Removal of aggregates

Abstract

To provide a method for dispersing an aggregated material in a preparation containing an influenza protein.SOLUTION: The method comprises subjecting the preparation to sonication. The present disclosure provides a method of dispersing aggregated material in a preparation comprising an influenza protein or virus, the method comprising subjecting the preparation to sonication. The present disclosure also provides a method of producing an influenza vaccine, the method comprising producing a preparation comprising inactivated or split influenza virions and sonicating the preparation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from Australian application no. 2018902497 entitled "Removal of agglomerates" filed on 10 July 2018, the entire contents of which are incorporated herein by reference.

[0002] Field The present disclosure relates to a method for dispersing aggregates in preparations containing influenza antigens, and in particular to the use of this method in the production of influenza vaccines. [Background technology]

[0003] background Influenza vaccines are considered the most effective method for preventing infection. The first influenza vaccines were whole-virus preparations [1]. The current manufacturing process for inactivated trivalent and quadrivalent influenza vaccines (TIV and QIV, respectively) is based on the chemical destruction or "splitting" of influenza viruses, which was introduced in the 1960s [2]. Chemical destruction (with detergents or solvents) has been found to reduce vaccine reactogenicity in many cases without compromising immunogenicity. Due to the high volatility of solvents, all commercially available influenza vaccines are detergent-destroyed or split. However, the concentration of detergent used to destroy whole virions exceeds acceptable limits within the vaccine and must therefore be reduced to an acceptable level.

[0004] The consequence of detergent removal is that the resulting split virions develop aggregates or aggregated material. The occurrence of aggregates is related to the strain and the level / type of surfactant used in the splitting process. Vaccines and other pharmaceutical products often contain either residual surfactant or additional surfactant / chemical materials to maintain the appropriate quality attributes of the vaccine. However, it has long been established that the presence of surfactants, particularly in the context of vaccination, makes antigens more soluble, enhancing immunogenicity and therefore reducing vaccine efficacy. Summary of the Invention [Means for solving the problem]

[0005] Abstract The present disclosure provides a method of dispersing aggregated material in a preparation comprising an influenza protein or virus, said method comprising subjecting said preparation to sonication.

[0006] The present disclosure also provides a method of producing an influenza vaccine, the method comprising producing a preparation comprising inactivated or split influenza virions and sonicating the preparation. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method of dispersing aggregated material in a preparation comprising an influenza protein, said method comprising subjecting said preparation to sonication. (Item 2) 2. The method of claim 1, wherein the preparation comprises influenza hemagglutinin. (Item 3) 3. The method of claim 1 or 2, wherein the preparation comprises split influenza virions. (Item 4) 4. The method of claim 3, wherein the preparation is substantially free of surfactants. (Item 5) 5. The method according to any one of items 1 to 4, wherein the sonication is carried out for a time and at an intensity such that at least 50% of the aggregates present in the preparation are dispersed. (Item 6) 6. The method according to any one of items 1 to 5, wherein the ultrasonic treatment is carried out at a speed of 80% amplitude. (Item 7) The sonication is carried out to deliver an energy of at least 90 Joules / mL. 7. The method according to any one of items 1 to 6, (Item 8) 8. The method of any one of items 1 to 7, wherein an H3N2 strain having a putative HA glycosylation site probability score (pGly score) of ≥ 16 requires sonication at ≥ 90 Joules / mL, and an H1N1 strain having a pGly score of ≥ 11 requires sonication at ≥ 90 Joules / mL. (Item 9) 1. A method of producing an influenza vaccine, the method comprising the steps of producing a preparation comprising inactivated or split influenza virions and sonicating the preparation. (Item 10) 9. The method of claim 8, wherein the vaccine comprises at least three different influenza strains. (Item 11) 9. The method of claim 8, wherein the vaccine is a monovalent vaccine. (Item 12) 9. The method of claim 8, wherein the vaccine is a tetravalent vaccine. (Item 13) 12. The method of any one of items 8, 9 and 11, wherein the vaccine comprises influenza A and influenza B. (Item 14) 13. The method of any one of items 8 to 12, wherein the vaccine is substantially free of aggregated material. (Item 15) 13. The method according to any one of items 8 to 12, wherein the ultrasonic treatment is carried out at a speed of 80% amplitude. [Brief explanation of the drawings]

[0007] [Figure 1] Repeatability of the optical density turbidity (ODT) assay for influenza virus vaccine (IVV) IVV Drug Matrix of strains A / Victoria / 361 / 2011 (H3N2), A / California / 07 / 2009 (H1N1), and B / Hubei-Wujiagang / 158 / 158 / 2009 (B Yamagata).

[0008] [Figure 2] Effect of sonication in terms of amount of delivered energy (Joules / mL) and heat (37°C, 30 min) on the dispersion of H3N2 A / Victoria / 361 / 2011 IVV Drug Matrix by ODT analysis.

[0009] [Figure 3] Effect of ultrasonic treatment in terms of input energy (amplitude) intensity on the dispersion of H3N2 A / Victoria / 361 / 2011 IVV Drug Matrix by ODT analysis.

[0010] [Figure 4] ODT results showing that ultrasonic treatment effectively disperses aggregates and maintains the dispersion state of H3N2 A / Victoria / 210 / 2009 IVV Drug Matrix over time compared to untreated and PS80-treated samples.

[0011] [Figure 5] Mean intensity particle size distribution (PSD) of (A) untreated, (B) PS80-treated, and (C) sonicated H3N2 IVV Drug Matrix of A / Victoria / 210 / 2009 analyzed by DLS over a 24-week period (n=5).

[0012] [Figure 6]Single radial immunodiffusion (SRID) analysis of untreated, surfactant (PS80)-treated, and sonicated IVV Drug Matrix (H3N2; A / Victoria / 210 / 2009) over 24 weeks.

[0013] [Figure 7] EM micrographs showing IVV Drug Matrix (MPH), IVV Drug Matrix after sonication (sonicated MPH), and IVV Drug Matrix in the presence of polysorbate 80 (MPH+PS80). All samples were analyzed by EM at 0, 1, 2, and 6 months.

[0014] [Figure 8] ODT results for four seasonal strains of influenza (A / Victoria / 361 / 2011 (H3N2), A / California / 7 / 2009 (H1N1), B / Hubei-Wujiagang / 158 / 2009 (B Yamagata), and B / Brisbane / 60 / 2008 (B Victoria)) before and after sonication (Son) at 0, 3, and 6 months.

[0015] [Figure 9] Mean particle size distribution (PSD) (n=5) of untreated (left) and sonicated (right) IVV Drug Matrix material for A / Victoria / 361 / 2011 (A, B), A / California / 7 / 2009 (C, D), B / Hubei-Wujiagang / 158 / 2009 (E, F), and B / Brisbane / 60 / 2008 (G, H) analyzed by DLS over a 6-month period.

[0016] [Figure 10] Sonication exposure time (min) across IVV Drug Matrix batch volumes (MPH volume mL) corresponding to ODT≧80% for various IVV Drug Matrix batch volumes of 60 ml, 500 ml and 1000 ml.

[0017] [Figure 11] Sonication input energy over processing time corresponding to ODT≧80% for various IVV Drug Matrix batch volumes of 60 ml, 500 ml and 1000 ml.

[0018] [Figure 12] Sonication input energy required to achieve ODT ≥ 80% using a flow-through device for batch volumes of IVV Drug Matrix (60 ml, 500 ml and 1000 ml).

[0019] [Figure 13] Linear relationship between the amount of aggregation in the IVV Drug Matrix (shown as % ODT) and the estimated number of glycosylation sites present on the HA molecule. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description This disclosure describes the use of sonication methods to effectively disperse aggregated material in influenza virus vaccine (IVV) drug substances or IVV drug products, hereafter referred to as IVV Drug Matrix. The feasibility of sonication was evaluated for the H3N2 strain of influenza virus because this influenza A substrain exhibits the highest levels of aggregation compared to non-H3N2 strains and influenza B.

[0021] One example of the present disclosure provides a previously unrecognized approach, as inhibition of protein aggregation is generally achieved by the addition of compatible excipients to the formulation. For example, excipients (e.g., sugars, polyols, amino acids, salts, polymers, and surfactants) can induce preferential interactions [Arakawa et al. (1991); Timasheff (1998)], increase the rate of protein folding [Wang et al. (1995); Frye and Royer (1997)], reduce solvent accessibility and conformational mobility [Kendrick et al. (1997)], and increase solvent viscosity [Jacob and Schmid (1999)]. (1999)] has been found to stabilize aggregates.

[0022] The present disclosure avoids the need for these additives, which can adversely affect the immunogenicity of the vaccine and can cause undesirable side effects in individuals receiving the vaccine.

[0023] In one example, the disclosure provides a method of dispersing aggregated material in a preparation comprising an influenza protein, the method comprising subjecting the preparation to sonication.

[0024] In another example, the disclosure provides a method of producing an influenza vaccine, the method comprising producing a preparation comprising inactivated or split influenza virions and sonicating the preparation.

[0025] Preparations for sonication can be whole virions, split virions, subunit vaccines, or recombinant vaccines. To facilitate filtration for sterility of the preparation, it is preferred that the percentage of aggregates be less than 10% in the final preparation.

[0026] In one example, the preparation contains influenza hemagglutinin, e.g., the preparation contains split influenza virions. In one example, the preparation is substantially free of surfactants. As used herein, the phrase "substantially free of surfactants" means having a level of less than 0.02%. For example, the surfactant level is less than 200 ppm. In a further example, the surfactant level is less than 50 ppm.

[0027] Typically, the ultrasonic treatment is carried out for a certain time and at a certain intensity so that at least 50% of the aggregates present in the preparation are dispersed. The ultrasonic treatment energy generated to disperse the aggregates can be delivered to the target IVV Drug Matrix by one of three methods: (1) the energy is directly transmitted through a sonicator probe suspended in the IVV Drug Matrix; (2) the IVV Drug Matrix passes through the vibrating tip of the sonicator probe tip enclosed in a flow-through device; or (3) the energy is indirectly transmitted through a metal or glass tube to the location where the IVV Drug Matrix passes. All ultrasonic treatment methods require the transmission of at least 89 joules / mL of energy to disperse at least 50% of the aggregates in the IVV Drug Matrix.

[0028] The vaccines produced by the methods of the present disclosure can be monovalent seasonal vaccines or monovalent pandemic vaccines. In another example, the vaccines of the present disclosure are multivalent vaccines, such as trivalent and tetravalent vaccines.

[0029] Vaccines produced by the methods of the present disclosure typically contain influenza A and influenza B antigens and are, for example, substantially free of aggregated material. In one example, the phrase "substantially free of aggregated material" means that more than 50% of the material (about 90 joules / mL sonication) is not agglomerated. In a further example, at least 60% (about 134 joules / mL sonication), 70% (about 178 joules / mL sonication), or 80% (about 223 joules / mL sonication) of the material is not agglomerated. In another example, at least 90% (about 267 joules / mL sonication) of the material is not agglomerated.

[0030] As described below, the disclosed method offers many unexpected benefits. First, the method is highly efficient at dispersing aggregates present in influenza antigen preparations. Surprisingly, these dispersed aggregates do not re-agglomerate despite prolonged storage (4°C). Furthermore, vaccines produced using the disclosed method have been shown to elicit stronger immune responses in a ferret model than vaccines containing the same antigens but that were untreated or treated with additives to disperse aggregates.

[0031] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations (e.g., "comprises" or "comprising") will be understood to imply the inclusion of a stated element or integer or group of elements or integers, and not the exclusion of any other element or integer or group of elements or integers.

[0032] Reference herein to any prior publication (or information derived therefrom) or to any publicly known matter is not, and should not be construed as, an acknowledgement or admission or any form of suggestion that the prior publication (or information derived therefrom) or publicly known matter forms part of the common general knowledge in the field to which this specification pertains.

[0033] All publications mentioned herein are incorporated by reference in their entirety.

[0034] It should be noted that, as used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent as well as two or more agents; reference to "a molecule" includes a single molecule as well as two or more molecules; etc. [Example]

[0035] method Sonication of IVV Drug Matrix Direct probe sonication method The IVV Drug Matrix was treated using the "direct" sonication method by immersing the sonicator horn / probe directly into the sample contained within the beaker.

[0036] Sonication was performed using a Branson Model 450 Sonifier® (Branson Ultrasonics), which consisted of four components including a power supply, a Model 102C converter, and a 0.5-inch tapped horn.

[0037] To evaluate the efficiency of the sonication process, a Bandelin SONOPULS Sonicator (Bandelin Electronic GmbH & Co. A Branson Sonicator (KG) was used. The setup consisted of a GM3200 Sonifier power supply, a UW3200 converter, an SH213G booster, and a TT13 13 mm titanium tip. Sonication of the IVV drug substance material was performed in a beaker according to the method described for the Branson Sonicator.

[0038] IVV Drug Matrix samples were prepared in 10 ml batches and placed into clean 30 ml beakers, which were secured on a clamp stand with the sonicator probe fully immersed in the sample solution (i.e., the distance between the sonicator tip and the base of the beaker was approximately 1 mm). Sonication was then carried out delivering a range of input energies while varying the rate (amplitude) of energy transfer:

[0039] The range of sonication energies delivered was measured as joules (energy) per milliliter of IVV Drug Matrix and included 0 joules / mL, 83 joules / mL, 165 joules / mL, 259 joules / mL, and 345 joules / mL.

[0040] To avoid overheating the sample, sonication was performed at least twice, with the beaker briefly placed on ice to cool between treatments. The final sonicated IVV drug substance sample was transferred to a plastic tube and stored at 2-8°C before further analysis.

[0041] Flow-through sonication method To evaluate the scalability of the ultrasonic treatment process, a Bandelin SONOPULS Sonicator (Bandelin Electronic GmbH & Co. KG) equipped with a flow-through device was used. Its configuration is GM3200. It consisted of a Sonifier power supply, UW3200 converter, SH213G booster, TT13 13mm titanium tip and DG 4 G flow-through processing vessel. Sonication of IVV drug substance material was circulated through the sonicator's flow-through device by a 520U peristaltic pump (Watson & Marlow, Australia).

[0042] Measurement of agglomerated materials Optical density turbidity (ODT) assay To assess the degree of non-aggregated material in the vaccine intermediate IVV drug substance, the level of recovered protein was determined using the optical density (OD) at A280 nm of the supernatant after application of mild centrifugal force (Tay et al.: Investigation into Alternate testing methodologies for characterization of influenza vaccine. Human Vaccine Immunotherapy 2015 11 (7) 1673-84). The percentage of protein in the pellet after centrifugation directly correlates with the degree of aggregated material in the sample, so a higher recovery in the supernatant corresponds to a greater proportion of dispersed protein. This assay was termed the optical density turbidity (ODT) method; protein recovery values ​​range from 0 to 100% (denoted here as % ODT) and increase with the amount of dispersed protein in the sample.

[0043] Several attributes of the assay were evaluated for three influenza strains (H1N1; A / California / 07 / 2009, H3N2; A / Victoria / 361 / 2011, and B; B / Hubei Wujiagang / 158 / 158 / 2009). Validation results showed repeatability (%CV) of 2.9%, 3.1%, and 3.1%, respectively; lot-to-lot variability (%CV) of precision of 8.8%, 6.5%, and 3.4%, respectively; intra-laboratory precision of 0.4%, 6.3%, and 0.2%, respectively; statistically insignificant differences between operators (p=0.81, 0.13, and 0.78, respectively); and predictable linearity between expected and observed results (R=0.9685). Representative %ODT profiles for replicate lots of IVV drug substance representing seasonal vaccine subtypes: H1N1, H3N2 and influenza B are detailed in FIG.

[0044] Furthermore, this assay correlated with alternative methods of aggregation characterization, including dynamic light scattering (DLS) and asymmetric field-flow fractionation (A4F). The low level of variation from the ODT analysis indicated that this assay is well suited for assessing aggregation in intermediate vaccine materials.

[0045] Dynamic Light Scattering (DLS) DLS particle size analysis was used as a complementary method to the ODT assay to further understand the aggregation characteristics within the sample. The DLS technique is based on the measurement of Brownian motion of proteins in solution, which is the random movement of particles due to collisions with surrounding solvent molecules. Brownian motion induces time-dependent fluctuations in the intensity of scattered light, which is measured by DLS, resulting in the particle size distribution (PSD) of the sample.

[0046] DLS measurements were performed using a Malvern Zetasizer Nano Series ZS (Malvern Instruments Ltd). Several properties affecting the Brownian motion of particles in solution were previously determined for each analyzed sample, including density (DA-100M Density Meter, Mettler Toledo), viscosity (Lovis 2000M Microviscometer, Anton Paar), and refractive index (30GS Refractometer, Mettler Toledo). Sample pretreatment included centrifugation at 8000 rpm for 1 minute to remove any extraneous material or precipitate, including large particles that may be unstable and interfere with subsequent analysis. The resulting supernatant components of each sample were then withdrawn and evaluated by DLS; each sample measurement was based on five replicates (n = 5) performed at a backscattering angle of 173° and equilibrated at 25°C for 3 minutes.

[0047] Influenza antigenicity assessment Single radial immunodiffusion (SRID) The SRID assay was performed as previously described [Williams et al., 1980]. Briefly, reference and test antigen materials were diluted 1:1, 2:3, and 1:3 in PBS containing 1% Zwittergent solution (Calbiochem, Darmstadt, Germany) and added to duplicate wells of an agarose gel containing polyclonal antisera. The gels were incubated in a humidified chamber for 72 hours, dried on a glass plate, and stained with Coomassie Brilliant Blue R-250 (Sigma). The circular zones of antigen-antibody precipitates were measured, and HA concentrations were calculated by parallel line bioassay in comparison with IZP standards (15). The validity of the test was confirmed using the "g" test (g ≤ 0.061) (16). Imaging by electron microscopy (EM)

[0048] Negative-stain electron microscopy (EM) was performed using the agar diffusion filtration method adapted from Hayat and Miller (1990). Three grids were prepared for each sample; IVV drug substances were diluted 1 / 50 with phosphate-buffered saline (PBS; pH 7.2) to provide a discontinuous monolayer. Samples (1 μL) were applied to formvar-coated copper electron microscope grids, which were then placed face-down on a 2% w / v agar plate. When the grid was placed on the agar plate (i.e., the liquid was absorbed by the agar), it floated on a drop of negative stain (2% w / v sodium phosphotungstate, pH 7.0). After 20 seconds, the grid was lifted, and excess stain was removed by contacting the edge of the grid with a small piece of torn Whatman No. 1 filter paper. The remaining thin film of stain was allowed to air dry before electron microscopy.

[0049] result Disruption of aggregated IVV drug matrix by ultrasonic treatment The most effective method for dispersing agglomerated material was determined using the H3N2 substrain of influenza, as it tends to form the highest levels of agglomerated material after detergent disruption. Several methods were evaluated to demonstrate whether agglomerated material after detergent disruption could be dispersed.

[0050] The first approach involved dispersing aggregates within A / Victoria / 361 / 2011, substrain H3N2, IVV drug substance material using high-frequency sound waves via sonication (Figure 2). A direct sonication method was used, in which localized, high-intensity ultrasonic energy could be delivered directly from a probe to a beaker containing the sample. Samples (prepared in 10 ml batches) were subjected to a range of sonication input energies, ranging from 83 Joules / mL to 345 Joules / mL, and the degree of dispersion was assessed by ODT assay. The results show a linear relationship between the amount of input energy and the level of disaggregated aggregates. That is, %ODT values ​​ranged from 50% to 80% after sonication, compared to 40% without treatment (Figure 2). Interestingly, the level of dispersion reached a maximum at 259 Joules / mL, from which no further increase in dispersion was observed. In contrast, mild heating and agitation of the IVV drug substance (30 minutes at 37° C. with agitation at 600 rpm) did not change the level of dispersed material.

[0051] The intensity at which energy was delivered to the IVV drug substance was explored by adjusting the sonication amplitude (0-100%) while keeping the exposure time constant at 0.70 s / ml for all samples. The ODT results showed a predictable trend, as previously observed, regarding the amount of energy delivered: the degree of dispersion in the IVV drug substance increased with sonication amplitude when the exposure time was held constant (Figure 3). The optimal rate of sonication energy delivery was achieved at 80% amplitude; beyond that, no further increase in the level of aggregate disaggregation was observed. These results suggest that 80% is the optimal amplitude (i.e., energy delivery rate) for disaggregating the aggregates.

[0052] Feasibility of Ultrasonication to Disperse IVV Drug Matrix Materials To be acceptable, inactivated vaccines require certain quality attributes: if a method is shown to disperse aggregated material, it is important that the material maintain this characteristic.

[0053] A 24-week study was conducted to evaluate the feasibility of sonication as a method to disperse aggregated material in the IVV Drug Matrix of H3N2 A / Victoria / 210 / 2009, both in the presence and absence of the surfactant polysorbate 80 (PS80). Multiple tests were used to monitor various characteristics of the samples, including ODT and DLS for aggregation assessment, single radial immunodiffusion (SRID) for antigenicity, and electron microscopy (EM) for morphological imaging.

[0054] Aggregation behavior by ODT and DLS The aggregation characteristics of untreated, sonicated, and PS80-treated IVV Drug Matrix were evaluated by ODT assay and DLS. ODT analysis showed that after sonication (at least 200 joules / mL), the level of dispersed material in the IVV Drug Matrix reached 80%, compared to 40% (at time 0) for the unsonicated material (Figure 4). This dispersion level remained constant over 24 weeks at 4°C, indicating an irreversible dispersion state (Figure 4). Furthermore, dispersion or further aggregation of the control (untreated) material did not increase over this time. Surfactant (0.1% The addition of surfactant (PS80) had no significant effect on the level of aggregates in this material compared to the initial level, thus indicating that the level of aggregation in the IVV Drug Matrix material is set after disruption with surfactant.

[0055] Along with the ODT assay, samples were analyzed by DLS to further characterize aggregation. For each sample, DLS measurements (n = 5) generated a particle size distribution (PSD) intensity, which indicates the relative intensity of light scattered by particles of various size populations. DLS results revealed a close correlation with ODT results for all three samples (Figure 5). For example, untreated and PS80-treated IVV Drug Matrix samples exhibited multimodal PSDs with peaks at 60 nm, 400 nm, and 7000 nm, thus indicating internal aggregates (Figures 5A and 5B). However, sonication produced a unimodal distribution with a single, clear peak at 300 nm, suggesting a uniform and well-dispersed sample lacking aggregates (Figure 5C). At each analysis time point, all samples produced reproducible PSDs, which remained unchanged over a 24-week period.

[0056] Antigenicity by SRID and EIA The levels of antigenic material were assessed by SRID to determine whether sonication or the addition of detergent affected influenza antigens. SRID analysis showed that the levels of antigenic material remained the same potency as untreated samples and remained constant over time, regardless of whether the material was sonicated or treated in the presence of detergent (Table 1, Figure 6). [Table 1]

[0057] Morphological imaging by EM EM imaging was used to examine the morphological appearance of the samples at 0, 1, 2, and 6 months (Figure 7). Significant differences were observed between the sonicated and control IVV Drug Matrix samples. The control / untreated IVV Drug Matrix (with and without PS80) contained significant amounts of aggregates throughout the entire 6-month time course, as represented by darker areas in the micrographs. In contrast, the sonicated IVV Drug Matrix contained fewer aggregates that were reduced in size, and the appearance of the material remained consistent over the tested period. These observations mirrored the results from both ODT and DLS analyses, in which the sonicated material was significantly more dispersed than the untreated or surfactant (PS80)-incorporated IVV Drug Matrix.

[0058] Applicability of sonication to all seasonal strains of influenza Although H3N2 exhibits the highest levels of aggregation compared to other seasonal strains (Figure 1), it is essential to demonstrate that this method can disrupt aggregates for all strains. Four seasonal strains were tested for dispersed aggregate levels before and after sonication (at least 200 joules / mL) over a 6-month period (Figure 8). In all virus preparations tested, application of sonication increased the level of dispersed material, which remained dispersed over the 6-month period. The increase in aggregate dispersion upon sonication appeared more pronounced for two influenza A substrains, H3N2 and H1N1, compared with two influenza B viruses derived from the Yamagata and Victoria lineages. For example, the level of dispersed material increased approximately 60-100% for the two A strains, compared with a 3-10% increase for the B strains.

[0059] The ODT results were further confirmed by data obtained from DLS analysis of all four influenza strains. The intensity PSDs obtained from five replicate measurements for each sample at 0, 3, and 6 months are shown in Figure 8. The untreated sample of A / Victoria / 361 / 2011 exhibited a multimodal profile, indicating the presence of aggregates of various sizes (Figure 9A). In contrast, the PSDs of A / California / 7 / 2009, B / Hubei-Wujiagang / 158 / 2009, and B / Brisbane / 60 / 2008 had a more unimodal structure, thus suggesting a more uniform particle population (Figures 9C, E, and G, respectively). After sonication, all four strains exhibited a distribution characteristic of a well-dispersed IVV Drug Matrix, free of any aggregates (Figures 9B, D, F, and H).

[0060] Stability and batch consistency of sonicated IVV Drug Matrix IVV Drug Matrix representatives of four vaccine candidate types / subtypes were sonicated to determine both the consistency and stability of applying controlled energy levels (Joules / mL) to achieve target aggregate dispersion levels (Table 2, Figure 8). The IVV drug material for each representative strain was divided into six subaliquots. Three of the six aliquots were independently exposed to at least 200 Joules / mL of sonication and stored at 2-8°C for up to six months, along with their non-sonicated control groups. Samples were taken from all groups at 0, 1, 3, and 6 months and analyzed by ODT for the level of aggregates present. For all sublots within the influenza A strain subtypes A / California / 07 / 2009 and A / Victoria / 361 / 2011, significant changes in the level of dispersed aggregates were observed in the sonicated samples compared to the non-sonicated controls. Excellent consistency existed between independently sonicated sublots for both representative influenza A strains. At time 0, all three sublots of the H1N1 and H3N2 strains met the target %ODT (>80%) after sonication (%CVs of 2.1% and 1.1%, respectively, well within the 10% limit). Less variation in the level of aggregate dispersion was observed in the lot representing the B strain due to the low content of aggregates present in the control group. After sonication, the B strain sublots exhibited the same level of batch-to-batch consistency as the lot representing the A strain (%CVs for B / Hubei Wujiagang / 158 / 2009 and B / Brisbane / 60 / 2008 at time 0 were 1.2% and 0.7%, respectively). Importantly, the sublots representing all four seasonal influenza strains maintained their elevated %ODT levels and batch-to-batch consistency over the entire 6-month time course, suggesting that aggregate disruption by sonication is permanent. [Table 2]

[0061] Linear relationship between the number of putative glycosylation sites on HA and the amount of sonication required The envelope glycoprotein hemagglutinin (HA) is a sialic acid receptor-binding protein of influenza viruses that allows them to bind to host cells and escape digestion once engulfed in endosomes. The globular head region of the HA molecule contains N-linked glycosylation sites that overlap with antigenic sites and are thought to be involved in masking these antigenic sites from binding by antibodies and major histocompatibility complexes (Skehel et al., 1984; Jackson et al., 1994). Furthermore, the structural complexity of N-glycans positively correlates with HA-receptor binding specificity (Tsuchia et al., 2002). The number of N-linked glycosylation sites in the globular head region of HA has increased during the evolution of H1N1 and H3N2 human influenza A viruses (Suzuki, 2011). We determined the relationship between the number of putative glycosylation sites on influenza A HA molecules and the level of aggregation present. The number of glycosylation sites was estimated by calculating a probability score using an algorithm available on the NetNGlyc 1.0 Server (http: / / www.cbs.dtu.dk / services / NetNGlyc / ). To generate a probability score, the HA protein sequence of the strain in question was entered into an algorithm submission panel and submitted for analysis. The software generates a table of putative glycosylation sites within the input sequence, scored with 1 to 3 plus (+) signs depending on the strength of the probability. The putative HA glycosylation site probability score (pGly score) is defined as the sum of the plus signs for a given output sequence. We propose that H3N2 strains with a pGly score of ≥ 16 require sonication at ≥ 90 Joules / mL, and H1N1 strains with a pGly score of ≥ 11 require sonication at ≥ 90 Joules / mL (where more than 50% of the material is not agglomerated). [Table 3] Alternative physical disruption methods for dispersing IVV drug substances To evaluate the unique ability of sonication to disperse the IVV Drug Matrix, other physical disruption methods were tested, including localized heating (microwave for 1 and 10 seconds) and shear force (Dounce homogenization using 25 and 100 strokes). In all cases, there was no significant difference in dispersion compared to the untreated material (Table 4). [Table 4]

[0062] conclusion The use of direct sonication was found to effectively disperse aggregates within the IVV Drug Matrix. Influenza strains within the subtype H3N2 exhibit the highest levels of aggregation. Process optimization demonstrated that the amount of energy delivered to the IVV Drug Matrix and the rate at which the delivery occurred were essential in controlling the level of aggregate disaggregation. Increasing the sonication speed (amplitude) and / or exposure time (seconds) resulted in increasing levels of aggregate disaggregation, correlated with a linear trend. A plateau in dispersion levels was observed after the ODT reached 97%, after which the aggregates were little or no longer dispersed (as measured by ODT). The use of sonication as a method to disperse aggregates in IVV drug substance was evaluated over a 24-week (6-month) time course, both in the presence and absence of a surfactant (PS80). Several characterization analyses, including ODT, DLS, and EM, revealed that the sonicated IVV Drug Matrix contained significantly increased amounts of dispersed material compared to untreated and surfactant-treated samples. The amount of sonication required to achieve the target level of aggregate dispersion was predictable and consistent between batches. Furthermore, the level of dispersion remained consistent throughout the duration of the feasibility study, indicating stable and permanent aggregate dispersion. Immunological evaluation by SRID confirmed that neither sonication nor surfactant treatment impaired the antigenicity of the IVV Drug Matrix.

[0063] This study strongly demonstrated the value of sonication as a simple, practical, and effective approach to improving the quality attributes of influenza vaccines containing highly aggregated IVV drug matrices. Furthermore, this method was shown to be applicable to all seasonal influenza strains. After sonication, increased levels of dispersed material were observed in the IVV drug matrices of H3N2, H1N1, and two influenza B strains, the Yamagata and Victoria lineages. This was well maintained over a 6-month period.

[0064] As an alternative to the direct sonication method designed for laboratory-scale studies, a continuous-flow sonication configuration was investigated for processing commercial volumes of IVV Drug Matrix. Briefly, the sonicator unit is powered by a high-frequency generator coupled to a 20 kHz converter; the connected booster horn is placed into a flow-through processing vessel containing a constantly recirculating sample at a specified flow rate.

[0065] To assess the scalability of the system, the effects of various processing parameters, including sonication intensity (amplitude) and product recirculation flow rate, were investigated to determine their impact on the efficiency of aggregate dispersion within the IVV drug substance. Using a constant recirculation flow rate through the sonicator of 120 ml / min and a fixed amplitude of 80%, a strong linear correlation was demonstrated between batch volume (60 ml, 500 ml, and 1000 ml) and the sonication time (over 60 minutes) required to reach an ODT threshold of 80% (R 2 =0.989). This trend also persisted between sonication energy (Joules) and time (R 2 ≥ 0.998), therefore, there was also a significant correlation between sonication energy and batch volume of IVV Drug Matrix (R 2= 0.981, Table 5 and Figures 10-12) was observed. This data suggests that the outlined sonication process is scalable with respect to IVV drug substance batch sizes. Furthermore, the above data suggest that a fixed input energy of at least 300 Joules / mL is sufficient to disassociate aggregates to ODT levels of ≥ 80%, regardless of volume in this system.

[0066] [Table 5]

[0067] We determined that there is a linear relationship between the number of predicted glycosylation sites on an influenza A HA molecule and the degree of aggregation found in the IVV Drug Matrix for that strain (Figure 13). Correlation coefficient values ​​(r values) were calculated for the relationship between predicted glycosylation sites and the degree of aggregation in the IVV Drug Matrix for 12 H3N2 strains produced between 2005 and 2017. The r value was 0.74, suggesting a moderately strong correlation between these two attributes. We propose that H3N2 strains with a pGly score of ≥16 require sonication at ≥90 joules / mL, and H1N1 strains with a pGly score of ≥11 require sonication at ≥90 joules / mL. References [Table 6-1] [Table 6-2]

Claims

[Claim 1] The invention described in this specification.

Images