Central laboratory

Central laboratory

Central laboratory (CL) is a core facility designed to offer a range of process development services necessary to develop and commercialize new products.

CL comprises 6 specialized labs (~ 600 square meters total) with state-of-the art equipment, storage areas, washing and sterilization room and offices for permanent staff.

Centralni Laboratorij 1

Microbiology laboratory staff:

Adriana Lepur, PhD, Head of laboratory, e-mail: adriana.lepur@biocentre.hr
Matea Skoblar, M.Eng., Junior associate, e-mail: matea.skoblar@biocentre.hr

Microbiology Laboratory is specialized in recombinant protein production and purification in a µg to mg scale. We offer production process development for biopharmaceuticals and biosimilars. We work with various microorganisms, optimize growth conditions, test different treatments and study metabolites and by-products. We offer bacteria and yeasts cell propagation and banking.

Microbiology Laboratory staff organizes workshops for groups or tailored to suit client’s specific needs.

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BACTERIA TRANSFORMATION

Chemically competent cells are transformed by heat shock in order to insert DNA of interest. Positive cells are selected and stored at – 80°C in glycerol for further use.

PLASMID PROPAGATION AND PURIFICATION

After transformation and propagation of competent E. coli, plasmid DNA is isolated (mini, midi or maxi prep) and analysed on agarose gel. Spectrophotometric DNA quantification is performed and A260/A280 absorbance ratio measured.

BIOMASS AND PROTEIN PRODUCTION

Recombinant protein production begins with cell transformation and cell cultivation in suitable conditions. Fermentation can be performed in a range from 0,1 to 30 L. Twelve 0,5 L and two 2 L bioreactors can be ran in parallel, before upscaling production to a 30 L bioreactor. In order to increase yield of a desired product, we implement different optimization strategies: selection of nutrient medium, carbon source, temperature, pH, aeration, chemical inducer concentration and overall processing time.

PROTEIN PURIFICATION

Downstream processing laboratory performs protein purification. The process starts with mechanical cell lysis and/or separation, followed by protein isolation and purification using preparative chromatography. Downstream processing also includes sample concentration and diafiltration (desalting and buffer exchange).

PROTEIN QUALITY CONTROL

Proteomics laboratory performs protein quality control. SDS – PAGE and western blot analysis are done throughout protein production process – from optimization to scale-up. Quality control includes protein quantification and LC – MS/MS protein identification.

MOLECULAR BIOLOGY

Microbiology laboratory offers isolation, purification and analysis of DNA and RNA. We perform mutagenesis, cloning, end-point and semi quantitative PCR. In addition to standard microbiology techniques, we use PCR to identify microorganism in various samples – probiotics, environmental samples, and food items (beer, wine, juices).

EQUIPMENT 

Bioreactors:

BIOSTAT Qplus 12 x 0.5L MO, BIOSTAT B Twin 2L MO, BIOSTAT Cplus 30L MO (Sartorius)

Homogenizers:

Panda Plus high pressure homogenizer (Gea Niro Savi); Bandelin ultrasonic homogenizer (Sonoplus).

NanoDrop One spectrophotometer (Thermo Scientific). Continuous flow centrifuge (Sorval Stratos).

Please visit our Downstream processing and Proteomics laboratories to see more available equipment.

Cell biology laboratory staff:

Marko Marjanović, PhD, senior associate, e-mail: marko.marjanovic@biocentre.hr

Cell Biology lab offers services in animal cell culture handling and expression of recombinant proteins on a laboratory and semi-industrial scale in separate or parallel bioreactor systems. We offer process development and cell bank maintenance, extensive number of biochemical and cell biology assays using the standard molecular biology methods (e.g. flow cytometry, PCR…).

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FLOW CYTOMETRY

Our flow cytometer (BD FACSVerse) is equipped with 3 lasers (405, 488 and 633 nm) and is able to measure up to 8 different parameters. We offer standard flow cytometry services: immunophenotyping (cell surface and internal), haematological analysis, immunodeficiency and transplant rejection testing, detection of specific human and animal markers, cell line validation, cell viability and apoptosis analysis, visualisation of fluorescent proteins (including quantification of transfection), measurement of proliferation markers, chemotaxis, actin polymerisation, DNA content quantification and cell cycle analysis, caspase activity, mitochondria depolarisation, detection of cellular and mitochondrial oxidative stress. We also offer flow cytometry data analysis in FlowJo software.

CELL CULTURE CULTIVATION AND LABORATORY SCALE ASSAYS

We offer services of human and animal cell cultures handling, including primary cultures, co-cultures, 3D cultures, generation of nurse-like and similar in vitro cell cellular models, cell bank maintenance, cytotoxicity and proliferation assays (MTT, XTT, MTS and similar), colony formation assay, etc.

CELL CULTURE CULTIVATION ON A SEMI-INDUSTRIAL SCALE

We offer cultivation of cell cultures in bioreactors of different volumes: 0.5L (up to 12 parallel bioreactors); 2L (2 parallel); or 30L bioreactor for the process optimisation and production of recombinant protein, medicinal drugs, cytokines, growth hormones, hematopoietic growth factor, antibodies, and similar.

PROTEIN ISOLATION AND PURIFICATION SERVICES ARE HANDLED BY OUR DOWNSTREAM PROCESSING LAB

Protein isolation includes cell lysis (high pressure and ultrasonic homogenisation), separation and purification of proteins. The lab also offers desalinisation, buffer exchange and concentration of samples.

PROTEIN QUALITY CONTROL IS HANDLED BY OUR PROTEOMICS LAB

Quality control includes SDS-PAGE and Western blot analysis, protein concentration quantification, LC-MS/MS protein mapping and identification.

MOLECULAR BIOLOGY TECHNIQUES

In addition to the services above, we offer DNA, RNA and protein extraction from cell cultures, blood, bone marrow, etc.; Western blot, PCR and ELISA assays; lentiviral and adenoviral vector particles production, cell transformation by transient or stable transfection, CRISPR/Cas9 system or similar.

EQUIPMENT

BD FACSVerse flow cytometer (3 lasers: 405, 488 and 633 nm); Bioreactors (Sartorius BIOSTAT QPlus 12 fold 0.5L CC, BIOSTAT B Twin 2L CC, BIOSTAT C Plus 30L CC); Hidex Chameleon microplate reader; Applied Biosystems SimplyAmp thermal cycler; Sorval Stratos continuous flow centrifuge, Beckman Coulter Vi-Cell XR cell counter.

Downstream processing laboratory staff:

Marina Markeš, M.chem., Head of laboratory e-mail: marina.markes@biocentre.hr
Marija Gunjača, M. Sc., Intern, e-mail: marija.gunjaca@biocentre.hr

Majority of our services revolve around biological material downstream processing, such as isolation of small molecules, biomolecules, or native and recombinant proteins from various samples. Services include process optimization on a laboratory scale, and transfer to a pilot production scale.

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PROCESSING OF COMPLEX MIXTURES

We use preparative liquid chromatography to separate biomolecules or small molecules from complex mixtures. Depending on the physiochemical properties of both, the sample and the analyte, we chose from a range of techniques: ion exchange chromatography, size exclusion chromatography, affinity chromatography, reversed – phase chromatography etc.

Complex mixtures can be fractionated based on a specific property, e.g. ionic strength, molecule size, and the obtained fractions can be analyzed and characterized further. By combining chromatography techniques, we can narrow down the isolation procedure to a couple of, or a single molecule for targeted analysis.

DIAFILTRATION AND CONCENTRATING

We use tangential flow filtration to concentrate a sample, or to exchange a buffer. We can process samples from half a liter to several tens of liters.

FROM A LABORATORY TO A PILOT SCALE

The Laboratory is equipped to accommodate processing on different scales – from modest sample quantities to a pilot production scale. We develop and optimize downstream processing procedure on a laboratory scale, and then scale up to a desired volume.

INSTRUMENTS AND EQUIPMENT

Preparative chromatography systems:

ÄKTA Avant 25, GE Healthcare Life Sciences

ÄKTA Avant 150, GE Healthcare Life Sciences

ÄKTA Pilot, GE Healthcare Life Sciences

Tangential flow filtration systems:

ÄKTA Crossflow, GE Healthcare Life Sciences

SARTOFLOW Advanced, Sartorius

Bioanalytical laboratory I (chemistry) staff:

Anita Šporec, PhD, Head of laboratory, e-mail: anita.sporec@biocentre.hr,
Iva Bebek, M.Sc., Senior associate, e-mail: iva.bebek@biocentre.hr,
Diana Nejašmić, M.Sc., Junior associate, e-mail: diana.nejasmic@biocentre.hr

Bioanalytical Laboratory I (chemistry) services portfolio include development, optimization, transfer and / or validation of analytical methods using spectroscopy (UV/VIS), liquid chromatography (HPLC-DAD-UV/VIS, HPLC-RID, HPLC-FLD, LC-MS/MS) and gas chromatography (GC-FID, GC-MS/MS).

Our team of experts develops new analytical methods from scratch, improve and expand existing methods, identify shortcomings, and troubleshoot and fix problematic analytical methods. Developed methods have been successfully implemented in a large number of projects and fields ranging from pharmaceutical and food industry, to biomedicine and biotechnology.

Our clients are both academics and industry experts, who, due to a lack of time, equipment or specific knowledge cannot solve complex analytical problems on their own. We provide fully established protocols for analytical methods development, validation and / or transfer, and we readily collaborate with our clients throughout the process.

Bioanalytical laboratory I (chemistry) organizes hands-on gas and liquid chromatography training. Workshop schedule and full description can be found online at BIOEDUCATION. In addition to general chromatography workshops, we provide tailored chromatography and mass spectrometry training, to suit client’s specific requirements and time schedule.

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INSTRUMENTS AND EQUIPMENT BASED SERVICES

Agilent Technologies HPLC-DAD-UV / VIS

Method development and validation for quantitative analysis of active substances (APIs) and impurities in finished pharmaceutical products.

Analysis of biologically active substances, preservatives, antioxidants, additives, water-soluble vitamins, and organic acids in plants and food.

Analysis of metabolites in biological samples (cell lysates, biomass).

Agilent Technologies HPLC-RID

Determination of sugars and carbohydrates, alcohols and higher alcohols in food and biological samples.

Molecular size analysis by size-exclusion chromatography (SEC).

Agilent Technologies HPLC-FLD

Analyses of naturally fluorescent compounds (e.g. polycyclic aromatic hydrocarbons), and non-fluorescent compounds by pre-column fluorescence derivatization (e.g. biogenic amines, amino acid content).

Agilent Technologies LC-MS/MS

Targeted analyses of traces of organic compounds in complex matrices.

Analysis of food quality and safety (e.g. acrylamide determination in different samples).

Analysis of impurities in pharmaceutical products.

Analysis of biological samples (e.g. small toxic molecules in biological fluids etc.).

Agilent technologies GC-FID

Quantitative analysis of e.g. alcohol content in different samples (e.g. disinfectants, alcoholic beverages etc.), fatty acids in food supplements, oil content in pharmaceutical emulsions.

Thermo Scientific GC-MS/MS

Determination of aroma profiles of food, beverages, spices and fragrances.

Determination of essential oils composition (lavender, lemon, etc.).

Analysis of volatile organic compounds (esters, alcohols, aldehydes, terpenes), trace impurities, and food contaminants.

Analysis of volatile lipophilic compounds in plant species, perfumes, etc. (e.g. menthol).

Method development for the determination of metabolic profiles; identification of compounds formed by metabolism of microorganisms, metabolites in biological fluids, fatty acids profile in serum.

Cary 100 UV/VIS spectrophotometer

Analysis of colour and bitterness of e.g. craft beer.

Determination of antioxidant activity, total phenols, and total carbohydrates in e.g. plants and food.

Bioanalytical laboratory II (proteomics) staff:

Dr. sc. Ana Butorac, Head of laboratory, e-mail: ana.butorac@biocentre.hr
Rea Bertoša, M.Eng., Junior associate, e-mail: rea.bertosa@biocentre.hr

BIOCentre’s Laboratory for proteomics provides full package of services: from a single protein characterisation to a complex proteomic studies (targeted and non-targeted protein analyses). Additionally, Laboratory for proteomics provides analysis of higher order protein structure.

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PROTEIN CHARACTERIZATION

Peptide mapping

Protein mass mapping involves protein molecule fragmenting using specific enzyme and analysing the resulting peptide mixture by mass spectrometry (MS). Analyses can be direct MS measuring or on-line connected to liquid chromatography (LC-MS). Peptide mass mapping can be extended to protein database search for sequence confirmation. Additionally, peptide mass mapping can also be extended to de novo sequencing.

Molecular weight analysis

The intact molecular weight MS techniques offer accuracy of measurement over a wide molecular weight range. Direct matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) is used for single protein analysis, and liquid chromatography with MALDI-MS detection is used for complex mixtures. Gel electrophoresis service offers you measuring of molecular weight and characterizing impurities in biopharmaceutical products. Additionally, gel electrophoresis can be extended to protein identification through in-gel digestion, tandem MALDI-MS analysis (MS/MS) and database search or with Western blot analysis.

Liquid chromatographic patterns

BIOCentre offers a wide range of LC techniques for protein/peptide analysis: Size Exclusion Chromatography (SEC), Reverse-Phase Liquid Chromatography (RP-HPLC) and Ion Exchange Chromatography (IEX).  Additionally, after LC separation we offer MS analysis.

Protein sequencing

Protein sequencing service offers MALDI top down sequencing (MALDI-TDS) and de novo sequencing. MALDI-TDS provides protein identification through database search and the simultaneous assignment of the N- and C-termini of up to 70 residues from either terminus. De novo sequencing is used for identification of unknown proteins. It is the process by which the amino acid sequence is deduced without prior knowledge of the DNA or protein sequence. Biocenter’s de novo sequencing service reveals structural information by laser induced fragmentation within a mass spectrometer (LIFT).

Amino acid analysis

Amino acid analysis services provide information of amino acid composition of proteins, peptides and other biological samples containing primary or secondary amino groups within molecular structure. Amino acid analysis allows quantitation of free amino acids, as well as quantitation of amino acids released from macromolecules.

PROTEOMICS ANALYSIS

Liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) services provide target and non-target proteome analysis

Non-target proteomics or “shotgun” proteomics service generally involves in-solution protein digestion using a suitable enzyme. Resulting peptides are separated by nano-LC and peptide fraction are collected. Separated peptides are analysed by MALDI-MS/MS and identified by protein database search. Non-target proteomics is focused on the cataloguing of proteins (from cells or tissues) or for determination of changes in protein expression between samples.

Targeted proteomic service targets specific peptides in a complex mixture and determines their presence. This analysis usually utilizes a triple quadrupole mass spectrometer (QQQ). Targeted proteomics is usually used for verification of knockdown targets, for quantification of a peptide/protein in a complex mixture and for a specific protein monitoring across samples or during a time course experiment.

HIGHER ORDER PROTEIN STRUCTURE

Circular dichroism (CD)

Circular dichroism services provide spectroscopic analysis for protein secondary structure assessment (alpha helix/beta sheets).

Differential scanning calorimetry (DSC)

Differential scanning calorimetry service provides to you protein thermodynamic parameters measurements (e.g. melting point determination) to screen protein stability.

EQUIPMENT 

Jasco Europe CD/LD spectropolarimeter, GE Healthcare Life Science differential scanning calorimetry, Thermo Fischer Sientific & Bruker Daltonics GmbH UHPLC – MALDI TOF/TOF, Agilent Technologies HPLC-DAD, Agilent Technologies HPLC-RID, Agilent Technologies HPLC-FLD, Agilent Technologies LC-(QQQ)-MS/MS

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The Washing and Sterilization Laboratory provides laboratory dishwashing service in professional Smeg washing machines, which have numerous washing programs for different types of glassware and different types of soiling. After washing, the dishes are dried or sterilized in a Pol-Eko Apparatura dry sterilizer.

The same unit also provides autoclaving – sterilization service in the Tuttnauer 66120-1 VEP autoclave, chamber volume 450 l. The autoclave has different programs, i.e. it has the ability to program the pressure, temperature and time period of the autoclave, depending on the type of material to be sterilized.

The resulting waste from the microbiology and cell biology laboratories is sterilized-autoclaved and thus reduced to a harmless level before disposal.