Annual report 2022

PPS has in the first year organized itself as one national infrastructure that offers competence and capacity within protein production. Major tasks have been to establish a common PPS website with a project application form where researchers can apply for PPS services, and the set-up of an accompanying project database. Already during the first year, 105 PIs applied for 141 different projects, showing the high demand for recombinant proteins from the Swedish scientific community. During 2022 PPS has completed 105 projects and 224 protein batches were delivered, which was possible because most staff and equipment was available from the start of PPS. Some additional members of staff have been recruited in 2022. To spread the information about PPS among Swedish researchers, we have presented our capabilities at several different conferences and meetings. The director and the management group of PPS worked together to get the joint activities up and running. In addition, a steering committee was established that could approve the strategic plan and other important documents for PPS. Overall, the PPS structure has taken form during the first year and we could immediately and successfully offer project support to many researchers.

The start-up of PPS has followed as outlined. At present no deviations are foreseen, and therefore we do not send an updated table. 2022 has been a year to get started and organized as a joint national infrastructure, both in its organization (see Module 1), the out-reach work (see module 2) as well as in the modules performing the protein production (see modules 3 and 4), with the aim of being fully operational from 2023 clearly within reach. Development of new capabilities within the modules will start or continue in 2023 and be gradually introduced and fully implemented in 2024.

A provisional management group had meetings already during 2021. In 2022, the director (Malin Bäckström, GU), and the steering committee were appointed (with a first meeting in June, in Gothenburg). The deputy director (Wolfgang Knecht, LU) and the node directors (Malin Bäckström, GU for Gothenburg, Tomas Nyman, KI, for Stockholm, Wolfgang Knecht, LU, for Lund and Uwe Sauer, UmU, for Umeå, with Hanna Tegel, KTH as assistant node director for Stockholm to have all universities represented) were then appointed. The agreement between the participating universities was approved. There is also a “Council of parties” for PPS, with representatives from the participating universities, with an advisory role before important decisions concerning PPS are taken by the steering committee. A scientific advisory committee (SAC) remains to be appointed. In the fall, the strategic plan for PPS 2022-2026, a settlement plan, a gender equality plan and the operational plan and budget for 2023 were written by the managements group and approved by the steering committee. The administrative functions have been appointed and both economy and communication/website related issues are now handled by the administration for the Core facilities at the Sahlgrenska Academy at GU. IT-related questions are solved with help from the IT-department at GU. A simple PDF application form was put on the early version of the PPS website and could be used already in the first half of 2022. This was later replaced by a web-based application connected to a database accessible to the management group. This fast implementation of the management of project applications was important as the modules 3 and 4 were ready to give user support, as most of the equipment and staff were already in place in the existing university labs and platforms. In September, all PPS staff met for a kick-off meeting with 25 participants, outside Gothenburg.

In this early phase of PPS, it is of utmost importance to reach out with information of our existence and the services that we can provide. During 2022, we have reached out primarily by:

• A first, temporary webpage (www.gu.se/pps) was set up in the spring of 2022 with information about PPS, instructions on how to apply for project support and contact information. This will be expanded and developed during 2023. A LinkedIn page (presently with 290 followers) and a Twitter account for PPS has also been started.
• Members of the PPS management group have presented PPS in oral presentations at six national and international conferences, the most important being the 25th Swedish Conference on Macromolecular Structure and Function in Tällberg 17-20 June, which was the official “launch” of PPS, and the Swedish PI-meeting organized by SFBBM in Gällöfsta 28-29 November 2022. In addition, a poster or roll-up about PPS have been presented at several meetings.
• Another important route for spreading the knowledge of PPS to the Swedish scientific community is through cooperation with other infrastructures, especially those who need proteins for the services they provide. For structural biology, the Integrated Structural Biology (ISB) platform at SciLifeLab is an important arena. The director of PPS is part of the extended management group as stakeholder for ISB. ISB, together with Infralife, gave the course ”Integrated Structural Biology” where the node directors for the Stockholm and Lund nodes of PPS participated. Proteins from PPS have during 2022 been used at other infrastructures, including the SciLifeLab platforms Autoimmunity Profiling Facility, Spatial Proteomics Facility, Drug Discovery and Development (DDD) and Cryo-EM, and at the Swedish NMR center, MAX IV, and the Chemical Biology Consortium Sweden (CBCS). In Lund, the PPS node shares labs with the FragMAX screening platform of MAX IV and the DEMAX platform of ESS, promoting daily interactions. Two nodes of PPS started a collaboration with the BioMS node in Gothenburg for mass spectrometry analysis as part of the quality control package that will be delivered on the recombinant proteins produced within PPS (see “Additional Comments: Quality control of proteins” below), and for glycoproteomic analyses and some other applications. Members of staff from the Stockholm node ran several projects at the Testa Center during spring 2022. PPS has an open dialogue with Testa Center and there will be good opportunities for future collaborations for scale-up of certain user projects.
• A user survey with 23 questions was sent out to 120 applicants and PIs who had used PPS in 2022. 40 replies (33%) showed that the users overall were very satisfied (>4.5 out of 5) with the services from PPS. Most users said they would use PPS again and the main reason for not using PPS again was that they now had all the protein they needed.

In module 3 (with its six submodules), the main part of the support and services in protein production are performed. This includes all steps from design of the expression plasmid, culturing of cells for the actual protein production in the expression system of choice, the purification and quality control.

Module 3.1 E. coli: Service and expert advice within recombinant protein production with E. coli as expression system within PPS has started with high activity in the first year at two geographical nodes, Stockholm and Umeå. Both participating labs had already ongoing protein production services and were well prepared when they joined the PPS. Many previous users are returning, and new ones are continuously added.

Module 3.2 P. pastoris: The work in the Yeast Protein Production (YPP) module producing proteins in the yeast Pichia pastoris started in March 2022 when a senior scientist was recruited to the infrastructure. During the first year, methods for both small-scale screening and large-scale growth have been established. One user project was performed and delivered, and several projects initiated.

Module 3.3 Insect cells (BEVS): All three modules (module 3.1, 4.1 and 4.2) making up the Lund node share the same laboratories and instruments as well as maintenance and support functions provided by LU. All modules had projects running during 2022, also fitting in relation to proportional staffing of the different modules. No development activities had been planned during 2022.

Module 3.4 Mammalian cells: Protein production in mammalian cells is performed within the Stockholm and the Gothenburg nodes. Both places have long traditions of protein production in mammalian cells and all protocols and equipment needed were available from the start of this infrastructure. A procurement for an additional chromatography system funded through PPS was done and implemented in early 2023. One new member of staff at each of the two nodes in this module have been recruited during 2022.

Module 3.5 Plant cells: This first year was used to plan the best set up of this protein production module within PPS. Some adjustments had to be made as some equipment will be custom made with expected delivery during March 2023. One new member of staff was recruited to run this module, and she will start 1^st of March 2023. With equipment and staff in place, the module will be able to reach full productivity during 2023.

Module 3.6 Cell-free: Cell-free protein synthesis (CFPS) allows the production of diverse protein samples on demand by utilizing the cellular transcriptional and translational activities within a test tube. In 2022, one person was recruited, and the focus has been on updating various aspects of the cell-free system. In addition, efforts have been initiated to attract users to this module.

Services within this module allow preparation of reagents and/or provide expertise for the subsequent performance of experiments at synchrotrons and neutron sources (in Sweden, particularly the MAX IV laboratory and, in the future, ESS). Module 4 features two specializations; Module 4.1 (Per)deuteration and Module 4.2 Molecular chaperones. These modules together with module 3.3 constitutes the Lund node of PPS and have been described together with module 3.3 above.

A common project application portal on the PPS website was established and applied projects end up in a common PPS project database The project database is still continuously improved with more and better functions, including the uploading of project reports and expression results (which will be further discussed and implemented in 2023).

PPS does not generate large amounts of data, but useful data should be collected in a uniform way. There is presently no need for additional e-infrastructure.

The activities within PPS have started as planned. Some new members of staff have been recruited during 2022, but most of the staff came from the already existing platforms. One position (in module 3.5) was vacant during 2022 but has been filled in early 2023.

Early in 2022, members for the steering committee were nominated and they had a first meeting in June 2022 and two more meetings in the fall. The steering committee has six members: Marjolein Thunnissen (MAX IV, LU) as chairman, Pär Nordlund (KI), Per Hammarström (LiU), Magnus Wolf-Watz (UmU), Lovisa Holmberg Schiavone (AstraZeneca) and Sabine Suppmann (Max Planck Institute of Biochemistry, Germany). All are either experts or users of protein production. Important first year-decisions have been to appoint node directors and members of the management group and to get important documents in place (strategic plan, settlement plan, gender equality plan and operational plan for 2023). They have also discussed user fees of the services performed, quality control methods for produced proteins, data collection and roles for the SAC.

The KPIs for PPS are defined as 1) the number of applied projects, 2) the number of completed projects, 3) the number of unique PIs for all applied and 4) completed projects and finally 5) the number of protein batches (samples) that were delivered. Numbers are reported for the individual modules as well as the total number. In 2022, PPS got 141 project applications and 105 of those (75%) were completed. The projects came from 105 unique PIs and 86 of those (82%) had completed projects. 224 protein batches were delivered. The number of users 2022 are slightly lower than the average values for 2016-2020 (that were shown in the application to VR in 2021), probably due to a lag time for users to apply to the new infrastructure. For the number of projects and samples, we cannot compare to earlier years as the definition of project and sample had not been agreed upon before. All modules had project applications except module 3.5 (plant cells) (see comments on this module above). We conclude that PPS had already in the first year a high number of project applications from many users.

The list of publications for 2022 contains 59 research articles published by our users. Practically all of them did not acknowledge PPS, but instead the individual platforms that have now formed PPS, as publication is connected to a time delay. VR encouraged us to include all publications acknowledging the individual platforms, as this will be a good start value. The number of publications for 2022 shows the demand of recombinant proteins from 
the research community, which is the rational that led to the formation of PPS.

PPS was mainly populated with staff from the existing platforms. The start values from September 2022 were around 60 % female employees and 50% women in the management and steering groups. This quite even gender distribution continues at different levels within PPS and this means that any measures do not primarily need to be taken here.

The KPIs for gender equality for 2022 are: 1) 65% female employees, 2) 43% women in the management group and 50% women in the steering committee, 3) 30% female users (unique PIs) and 4) 45% of applied projects and 29% of completed projects were from women. 34% of protein batches were delivered to women. The proportion of projects from women falls well into the span of the typical distribution of applicants based on project applications to VR in the areas medicine and technology (a span of 25% - 50% women as applicants). When looking at the applicants who are not themselves PIs for the projects, 42% of applied projects and 45% of completed projects came from women. We did not have any external educational or user activities this year. For the next year we are planning to intensify our outreach activities. his should include identifying the forums where we can make a difference, such as workshops and women's networks in science, as outlined in the gender equality plan.

A risk assessment was done as part of the application. This risk assessment is considered by us as still valid. Some mitigation measures for “Failure to follow new trends in the field/Ensure that qualified personnel stay within PPS” have been anchored in the strategic plan and operational plan 2023. An event very appreciated by our staff was the startup meeting for PPS staff. All other points identified in the risk assessment relate very much to the KPI 2022 and their further development into 2023. Depending on this development, mitigation measures will be taken, if necessary.

To ensure that the users of PPS will receive materials of highest quality, PPS will follow the guidelines issued by The Association of Resources for Biophysical Research in Europe (ARBRE-MOBIEU) and Protein Production and Purification Partnership in Europe (P4EU) regarding quality control. This means that methods to ascertain the purity, identity and homogeneity/monodispersity of the proteins need to be in place. In 2022, an inventory of the methods already in use at the different nodes has been performed. Protein analysis by SDS- PAGE is performed in all labs and is normally included in the user fee. For identity, different mass spectrometry analyses can be performed. For monodispersity, different variants of size exclusion chromatography, either as the last purification step, a separate analytical run or SEC connected to multi-angle light scattering (SEC-MALS), or other biophysical methods such as mass photometry, can be used. As these analyses often both require more protein and are more work-intense, it will be more costly for PPS and/or the user. The discussion around the balance between required quality control analyses and costs is ongoing and will be continued in 2023. Another important quality parameter is to provide instructions to the users what information needs to be included in future publications (sequences, purification procedure etc), and this information will be clearly available to the users in the project reports.