Central laboratory

Cell biology laboratory staff:

Dora Markulin, PhD, Head of laboratory, e-mail: dora.markulin@biocentre.hr 
Mihaela Kordun, Mag.Biol.Mol., Associate, e-mail: mihaela.kordun@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, qPCR).

CELL CULTURE CULTIVATION AND LABORATORY SCALE ASSAYS 

We offer services of human and animal cell cultures handling on a laboratory scale: 

• adherent cell lines and cell lines in suspension 
• primary cultures, co-cultures, 3D cultures 
• cytotoxicity and proliferation assays (MTT) 
• mycoplasma testing 
• cell bank storage 

CELL CULTURE CULTIVATION ON A SEMI-INDUSTRIAL SCALE 

We offer cultivation of cell cultures on a semi-industrial scale: 

• cell cultivation in bioreactors of different volumes: 0.5 L (up to 12 parallel bioreactors); 2 L (2 parallel); or 30 L bioreactor 
• process optimisation 
• production of recombinant proteins, cytokines, growth hormones, hematopoietic growth factor, antibodies, and similar 

FLOW CYTOMETRY 

We offer flow cytometry analysis (3 lasers, BD FACSVerse; 405, 488 and 633 nm): 

• cell viability tests 
• apoptosis / necrosis cell death analysis 
• measurement of proliferation markers 
• cell cycle analysis 
• cell line validation using cell-specific markers 
• detection of fluorescent proteins 
• quantification of transfection 
• FCS data analysis in FlowJo software 

MOLECULAR BIOLOGY 

We offer an array of standard molecular biology tests: 

• DNA, RNA, and protein extraction from tissues, cell suspensions, plants, and similar 
• PCR analysis 
• qPCR analysis 
• ELISA 
• Western blot 
• Lentiviral and adenoviral vector production 
• Stable and transient cell transformation 
• CRISPR/CAS9 genome engineering 

EQUIPMENT

Cytometer 

BD FACSVerse flow cytometer (3 lasers: 405, 488, and 633 nm) 

Bioreactors 

Sartorius BIOSTAT Qplus 12 fold 0,5 L CC 

Sartorius BIOSTAT B Twin 2 L CC 

Sartorius BIOSTAT C Plus 30 L CC 

Other 

Agilent AriaMx Real-Time PCR System 

Applied Biosystems SimplyAmp thermal cycler 

Hidex Chameleon V microplate reader 

Invitrogen Countess 3 Automated Cell Counter 

Sorvall Stratos continuous flow centrifuge 

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,
Olivera Platiša-Cerovski, Mag. Chem., Senior Associate, e-mail: olivera.platisa-cerovski@biocentre.hr
Diana Nejašmić, M. Eng., Senior 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.

Analytical services for the pharmaceutical, chemical and cosmetic industries

Method development and validation of analytical methods using gas and liquid chromatography as well as providing analysis for:

• Quantification and identification of active pharmaceutical ingredients (APIs), preservatives, antioxidant and impurities in raw materials, intermediates, and finished products
• Quantification and identification of excipients: polyethylene glycols (PEGs), alcohols and higher alcohols, as well as fatty excipients for ointments and gels (e.g. lanolin, white soft paraffin, yellow paraffin, vegetable oils such as castor oil and corn oil)
• Analytical methods for cleaning validation with the purpose of detecting and quantifying residues of active ingredients or other components on equipment surfaces (e.g. inox, enamel, polypropylene glycol, ethylene glycol, etc.)
• Quantification of active components from plant extracts used for oral solutions and syrups
• Quantification and identification of unknown volatile impurities using gas chromatography with headspace or liquid sample injection coupled with mass spectrometry
• Quantification and identification of impurities from contact packaging (rubber materials) as potential leachable using gas and/or liquid chromatography coupled with mass spectrometry
• Titrimetric determination of acid number, hydroxyl value, iodine value, peroxide value, saponification value, ester value in raw materials for pharmaceutical and cosmetic products
• Quantitative analysis of alcohol content by volume or alcohol content by mass as well as impurities in raw materials and finished products of disinfectants
• R&D analytical support during product development and formulation by analysing bulk drug materials, intermediates, drug products and impurities, as well as during out-of-specification investigations and stability studies

Analytical services for plant materials analyses

• Method development and analysis of bioactive components such as natural insecticides, water soluble vitamins, fatty acids, carbohydrates, polyphenols, carotenoids, condensed tannins, total and organic acids, chlorophyll and antioxidative activity by DPPH method
• Aroma profile analysis of plant species and essential oils by gas chromatography coupled with mass spectrometry
• Analyses for different plant parts such as leaves, pulp / fruits, pits or seeds by different extraction methods (e.g. in olives, prickly pear, tangerines, lettuce, beans or strawberries

Analytical services for food and beverages analyses

• Craft beer analysis, including pH determination, colour, bitterness, alcohol by volume and alcohol by mass
• Liquor and spirits analysis, including alcohol by volume, alcohol by mass and aroma profiling by gas chromatography coupled with mass spectrometry
• Analysis of acrylamide in a variety of fried food samples such as chips, coffee, waffles and cookies by liquid chromatography coupled with mass spectrometry
• Analysis of biogenic amines (histamine, cadaverine, putrescine, spermine, spermidine, tryptamine, tyramine, 2-phenyl-ethylamine) in fish and fish products by liquid chromatography with fluorescence detection
• Analysis of spices, seasonings and flavourings: identification of unknown volatile impurities and quantification of known active components by gas chromatography with flame ionisation detector (GC-FID) or coupled with mass spectrometry (GC‑MS/MS)
• Analysis of protein powders: condensed tannins and trypsin inhibitor activity (TIA)
• Aroma profiling of fresh meat (e.g. lamb, pork, beef), meat products (e.g. sausages), and delicatessen (Croatian traditional meat products, e.g. buđola, šokol, etc.) by gas chromatography coupled with mass spectrometry (HS-SPME-GC/MS/MS)
• Analysis of fermented beverages (e.g. kombucha fermented tea): assay of sugars, alcohols, phenols and vitamins
  

Analytical services for biological samples analysis

• Analysis of volatile and non-volatile metabolites as organic acids, sugars or higher alcohols from cell lysates or biomass
• Method development and analysis of metabolic profiles by identifying compounds formed by different microorganism metabolic pathways
• Analysis of known toxic metabolites in biological fluids (e.g. rat urine) by liquid chromatography coupled with mass spectrometry
• Fatty acid profile: analysis of 37 individual fatty acids in human serum by gas chromatography coupled with mass spectrometry

Analytical services as a part of clinical research

• Quantification of residual dimethyl sulfoxide in cryopreserved tissues after rinsing and thawing of the tissue by gas chromatography coupled with mass spectrometry
• Currently in process: method development for quantification of residual antibiotics after rinsing stored tissues by liquid chromatography coupled with mass spectrometry

INSTRUMENTS

Agilent Technologies HPLC-DAD-UV/VIS

Agilent Technologies HPLC-RID

Agilent Technologies HPLC-FLD

Agilent Technologies LC-MS/MS

Agilent Technologies GC-FID

Thermo Scientific GC-MS/MS

Cary 100 UV/VIS spectrophotometer

Cary Eclipse fluorescence spectrometer

Bioanalytical laboratory II (proteomics) staff:

Vanja Kelava, Mag. Appl. Chem., Senior Associate, e-mail: vanja.kelava@biocentre.hr
Rea Bertoša, M.Eng., Associate, e-mail: rea.bertosa@biocentre.hr
Željka Stanečić, M. Eng., Junior Associate, e-mail: zeljka.stanecic@biocentre.hr

The Bioanalytical laboratory II (proteomics) services are focused on protein characterization and the analysis of complex protein mixtures using both targeted and untargeted approaches. Additionally, our laboratory provides higher-order protein structures analysis. We consistently organize practical workshops and offer personalized trainings for our clients.

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, typically involves in-solution or in-gel protein digestion using a suitable proteolytic enzyme. The resulting peptides are then separated using nano-LC coupled to a TripleTOF 6600+ mass spectrometer.

We employ two approaches for generating proteomic data: data-dependent acquisition (DDA) and data-independent acquisition (DIA). The DDA approach is primarily focused on cataloguing proteins from the sample and for building of spectral libraries. On the other hand, the DIA approach is specifically designed for determining 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

Sciex TripleTOF® 6600+ System; 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.