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.
Microbiology laboratory staff:
Maja Marinović, PhD, Senior Associate, e-mail: maja.marinovic@biocentre.hr
Matea Zelenika, M.Eng., Associate, e-mail: matea.zelenika@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.
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 0.5 L MO, BIOSTAT B Twin 2 L MO, BIOSTAT Cplus 30 L MO (Sartorius)
Homogenizers
Panda Plus high pressure homogenizer (Gea Niro Soavi); Bandelin ultrasonic homogenizer (Sonoplus).
Other
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:
Dora Markulin, PhD, Senior Associate at Cell biology laboratory, e-mail: dora.markulin@biocentre.hr
Marta Žužić, PhD, Senior Associate at Cell biology laboratory, e-mail: marta.zuzic@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…).
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: 0.5 L (up to 12 parallel bioreactors); 2 L (2 parallel); or 30 L 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
Cytometry
FACSVerse flow cytometer – 3 lasers, 8 colours (Becton-Dickinson); Vi-Cell XR cell counter (Beckman Coulter).
Bioreactors
BIOSTAT Q Plus 12 x 0.5 L CC; BIOSTAT B Twin 2 L CC; BIOSTAT C Plus 30 L CC (Sartorius).
Other
Chameleon V microplate reader (Hidex); SimplyAmp thermal cycler (Applied Biosystems); Continuous flow centrifuge (Sorvall Stratos).
Downstream processing laboratory staff:
Marina Markeš, M.Sc., Head of laboratory, e-mail: marina.markes@biocentre.hr
Marija Ivić, M. Sc., Associate, e-mail: marija.ivic@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.
DOWNSTREAM PROCESSING
The majority of our services revolve around biological material downstream processing – the isolation of small molecules, biomolecules, native and recombinant proteins from various samples. Our services include process optimisation on a laboratory scale, and transfer to a pilot production scale.
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.
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,
Jasna Jakovljević, Mag. Chem., Senior Associate, e-mail: jasna.jakovljevic@biocentre.hr,
Diana Nejašmić, M. Eng., Associate, e-mail: diana.nejasmic@biocentre.hr
Our team of experts develops new analytical methods from scratch, improves and expands existing methods, identifies shortcomings, and troubleshoots and fixes problematic analytical methods. The methods we have developed have been successfully implemented in a large number of projects and fields ranging from the pharmaceutical and food industries to biomedicine and biotechnology.
Our clients are academics and industry experts who, due to a lack of time, equipment or specific knowledge contract our laboratory to solve complex analytical problems. 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.
EQUIPMENT BASED SERVICES
HPLC-DAD-UV/VIS (Agilent Technologies)
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).
HPLC-RID (Agilent Technologies)
Determination of sugars and carbohydrates, alcohols and higher alcohols in food and biological samples.
Molecular size analysis using size-exclusion chromatography (SEC).
HPLC-FLD (Agilent Technologies)
Analyses of naturally fluorescent compounds (e.g. polycyclic aromatic hydrocarbons) and non-fluorescent compounds using pre-column fluorescence derivatisation (e.g. biogenic amines, amino acid content).
LC-MS/MS (Agilent Technologies)
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).
GC-FID (Agilent Technologies)
Quantitative analysis of e.g. alcohol content in different samples (disinfectants, alcoholic beverages, etc.), fatty acids in food supplements, oil content in pharmaceutical emulsions.
GC-MS/MS (Thermo Scientific)
Determination of aroma profiles of food, beverages, spices and fragrances.
Determination of the composition of essential oils (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 through the metabolism of microorganisms, metabolites in biological fluids, fatty acids profiles in serum.
Cary 100 UV/VIS spectrophotometer (Agilent Technologies)
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, PhD, Head of laboratory, e-mail: ana.butorac@biocentre.hr
Rea Bertoša, M.Eng., Associate, e-mail: rea.bertosa@biocentre.hr
Vanja Kelava, Mag. Appl. Chem., Senior Associate, e-mail: vanja.kelava@biocentre.hr
Željka Stanečić, M. Eng., Junior Associate, e-mail: zeljka.stanecic@biocentre.hr
PEPTIDE MASS FINGERPRINTING OR PEPTIDE MAPPING
Involves protein molecule cleavage using a specific proteolytic enzyme and analysing the resulting peptide mixture using mass spectrometry (MS). Analyses can be subjected to direct MS measuring or on-line connected to liquid chromatography (LC-MS). Peptide mass fingerprinting can be extended to a protein database search for sequence confirmation. Additionally, peptide mass fingerprinting 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. Our gel electrophoresis service offers molecular weight measuring and characterisation of 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
We offer a wide range of LC techniques for protein/peptide analysis: size exclusion (SEC), reversed-phase (RP) and ion exchange chromatography (IEX). Additionally, after LC separation we offer MS analysis.
PROTEIN SEQUENCING
Involves MALDI top down sequencing (MALDI-TDS) and de novo sequencing. MALDI-TDS provides protein identification through a database search and simultaneous assignment of the N- and C-termini of up to 70 residues from either terminus. In de novo sequencing for the identification of unknown proteins, the amino acid sequence is deduced without prior knowledge of the DNA or protein sequence. We offer a de novo sequencing service that reveals structural information using laser induced fragmentation within a mass spectrometer (LIFT).
AMINO ACID ANALYSIS
Provides information on the amino acid composition of proteins, peptides and other biological samples containing primary or secondary amino groups within a molecular structure. Amino acid analysis allows quantitation of free amino acids, as well as quantitation of amino acids released from macromolecules.
ELLMAN’S TEST
Ellman’s test is used to determine the concentration of free thiol groups in proteins by spectrophotometric measurement at 412 nm. Ellman’s reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) reacts with a free thiol group to give a disulfide and TNB. The target of DTNB in this reaction is to conjugate the base of the free thiol group, the resulting TNB is colored species in the reaction. The concentration of free thiol groups in the sample is determined using a standard curve containing known concentrations of compounds containing thiol groups, such as cysteine.
ISOELECTRIC FOCUSING OF PROTEINS
Isoelectric focusing enables the separation of proteins in a gel based on their isoelectric point. Isoelectric focusing is performed on polyacrylamide gels with a pH gradient. Protein bands obtained by isoelectric focusing can be further analyzed with a MALDI-TOF/TOF instrument for the purpose of protein identification.
PROTEOMICS ANALYSIS
Our liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) services provide targeted and non-targeted proteome analysis.
Our non-targeted proteomics or “shotgun” proteomics service generally involves in-solution protein digestion using a suitable proteolytic enzyme. The resulting peptides are separated using nano-LC and peptide fractions are collected. The separated peptides are analysed using MALDI-MS/MS and identified using a protein database search. Non-targeted proteomics is focused on the cataloguing of proteins (from cells or tissues) or on the determination of changes in protein expression between samples.
Our targeted proteomics service targets specific peptides in a complex mixture and determines their presence. This analysis usually utilises a triple quadrupole mass spectrometer (QQQ). Targeted proteomics is used for verification of knockdown targets, for quantification of a peptide/protein in a complex mixture, and for monitoring a specific protein across samples or during a time-course experiment.
HIGHER ORDER PROTEIN STRUCTURE
Circular dichroism (CD)
Spectroscopic analysis for protein secondary structure assessment (alpha helix/beta sheets).
Differential scanning calorimetry (DSC)
Provides protein thermodynamic parameters measurements (e.g. melting point determination) to screen protein stability.
EQUIPMENT
Mass spectrometry
UHPLC-MALDI TOF/TOF (Thermo Fisher Scientific & Bruker Daltonics); LC-(QQQ)-MS/MS (Agilent Technologies).
Liquid chromatography
HPLC-DAD (Agilent Technologies); HPLC-RID (Agilent Technologies); HPLC-FLD (Agilent Technologies).
Other
CD/LD spectropolarimeter (Jasco Europe); Differential scanning calorimeter (GE Healthcare Life Science).
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.