Fig 1.
Overview of the process flows, starting with inoculation of 25.000 eggs and resulting in 6 vaccine products.
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.
Table 1.
Overview of the vaccine bulk products produced including the differences in the applied unit operations.
Table 2.
Parameters of the bulk material quantified/determined, including specification and reference to method used.
Table 3.
The main characteristics of the starting material before (fraction 1.2) and after ZUC, i.e, fraction 2.1F and 2.1B.
Haemagglutin (HA) and total protein concentration are similar; ovalbumin concentration after ZUC in citrate buffer is lower than after ZUC in phosphate buffer.
Table 4.
The main characteristics of the products from the six different downstream processes.
If applicable the requirements are listed. All products comply with the requirements, except product 5.1FE that has low antigenic HA content
Fig 2.
SDS PAGE of reduced and reduced plus de-glycosylated samples as a fingerprint of principle proteins present.
Lanes M were loaded with marker proteins, with the corresponding molecular weight presented to the left. The fraction sample identity (Fig 1) 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 [22]. 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
Table 5.
The recoveries after each unit operation for the six downstream processes, based on total protein (tot.protein) and HA quantities, relative to the fraction after zonal ultracentrifugation (Fig 1, fraction 2.1F and 2.1B).
Noteworthy is the difference in HA recovery versus total protein recovery after sterile filtration of the ether split formaldehyde inactivated product FE: 32% versus 72% in product 5.1 after SF, which cannot be attributed to the test variation of 7.5% for total protein and 20% for SRID test.
Fig 3.
SDS PAGE of samples (reduced) taken during removal of Triton of BPL inactivated virus (3.3BT).
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.
Fig 4.
DLS results of fraction before split and after split, before and after sterile filtration.
Left panel presents purified live influenza virus fraction 3.0 (Fig 1). 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.
Table 6.
DLS results of products before and after split and before and after sterile filtration (SF).
The radius of the particles was measured before and after splitting (if applicable) and before and after SF. The results of the BPL inactivated fractions are given in the columns to the right, while the results of the formaldehyde inactivated fractions are presented in the columns to the left.
Fig 5.
Representative electron microscope pictures of influenza virus particles, before and after split.
Enlargement pictures to the left 300.000x, pictures to the right 400.000x.Top row whole virus (Fig 1, fraction 3.0), bottom row left panel ether split formaldehyde inactivated virus (Fig 1, fraction 5.1FE), bottom right panel BPL inactivated Triton split virus (Fig 1, 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.
Fig 6.
Graphs presenting the stability of vaccine bulk products, based on haemagglutinin concentration.
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.
Table 7.
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.