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Why a Paradigm Change CHRETO's single-use purification technology offers a paradigm change for the biopharmaceutical community by  Facilitating a fast, easy-to-use, scalable and cost efficient recovery of large biomolecules, in a single-use set-up  Increasing productivity and reducing costs  Introducing the first truly single use technology for the affinity purification of monoclonal and polyclonal antibodies (mAbs and IVIGs)

"The need for better single-use and disposable systems in downstream processing is growing…The move toward disposable upstream bioprocessing will push downstream operations toward innovative alternatives that may include membrane adsorbers and prepacked single-use chromatography columns."

"Disposable Chromatography: Options Are Increasing" Genetic Engineering News, Dec. 10, 2013

The principle of the CHRETO TechnologyTM is straightforward The DAP molecules bind specifically to the target molecules in solution and form complexes. The complexes are separated on a support in the form of a membrane. After washing away impurities, the complex is dissolved and the target molecule can then be collected.  This procedure allows the process to be membrane based as well as single use which gives a long list of advantages over the current column technology based on protein A, without compromising process yield and product quality. CHRETO TechnologyTM is a single-use, membrane-based affinity purification technology that will revolutionize downstream processing for the Biopharmaceutical industry. The technology is based on a DUAL AFFINITY PROTEIN (DAP) molecule which is constructed combining bindings domains from protein A (alpha-helix structures in blue, below) that binds to the Fc region of antibodies and streptavidin (beta-sheet structures in yellow, below) that displays high affinity for biotin (structure in red at the bottom of streptavidin). Reaction conditions in CHRETO TechnologyTM are radically different, much faster and more efficient than current column-based approaches. The figure below depicts a DAP monomer.

$The proprietary CHRETO TechnologyTM is a single-use kit format with a recombinant “Dual Affinity Protein” (DAP) molecule binding to the target protein in solution, a generic membrane support, buffers, assays and ancillaries. The kit package format strongly supports the biopharmaceutical industries’ need for manufacturing process simplicity, ease of use, fast set-up and linear scalability from R&D to pilot and full manufacturing scale.   Advantages for the Biopharmaceutical Industry (mAb production) CHRETO TechnologyTM avoids the technical and practical shortcomings of the 30 year old column affinity chromatography technology. Especially the bottleneck issues, packaging of large diameter columns with difficult-to-control flow properties as well as need for repeated extensive quality controlled cleaning and validation steps. Advantages for the Plasma Fractionation Industry (IVIG production) CHRETO TechnologyTM avoids the technical and practical shortcomings of the 60 year old Cohn fractionation technology and provides novel business opportunities due to improved yields, higher purity and better safety. The price competitive CHRETO TechnologyTM will enable closing the gap between market demand and product availability of gamma-globulins. The plasma fractionation industry can significantly increase earnings when they implement CHRETO TechnologyTM in their production.$

CHRETO TechnologyTM process overview illustrating the catch, clean and collect steps. See details below (How does it work?).

The characteristics of the DAP molecule include 1) easy elution and no leakage, 2) specific binding to product (to secure selective reaction with target protein(s) and easy dissolving of formed DAP-target complexes and 3) tight binding to solid support (to secure immobilization and final removal of DAP molecules from product stream)

The purification process consists of three steps: catch, clean and collect.

Catch. The DAP molecule reacts in solution with the feed stream containing the target molecule(s) e.g., an antibody.

Clean. Impurities including HCP (host cell proteins) and DNA are removed followed by dissociation of the DAP-antibody complex and capture of DAP to a biotin support.
Collect. The purified antibody is collected and the membrane cartridge is discharged.

Sounds good in theory, but does it really work?

Time (h)

Below, two examples of the application of the technology are shown. 1. mAb purification CHRETO TechnologyTM was used to purify a monoclonal antibody (mAb) using directly the clarified cell broth.

The left-hand figure (above) shows the chromatogram profile, with two major protein peaks representing the fractions containing the flow through after reaction with DAP (Fraction 7 and Lane 7 on the SDS gel, right panel above), and the fraction containing the purified mAb (Fraction 8 and Lane 8 on the SDS gel above). Lane 3 shows purified DAP; Lane 4 shows the feedstock used and Lane 6 show the same sample run without addition of DAP. Lanes 1 and 10 include the MW marker.

2. Plasma Fractionation CHRETO TechnologyTM was used to purify gamma globulins from human plasma. Initial results showed recovery of 6 g of gamma globulin/L plasma achieved in our test and represents a 1.5-fold increase in yield compared to industry standard of 4 g/L plasma.

The left-hand figure (above) shows the chromatogram profile, with three major protein peaks representing, from left to right: proteins not bound to DAP, proteins washed away from DAP-target complexes and the elution of the DAP-purified IVIG, respectively. Arrows indicate the fractions that are further analysed by SDS-PAGE (figure on the right). Lanes 7-9 on the SDS-PAGE contain highly purified gamma globulins (right-hand panel above).

High purity was obtained using a single purification step for both mAb purification and plasma fractionation.

Background CHRETO was founded in 2010, as a spin out of Novozymes, by the inventor Jan Kyhse- Andersen and Torben Jørgensen to develop and commercialise a novel protein purification technology for bio-manufacturing. CHRETO is based on a set of fundamental values Creativity, Honesty, Respect and Trustworthiness The company acquired in 2014 the rights to the original IP related to protein purification technology from Novozymes. A prototype of the technology has been developed and thoroughly tested by industry experts and a leading pharmaceutical company in Europe during 2014 - 2016.

Our vision is that CHRETO TechnologyTM will radically improve the process efficiency of downstream processing of large biomolecules. We believe that CHRETO TechnologyTM will bring a true revolution to biomanufacturing, which has been sought by the industry for decades. Our goals are: 1) to have the first product on the market in 2018 and 2) to position CHRETO as the preferred purification technology for monoclonal antibodies and IVIG going forward.

CHRETO has been supported by funding from a Eurostars project, a collaboration agreement with a major European pharmaceutical company and Skeikampen Holding ApS together with national grants.   In September 2016, Biotage AB and Brohuvudet AB invested a total of DKK 20 million (15 + 5, respectively) and Innovationsfonden invested DKK 4.6 million in CHRETO to secure development of the proprietary purification technology. Shareholders (77%) Jan Kyhse-Andersen Holding ApS Biotage AB Novozymes A/S Brohuvudet AB

Board of Directors

Lars Bäckman - Chairman of the board

Lars Bäckman holds the position as VP of Corporate Development with Biotage AB since January 2007 and he is also the General Manager of Biotage subsidiary MIP Technologies. Prior joining Biotage, Lars Bäckman held the position as SVP of Corporate Development at Affibody. He has previous experiences acting as a lawyer at the Swedish law firm Hamilton & Co, specialized in Corporate Finance and Mergers & Acquisitions, and from Venture Management at the Business Development firm Googol. Mr. Bäckman holds a LLM from the Stockholm University and was born in 1961.

Kim Bjørnstrup - Member of the board

Kim has a management carrier of more than 25 years in the pharmaceutical industry. He graduated from Copenhagen University in 1984 and was licensed as a Lawyer in 1987.

From 1987 to 1992, he was Internal Legal Counsel to Lundbeck AS and Later Coloplast AS.

From 1992 to 2010 he worked at Octapharma AG in Switzerland, a swiss biotech, initially as Licensing manager and the last 10 years Vice Chairman /CEO. The Company had revenues exceeding one billion EURO and 4000 employees.

In 2010, he re-started Xeltis AG a Swiss biotech with a group of investors, specialising in self-generating heart valves.

From 2013 to 2014, he was the CEO of BPL UK a company with revenues of 300 million EURO and 2000 employees.

From 2015 he worked as CEO at Assistance HR Partners AS, where he I s co-owner, a company specialising in management training recruitment and temporary work.

Mikael Bundgaard-Nielsen - Member of the board

Mikael Bundgaard-Nielsen holds a position as VP for Product & Process Development with Novozymes A/S since 2013. Prior to this position Mr. Bundgaard-Nielsen (2008-2012) was Managing Director for Novozymes Biopharma Sweden AB (now Repligen Sweden AB) in Lund, that manufactures affinity ligands and active pharmaceutical ingredients according to cGMP. He has additional experience

from various leadership positions within research, development and manufacturing in Novo Nordisk A/S, Pharmexa A/S and

Novozymes A/S.

Mr. Bundgaard-Nielsen holds a Master of Science in Chemical Engineering and a Graduate Diploma in Business Administration. He was born 1969.

Sussi Wetterlin - Member of the board

Sussi is CEO of BASTAonline, a company focused on the substitution of chemicals of concern in the Building and Construction sector.

Sussi has more than 20 years of experience from the chemical industry within the environmental and commercial area. Sussi has extensive managerial experience with proven track record of leading international sales organisations during build-up, growth and turnaround phases. She held a position as European Sales Director in the Wood Industry sector with focus on the emerging markets in Eastern Europe and Russia.

Sussi has a M.Sc in Toxicology from Karolinska Institutet in Stockholm

Svend Licht - Member of the board

Svend is Chief Commercial Officer in Albumedix A/S, with responsibility for commerical activities, customer technical support, IP, regulatory and legal matters.

Svend brings more than 25 years managerial experience from international positions in Sales, Marketing, Business Development and M&A. His broad professional background and strong business skills have previously been utilized in companies such as Novo Nordisk and Novozymes through various positions, most recently as Global Head of Sales & Business Development in Novozymes Biopharma. Svend Erik Licht also serves on a number of life science boards.

Svend holds a B.Sc. in chemical engineering from the Technical University of Denmark, a B.Sc. in foreign trade from Copenhagen Business School and an executive MBA with distinction from Ashridge Management College in UK.

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Jan Kyhse-Andersen - Chief Executive Officer

Jan is inventor of the DAP technology and founder of CHRETO. Jan has more than 25 years entrepreneurial and managerial experience from the biotechnology and diagnostic industry. Jan held a position as senior manager in Business Development at Novozymes A/S, prior to the management spin-out of the DAP technology. Jan has been supervisory Board Member in a privately equity fund, Life Equity Sweden KB for several years and prior to joining Novozymes A/S, Jan was Vice President, R&D and Production at Vir Biosensors A/S (SPR biosensor).

Jan started his career as post doc at the Technical University of Denmark and then joined DAKO A/S where he had several managerial positions in Diagnostic assay development, Sales and Marketing, R&D manager and subsequently Director of Antibody Manufacturing.

Jan is also the inventor of Semi Dry Electroblotting, which significantly improved the Western blotting procedure. Jan was founder of JKA-BIOTECH and co-founder of Kem-En-Tec A/S and was the first to commercialize the Semi Drying Electroblotting apparatus.

Jan holds a M.Sc. and a Ph.D. in Chemical Engineering from Technology University of Denmark.

Jens Ingwersen - Administrative Director

Jens has a management career of more than 25 years in the Danish pharmaceutical industry (Animal Health). He graduated from Copenhagen University in 1976 as Master of Agric. Science

From 1986 to 2005 he was employed by LEO Pharma. In a position as CEO of LEO Animal Health he conducted a management buy out of the Animal Health business unit. Completion of this transaction took place April 2005.

Since end of 2006 he has held several positions as interim manager in biotech companies. Amongst those were Upfront Chromatography providing EBA (Expanded Bed Adsorption) technology, where he held a position as commercial Director.

Susanne Andresen - QA/RA Director

Susanne has a background in Quality Management and Research & Development from various Danish and International companies. She graduated from the Technical University of Denmark in 1998 as Cand. Polyt in Chemical Engineering.

Susanne has extensive R&D experience from leading establishments including The Danish Cancer Society and Coloplast. For the past 10 years she has been engaged in Quality Management of QC laboratories in the areas of Food and Medical Device leading to extensive knowledge in ISO and FDA regulations and European and US Pharmacopoeias.

Ann Dorrit Enevoldsen - Membrane Technology Manager

Ann has a background in Membrane Technology. She graduated from the Technical University of Denmark as a Chemical Engineer in 2003 and completed an industrial PhD in Membrane Technology at the Technical University of Denmark and Novozymes in 2008.

Ann has worked with Research and development of Membrane technology for more than 10 years within various fields as water treatment, development of non-alcoholic beverages and enzyme production. She has experience in ultrafiltration, microfiltration, membrane materials and characterization and design of membrane systems. She is experienced in managing R&D projects from various projects including work package leader in a FP7-EU project concerning development of functional membranes.

Anders Bundgård Sørensen - Protein Science Manager

Anders has a background in peptide self-assembly, protein chemistry and is a Cold Spring Harbour trained structural biologist.

He graduated from Aalborg University as part of the Elite M.Sc. program in Molecular Biotechnology in 2012 and completed his Industrial Ph.D. at Novo Nordisk A/S and Aalborg University in 2016.

Anders has extensive experience working with proteins, ranging from design, expression, purification, characterization to structural biology methods. His track-record includes several publications in peer-reviewed international journals and hands-on experience with managing CMC projects at Hejnaes Consult AB.

Recently Anders has had practical insights on how to leverage strategic alliances in biotech startups from his time at Copenhagen Business School (M.Sc. BA in bio entrepreneurship) and Evaxion-Biotech Aps.

Click on the names to see CV

Get in touch with us to hear more about DAP technology and its exciting potential for the biopharmaceutical and plasma fractionation industries.

Tel: +45 22 92 80 82

Email: info@chreto.com

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CHRETO :: Lejrvej 17 :: DK-3500 Værløse :: Denmark :: tel: +45 22 92 80 82 :: Email: info@chreto.com