that no major protein is lost during removal of Triton.</p

DLS results of fraction before split and after split, before and after sterile filtration.

<p>Left panel presents purified live influenza virus fraction 3.0 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>). Right panel, red solid curve, presents results of fraction 3.3BT, BPL inactivated virus, after splitting and removal of Triton. Clearly two populations are present indicating the splitting of the virus was effective. After sterile filtration of this fraction (5.1BT, red dotted curve), significantly less volume % of large entities is present.</p

Graphs presenting the stability of vaccine bulk products, based on haemagglutinin concentration.

<p>Y-axis: HA μg/mL by SRID at t = 0 is 100%; X-axis: duration in months. The left graph presents the six vaccine bulks over a period of 5 months. Given the HA test variation of 20% and the limited data set, it can be concluded that the ether split formaldehyde inactivated product 5.1FE has least stability. The right graph presents data from influenza vaccine batches prepared at Intravacc (inactivation with BPL and splitting with Triton): stability over a period of twelve months for four WIV products and one Triton split product. The product stabilities are in the same range as in the left panel except for product 5.1FE.</p

Overview of the main characteristics of the BPL inactivated, Triton split bulk produced at Cantacuzino Institute, Romania and the average of clinical batches produced at Intravacc, The Netherlands.

<p>Overview of the main characteristics of the BPL inactivated, Triton split bulk produced at Cantacuzino Institute, Romania and the average of clinical batches produced at Intravacc, The Netherlands.</p