Your reliable partner for the research and regulatory ... · ICH Topic Q6B Specifications: Test procedures and acceptance criteria for biotechnological / biological products, Step

BIOLOGICALS

Your reliable partner for the research and regulatorydevelopment of innovative biologicals, biosimilars and vaccines

KYMOSBIOTECH

KYMOS BIOTECH DIVISION

KYMOS is a 10-year-old company, focused on bioanalysis and analytical development of innovative and generic small molecule drugs for the pharmaceutical, veterinary and fine chemical sectors.

Consistently with its efforts to provide their customers with integrated solutions, KYMOS created a biotech division in January 2012, located in a new laboratory at the Barcelona Science Park (PCB). This new division is the result of a deal with IPSEN, a global biotechnology pharmaceutical group. Thanks to this agreement KYMOS obtained the necessary equipment, know-how and personnel with a wide experience in the development of biotechnological products.

The laboratory is equipped with state-of the-art technologies both in LC-MS/MS and immunology and the team had worked over many years in the development of peptides and proteins. With the implementation of this new area, Kymos extended its activity to biotech companies who develop innovative biopharmaceutical drugs, biosimilars and vaccines.

BIOLOGICAL DRUG DEVELOPMENT

With the development of biotechnology, disease treatment has suffered major transformations both from the point of view of the therapeutic approaches and from the type of molecules involved.

Latest-generation pharmaceuticals are moving from exogenous synthetic small molecules to high-molecular weight endogenous related biomolecules. Some examples of this new generation of biopharmaceuticals are hormone peptides, recombinant proteins, monoclonal and therapeutic antibodies, fusion proteins, oligonucleotides, silent RNA, recombinant vaccines, etc.

Due to the special properties of biopharmaceuticals such us their high molecular weight, high structural complexity and mode of action, the regulatory requirements for these molecules differ a lot from the requirements for small molecules. Indeed, a large list of tests are required to characterize the quality of the biopharmaceutical drug, which is known as the analytical fingerprint.

In addition, pharmacokinetic studies require specific bioanalytical methods like immunological or innovative mass spectrometry methods. Whereas metabolism plays a very limited role in the biotransformation of these molecules, immunogenicity has turned into the major safety issue for them. Because of this, both FDA and EMA have published several guidelines to describe how to detect and study the development of spontaneous antibodies against the biopharmaceutical drug.

KYMOSBIOTECH

KYMOS BIOTECH SERVICES

PROTEINS AND PEPTIDESKymos provides complete bioanalytical solutions from early stages of candidate selection to post-marketing commitments of marketed drugs for both innovative biologicals and biosimilars:

Characterization and Quality Control (ICH Q6B)• Structural characterization: amino acid sequencing and peptide mapping, N- and C- terminal sequencing, disulfide bridges and glycosylation profile.• Physico-chemical properties: molecular weight, isoform pattern, extinction coefficient, electrophoretic pattern, liquid chromatographic pattern and spectroscopic profile.• Process related impurities: host cell proteins, antibiotics, processing reagents, heavy metals, solvents, etc .• Product related impurities: truncates and aggregates, modified forms (deamidated, isomerised, oxidised, etc.).• Biological characterization: biological activity and receptor binding• Stability studies for drug substances and drug products.

Including development, validation and analysis of different batches, full characterization of a standard batch, setting-up substance specifications and comparability of biosimilars.

Pharmacokinetic and Pharmacodynamic studies• Pharmacokinetic preclinical studies including management of animal housing• Development and fit-to-purpose validation of biomarkers from pharmacodynamic studies and Translational Research• Development, validation and sample analysis of biological drugs on plasma or serum from clinical studies • PK calculations and reports

Immunogenicity studies• Binding antibodies assays: screening, confirmatory, titration and isotyping.• Neutralizing antibodies assays.• Cellular immune response assays

VACCINESKymos provides analytical support in characterization and quality control testing of non-living vaccines (or without special biosafety requirements) in human and veterinary products:• Ingredient characterization: - Antigens - Adjuvants - Preservatives and other excipients• Potency assays of the antigens: - In vivo immunization in different species - Relevant antigen determination by immunoassays - Activity determination by competitive immunoassays• Characterization of In-House Reference Preparations (IHRP)

KYMOS TECHNOLOGICAL PLATFORMS

KYMOS has set up two main technological platforms to provide its partners with comprehensive support to its partners for the development of biopharmaceutical drugs which operate under GLP and GMP.

BIOLOGICAL MASS SPECTROMETRY PLATFORM

Application of LC-MS/MS to peptide and protein analysis in biological fluids is a powerful technique, its major advantages being specificity, flexibility and sensitivity. KYMOS has a wide experience in peptide drug determination in biological fluids and is now applying its knowledge to the analysis of proteins using two approaches depending on the size of the protein:

• Quantitation of the intact drug for small proteins (up to 8-9 KD), peptides, pegylated peptides, etc. The procedure involves drug purification and subsequent quantitation by LC-MS/MS analysis.

• Quantitation of the protein using selected diagnostic hydrolytic peptides. This approach applies to proteins, independently of its size, pegylated proteins, etc. In this case, after a major endogenous protein depletion step, the protein is submitted to enzymatic hydrolysis, like trypsin hydrolysis, selection of one or more fragment peptides and LC-MS/MS quantitation of these peptides.

This methodology is particularly useful at the early steps of the research where the final primary structure of the protein is not well defined and LC-MS/MS allows to cope with this changes easily.

IMMUNOLOGIC ASSAY PLATFORM

Binding assays based on immunological methods are nowadays widespread analytical methods for protein analysis. The immunological assays available at KYMOS are:

Radioimmunoassay (RIA)RIA methods based on the interaction between the antibody, the protein/peptide and the protein/peptide labelled with a radioactive isotope (i.e.125I) fully developed and validated for determination of drug levels and anti-drug antibodies.

Availability of animal house and radioactivity lab to carry out auxiliary activities such as obtaining the antibodies and the synthesis of the radiotracers needed for the RIA analysis.

Enzyme-linked immunoassay (ELISA) ELISA methods are developed and validated for drug level determination and immunogenicity studies in all its types (direct, indirect, sandwich, etc.) and using several detection modes (colorimetric, fluorescence, time resolved fluorescence, etc.). Methods can be developed ex novo or adapted from commercially available kits.

Electrochemiluminiscence (ECLA)ECLA detection, operating under the Meso Scale Discovery technology, uses labels that emit light when electrochemically stimulated. Labelled tracers are stable, non-radioactive and offer a choice of convenient coupling chemistries. KYMOS develops and validates ECLA methods for antibody detection and protein level measurement in biological fluids, including the synthesis of the required labelled tracers.

Cell-based assays for neutralizing antibodies

In immunogenicity studies, the neutralizing capacity of antibodies present in positive samples needs to be established using cell-based assays. A recommended approach is to set up a potency test for the biopharmaceutical drug and study if the addition of the sample containing the antibodies causes decrease of the initially observed potency. Kymos can provide this kind of studies to complete the study of the inmune response to certain biological drugs.

LC-MS/MS (6 units), AB SCIEX API 3000, 3200, 4000 (2), 4000 QTRAP & 5500 QTRAPNano electrospray sourceTwo dimensions nano HPLC, Tempo MDLCHPLC/UHPLC: Agilent 1200 and Waters AcquityOn line solid phase extraction system, Symbiosis PharmaGas chromatograph (FID and ECD) and head spaceWIZARD gamma counterVICTOR2 multilabel counter SECTOR Imager 2400 Meso Scale DiscoveryGuava PCA-96 flow cytometerElectrophoresis: One and two dimensions, Western BlotIsogen Proxima imaging systemPharmacia PhastSystem high speed electrophoresisAgilent 2100 Bioanalyzer: microfluidic-based platform for cell, DNA, RNA and protein analysisAgilent 3100 Offgel fractionator for protein purification based in isoelectric focusingATKA fast protein liquid chromatography systemZephyr liquid handler workstation for automatic sample processing

QUALITY

KYMOS is GLP certified to perform bioanalytical and immunological studies and it is GMP certified for quality control testing.

All the studies are carried out following the EMA and FDA current guidelines and other relevant papers in this field:

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EQUIPMENT AND TECHNOLOGIES

Because of KYMOS’ privileged emplacement at the Barcelona Science Park, one of the most important sites of Biomedical and Biotechnological Research in Europe, we have available a wide range of state of the art technologies like: Biacore, NMR, Orbitrap MS, MALDI-TOF/TOF, etc.

ICH Topic Q6B Specifications: Test procedures and acceptance criteria for biotechnological / biological products, Step 5 (CPMP/ICH/365/96)

FDA Guidance for industry: Bioanalytical method validation, May 2001 .

EMA Guideline on bioanalytical method validation,(EMEA/CHMP/EWP/19221/2009)

DRAFT FDA Guidance for industry: Assay development for immunogenicity testing of therapeutic proteins, December 2009.

EMA Guideline on immunogenicity assessment of biotechnology-derived therapeutic proteins, (EMEA/CHMP/BMWP/14327/2006)

Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. Mire-Sluis AR et al., Immunol. Methods, 289:1-16 (2004)

Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics. Gupta S et. al., J. Pharm. Biomed. Anal. 55(5):878-88 (2011).

OTHER SERVICES

PHARMACEUTICALS / SMALL MOLECULES

Bioanalysis• Bioanalysis for innovative drugs and biomarkers• Bioequivalence studies for gener ic products• Pharmacokinetics data analysis and reports

Analytical Development• Characterization of drug substances• Development and validation of analytical methods• Pharmaceutical development analytical support

Microbiology• Microbiological contamination of non-sterile products• Suitability and validation• Challenge test and Bacterial Endotoxines• Microbiological assay and validation

Stability Studies• ICH Stability Studies (API's and medicinal products)• Photostability and in use stability• On-going Stability Studies

Quality Control• Quality Control testing for API's and medicinal products• GMP certificates of analysis• Retest for batch release (non-European products)

Process Analysis• NIR & RAMAN analysis of ingredients• NIR applications for drug product batch release• QbD training and consultancy