Joint initiative on the quality of recombinant proteins ...

Dr. André MatagneCentre for Protein Engineering

Protein Factory & Robotein® facilitiesUNIVERSITY OF LIEGE

Joint initiative on the quality of recombinant proteins and peptides

Quality control of purified proteins to improve research data reproducibility





Clemens Fraikin(Jasco Benelux B.V.)













QC team of ARBRE and P4EUNick Berrow - Institute for research in Medicine, Barcelona, Spain (P4EU)

Maria Garcia-Alai - EMBL Hamburg, Germany (ARBRE-MOBIEU)

Stefan Knauer - Bayreuth University, Germany (ARBRE-MOBIEU)

Mario Lebendiker - Hebrew University of Jerusalem, Israel (P4EU)

Blanca Lopez-Mendez – University of Copenhagen, Denmark (ARBRE-MOBIEU)

Ario de Marco - University of Nova Gorica, Slovenia (P4EU and ARBRE-MOBIEU)

André Matagne – University of Liège, Belgium (ARBRE-MOBIEU)

Annabel Parret - EMBL Hamburg, Germany (P4EU)

Bertrand Raynal - Pasteur Institute, Paris, France (ARBRE-MOBIEU)

Kim Remans - EMBL Heibelberg, Germany (P4EU)

Stephan Uebel - MPI for Biochemistry Munich, Germany (ARBRE-MOBIEU)

• A lot of time and resources is spent on poor quality samples

• The best experiments in the world will turn rubbish into expensive rubbish

Our analysis of the situation (as core facilities) in different European institutions


Improving the quality of the samples is essential to enhance the quality (i.e. accuracy, reproducibility) of the results we produce

• The best experiments in the world will turn rubbish into expensive rubbish



“It is the way we have prepared samples in the lab for ten years….”“But some experiments have worked with this sample…”“SEC? DLS? MS?... My boss thinks it is a waste of time…”“I’ll do the experiment anyway - it may work…”“I don’t know how to do it…”“I don’t have time…”


The international press

Freedman LP, Cockburn IM, Simcoe TS (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol 13(6): e1002165.(using 2012 data) 459 citations on April 19th, 2021 (source: Scopus)

(56.4109)

Tremendous economic impact

Freedman LP, Cockburn IM, Simcoe TS (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol 13(6): e1002165.(using 2012 data) 459 citations on April 19th, 2021 (source: Scopus)

(56.4109)

$10.4 billion

worth of

research!!!



70% of 1576 researchers had been unable to reproduce another’s experiment and over half had even been unable to reproduce theirownBaker, M. (2016) 1500 scientists lift the lid on reproducibility, Nature 533, 452-544



Free, T. (2020) The ABCs of reproducibility; effectingattitudes, behaviors and change (2020), BioTechniques, 69, 359-361

[5] Knudtson, K. et al. (2019) Survey on scientific shared resourcerigor and reproducibility, J. Biomol. Tech, 30, 36-44.[6] Baker, M. (2016) 1500 scientists lift the lid on reproducibility, Nature 533, 452-544

(…)



Free, T. (2020) The ABCs of reproducibility; effectingattitudes, behaviors and change (2020), BioTechniques, 69, 359-361

5] Knudtson, K. et al. (2019) Survey on scientific shared resourcerigor and reproducibility, J. Biomol. Tech, 30, 36-44.[6] Baker, M. (2016) 1500 scientists lift the lid on reproducibility, Nature 533, 452-544

359-361

(…)

Are we wasting 50% of life science and pre-clinical research buget? What can we do?

✓ Improve experimental design/data analysis!



✓ Improve QC and characterization of the‘protein’ samples (biological reagents)




✓ Raise the educative and basic question: howmuch can I trust my protein? What should I know?





✓ Check that we are working with:

the correct protein





✓ Check that we are working with:

the correct protein

that it is pure/homogeneous

it has right fold/activity





✓ Check that we are working with…

the correct protein

t

✓ Report the results of each control in publications





✓ Check that we are working with…

the correct protein

that it is pure/homogeneous

it has right fold/activity

✓ Report the results of each control in publications

✓ Encourage more extensive use of supplementary data


Our proposal:

minimal information to provide in publications

Protein identity and production parameters➢ protein name and complete sequence, by providing a

NCBI or UniProt accession number, cloning strategy and the source of the DNA (species)

➢ expression vector and host strain, including the tags and cleavage sites used, with the full amino acid sequence of the final protein

➢ Expression and purification protocol, namely the detailed description of all the production steps

Our proposal:






Protein concentration (A = log I0/I = e280·C·l)Specifying the method used for quantification and themolar extinction coefficient at 280 nm, if applicable

Our proposal:






Protein concentration (A = log I0/I = e280·C·l)Specifying the method used for quantification and themolar extinction coefficient at 280 nm, if applicable

Storage conditionsi.e. final buffer composition (pH, buffers, salts and additives), storage temperature and, when applicable, freezing or lyophilization conditions

Our proposal:


PurityChecked by SDS-PAGE, Capillary Electrophoresis (CE) orReverse Phase Liquid Chromatography(RPLC)

Our proposal:

minimal quality control tests to be performed

Homogeneity (size distribution)checked preferably by Size Exclusion Chromatography (SEC) and/or Dynamic Light Scattering (DLS), or by SEC in combination with Multi Angle Light Scattering (SEC-MALS), Field Flow Fractionation (FFF) or FFF-MALS, or analytical ultracentrifugation (AUC)


Our proposal:


Homogeneity (size distribution)checked preferably by Size Exclusion Chromatography (SEC) and/or Dynamic Light Scattering (DLS), or by SEC in combination with Multi Angle Light Scattering (SEC-MALS), Field Flow Fractionation (FFF) or FFF-MALS, or analytical ultracentrifugation (AUC)

Identity and integritychecked preferably by intact protein mass (MS), peptide mass fingerprint (MS), or Edman sequencing (correct sequence? no proteolysis? no undesired modifications? expected PTMs?)


Our proposal:


UV absorbance spectrum between 340 nm and 240 nm

to check nucleic acid content, absence of aggregates and measure [P]Mandatory if protein binds nucleic acids

Our proposal:

extended quality control tests



Conformational stability/folding stateChecked by Circular Dichroism (CD), Fourier Transform Infra Red (FTIR), NMR, Differential Scanning Calorimetry (DSC); “spectral signatures” and “thermal denaturation signatures” (very useful for lot-to-lot consistency)

Our proposal:





Homogeneity (charge distribution, conformational species)Checked by analytical Ion Exchange Chromatography (IEX), analytical Hydrophobic Interaction Chromatography (HIC), Isoelectric Focusing (IEF)

Our proposal:






Protein competent fraction, i.e. the relative amount of active protein Measured as specific activity, by active-site titration or other suitable methods (to measure biological function)

Our proposal:







Optimization of storage conditions (buffer, pH, etc.)Minimize aggregation, improve solubility and stability (also activity!)

Our proposal:







Optimization of storage conditions (buffer, pH, etc.)Minimize aggregation, improve solubility and stability (also activity!)

Batch-to-batch consistency Mandatory if more than one batch is to be usedUse some of the methods listed for minimal/extended QC

Our proposal:


The minimum quality control relies on five(first-line) methods only:

• SDS-PAGE

• UV-Visible spectrophotometry

• Mass spectrometry

• Size exclusion chromatography

• Dynamic light scattering

Raynal et al. (2014) Microbial Cell Factories 13, 180

Minimum quality control guideline(recommended tests)


• SDS-PAGE








• SDS-PAGE








• SDS-PAGE








• SDS-PAGE








• SDS-PAGE








• SDS-PAGE


• MS

• SEC-MALS


=>=>=> cost is less than 200 µg of protein

~10 µg (coomassie blue staining)

(~400 µg)

~60 µg (Mr 105, i.e. 0.6 nmole)

~100 µg

(~5 µg)


31 institutions

43 laboratories

188 samplesGives a 90% confidence level in our analysis, with a 6% margin of error

Collection of experiment datafor statistical purposes

6%20%

26% 21%

53%

74%

69%

31%

Survey of protein QC procedures

1/3 samples tested for purity, homogenityand identity/integrity failed at least one ofthem

Correlation between QC results and satisfactory results obtained in downstream applications

Out of these 130 samples, among those that passed the three criteria (purity, more than 90% assessed by SDS-PAGE, aggegation state by DLS or SEC, identity by intact mass or peptide mass fingerprint), 74% yielded satisfactory results, 20% suceeded only partially and 6% failed in downstream applications

In contrast, most (ca. 80%) of the samples that failed one or more of the minimal QC tests didnot give satisfactory results in downstream applications

6%20%

26% 21%

53%

74%

69%

31%

Survey of protein QC procedures

1/3 samples tested for purity, homogenityand identity/integrity failed at least one ofthem

Success in downstream applications (130 samples)

Guidelines with minimal data that needs to be known before using a protein

So what is our proposal?


Identity HomogeneityPurity Integrity



✓ our guidelines are the result from the collective expertise of ca. 150 laboratories





✓ lay the groundwork for the standardization of procedures and reproducibility of data in any laboratory that produces recombinant proteins






✓ encourage the whole scientific community (researchers, editors and funding agencies alike) to implement this QC methodology







✓ raise awareness amongst colleagues and collaborators








✓ reverse the trend for less and less information available in mainsteam journals on theproduction and purification of laboratory reagents









✓ lobby editors to encourage/oblige a more extensive use of supplementary data and also the report of the results of each check-point in research papers









✓ lobby editors to encourage/oblige a more extensive use of supplementary data and also the report of the results of each check-point in research papers

✓ improve reproducibility between labs and increase confidence in published data



The QC team of ARBRE MOBIEU and P4EU

Nick Berrow - Institute for research in Medicine, Barcelona, Spain (P4EU)




Blanca Lopez-Mendez - University of Copenhagen, Denmark (ARBRE-MOBIEU)


André Matagne - University of Liège, Belgium (ARBRE-MOBIEU)




Stephan Uebel - Max-Planck Institute for Biochemistry, Munich, Germany (ARBRE-MOBIEU)

On behalf of…













On behalf of…

list of recommended tests (QC Guidelines)













On behalf of…

[email protected]

list of recommended tests (QC Guidelines)

Acknowledgement (Dankjewel!!!)

Titel der Präsentation (Editierbar im Folienmaster), Ort, 20.04.2021 (Editierbar im Folienmaster)Sabine Suppmann, Biochemistry Core Facility 9th P4EU meeting, Nov30

Network of more than 100 members from > 40 protein facilities, mainly but not exclusively in Europe

▪ Started in 2010 (Hüseyin Besir, EMBL)

▪ Share expertise and information

▪ Exchange materials & protocols

▪ New tools & technologies

▪ Establish standards for P related work

▪ Benchmarking and SOPs

▪ Enable collaboration

▪ Access to external facilities

▪ Training of staff/usersfrom Hüseyin Besir, CTLS 2014

(2015)

A large communityhas already joined

More than 170

resource labs/infrastructures/facilities

from 30 different european countries

working in very different contexts ,

some more "research topic oriented"

and others

more "technology oriented"

http://arbre-mobieu.eu/

http://arbre-mobieu.eu/

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Joint initiative on the quality of recombinant proteins ...

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