Summary of the context and overall objectives of the project

In the vaccine industry, downstream processing is of extreme importance. Prophylactic vaccines aim at protecting healthy people, so any contaminant has to be removed with the most drastic measures. Such « negative » approach comes at the expense of the recovery of product: yields are poor, thereby increasing product cost. Processes are also complex, since they rely on multiple eliminations rather than on recovery of the unique product of interest. Technically, this is mostly due to the lack of specific capture systems that would allow direct, « positive » separation of the vaccine from its environment. In addition, for developing countries, the pressure on costs is acute, and local production is a way to try to reach the 1$ per dose target. In this context, sufficient water availability is a major issue, as it is a sensitive ingredient in bioproduction.
DiViNe will tackle these cost and environmental issues with technological answers. The partners will combine two major Nano/biotechnology innovations to develop an integrated purification platform amenable to the different types of vaccines: glycoconjugates, protein antigens and enveloped viruses. We will implement Nanofitins (novel affinity capture ligands) and Aquaporin-based membranes (energy-saving nano-biomimetics used in the cleantech industry), for a « positive » purification approach. High yields are expected (data from antibody purification with Nanofitins), at affordable “costs of goods” and with a sustainable approach for water recycling.
GSK Vaccines brings to the Consortium a broad range of targets, and identical strategies can be applied for biopharmaceuticals in general. The custom affinity capture processes being developed as a sustainable platform is therefore economically relevant, in a very large market. Beyond the technical partnership, the project is a first run for the partners to structure the platform as a commercial offer for downstream processing of biologics.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The DiViNe project is targeting three different families of vaccines: Family 1 (TF1): Carrier proteins used for glycoconjugates vaccines, extracted from live bacteria and for which only traditional purification technologies are applied with a low yield, Family 2 (TF2): Protein antigens that are particularly hard to separate from by products such as truncated forms, thereby requesting highly specific capture and Family 3 (TF3): Fragile enveloped viruses that are not amenable to usual purification systems. Since these target families will be covered chronologically in the course of the DiViNe project, the first 18-month focused on TF1, even though some work has already started regarding TF2 and TF3.
One arm of the DiViNe project, as clearly shown in 2.1.1. (GA Part B) is to obtain Nanofitins against the targets and evaluate them in combination with a chromatographic support, as covered by WP1 and WP3. TF1 was provided by GSK as well as bought from commercial source. Affilogic used Ribosome display technology to select anti-TF1 Nanofitins and clones were assessed for different parameters: affinity and specificity for TF1 in ELISA and Bio-layer interferometry, stability in sodium hydroxide, manufacturing yields and in silico immunogenicity score. The three most interesting Nanofitins were chosen and a unique C-terminal cystein was added to enable regio-selective conjugation to the chromatographic resin by Merck. Affilogic started screening of Nanofitins against TF2, also provided by GSK, based upon the same selection process.
Epoxy activation was chosen out of four different activation chemistries evaluated by Merck. Binding assays were developed by Merck with immobilized model Nanofitin and its target and then adapted to anti-TF1 Nanofitins. Two promising anti-TF1 Nanofitin candidates with slightly different profiles were identified and will be further assessed in the next 18-month Period to identify one Nanofitin candidate to perform the pilot-scale DSP.
The complementary arm of the DiViNe project performed by Aquaporin in WP2 is to adapt a Forward Osmosis (FO) process for reducing volume of waste water from vaccine production fermentation processes. Broths were provided by GSK for TF1 and TF2 and by iBET for TF3. Both flat sheet membranes and hollow fibre modules have been assessed. Short (using TF1, TF2 and TF3) as well as long term experiments (using TF2) have been done using a semi-automatic laboratory scale FO plant.
To anticipate the QC-controlled full-scale runs of purification to be performed in WP4, iBET started to implement QC methods for benchmarking of such novel technology. GSK provided technical information regarding QC methods used for the characterization of TF1 to identify equivalent methods currently implemented at iBET.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

One objective of the DiViNe project is to obtain Nanofitins against the targets and evaluate them in combination with a chromatographic support. This part of the work involved, as regards TF1, first GSK for provision of the target then Affilogic for discovery and early characterization of Nanofitins binding to TF1 and lastly Merck for immobilization of the best Nanofitin candidates to chromatographic resins. This platform-approach, from target to affinity material, implemented during the first 18-month period for TF1, illustrates the custom platform that will be available by the end of the DiViNe project for commercial service in the biopharmaceutical field at large.
Such process should be simple enough to be readily adopted by any existing manufacturing facility, and compatible with the ultimate 1$ per dose vaccines. Yet, as no concession will be made to quality, this will be achieved by augmenting the yield while keeping the highest standard of purity. Thus the progress of DiViNe will constantly be confronted to the needs of emerging countries in terms of enhancing access to vaccines. To this end, Vito di Cioccio, from GSK Vaccines Institute for Global Health, Odile Leroy, from European Vaccine Initiative and Katey Owen, from The Bill and Melinda Gates Foundation constitute the External Experts Advisory Group, which already met to assess concept in April 2015.