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.

BIOCentre’s Microbiology lab specializes in two types of services – protein production and training. Microbiology lab performs expression (µg to mg scale), purification and process development of recombinant proteins, biopharmaceuticals and biosimilars.

Microbiology lab offers hands-on-training courses on separate and parallel bioreactor systems (BIOSTAT Qplus 12 x 0.5L, B 2 x 2L, Cplus 30L), various cloning and general microbiology lab techniques. We also provide bacteria and yeast cell banking.

BACTERIAL CELL TRANSFORMATION
Transforming competent E.coli cells by heat shock with an expression vector carrying the target DNA, encoding the protein of interest. Selection of the transformed E.coli on selective agar plates and preparation of glycerol stocks for further use.

PLASMID PROPAGATION AND PURIFICATION
The competent E.coli cells are transformed with the desired template DNA provided by customer. Extracted DNA prep (mini, midi or maxi) quality control includes separation on agarose gel, A260/280 ratio, and quantification.

PROTEIN EXPRESSION IN E. COLI OR YEAST
Recombinant protein expression involves cultivation of transformed cells in desired conditions and scale: 1L shaker flask, 0,5L fermenter (up to 12 in parallel), 2L fermenter (up to 2 in parallel), or 30L fermenter. Preceding the large scale production, we offer an analytical (small scale of 100mL) expression of recombinant protein. To maximize the content of soluble recombinant proteins we optimize the expression conditions including choice of medium, carbon source, temperature, pH, aeration, inducer concentrations, and length of induction.

PROTEIN DOWNSTREAM PROCESSING is done in our DOWNSTREAM PROCESSING LAB
Downstream processes include cell lysis (high pressure and ultrasonic homogenizers), protein separation and purification. Desalting, buffer exchange and sample concentration can also be done.

PROTEIN QUALITY CONTROL is done in our BIOANALYTICAL PROTEOMICS LAB
SDS-PAGE and western blot analysis of the recombinant protein samples from analytical or production scale. NanoDrop quantitation, LC-MS/MS peptide mapping and protein identification.

MOLECULAR BIOLOGY
DNA, RNA and protein extractions from microbial cells. Protein SDS-PAGE and western blotting. PCR detection and identification of microorganisms in different samples e.g. probiotics, beer spoilers, and other food and beverages.

EQUIPMENT AND INSTRUMENTATION
Sartorius BIOSTAT Qplus 12 fold 0.5L MO, BIOSTAT B Twin 2L MO, BIOSTAT Cplus 30L MO; high pressure homogenizer Panda Plus, Geo Niro Savi; ultrasonic homogenizer Bandelin, Sonoplus; NanoDrop One, Thermo Scientific; Sorval Stratos continuous flow centrifuge.

Flow cytometry (BD FacsVerse, FACSuite, 3 lasers, 8 fluorochromes per sample)

  • Immunophenotyping
  • Hematologic analysis (Leukemia and lymphoma phenotyping, leukocyte surface antigens, identification of prognostically important subgroups, Hematopoietic progenitor cell enumeration), systemic mastocytosis, Reticulocyte enumeration, Autoimmune and alloimmune disorders (anti-platelet antibodies IgG, IgM, anti-neutrophil antibodies IgG, immune complexes), feto-maternal hemorrhage quantification, erythrocyte surface antigens, assessment of leukocyte contamination of blood products (Forward and side scatter, leukocyte surface antigens), paroxysmal nocturnal hemoglobinuria
  • Immunodeficiency studies, transplantation rejection
  • Specific human or animal cell surface or intra-cellular markers
  • Cell line validation
  • Apoptosis/viability monitoring/tests
  • Visualization of fluorescent proteins
  • Measurement of proliferation markers
  • Chemotaxis
  • Actin polymerization
  • DNA content analysis
  • Caspase activity

Cell culture

  • Human and animal cell lines (adherent or in a suspension)
  • Primary cultures
  • Co-cultures, 3D cultures
  • Generation of Nurse-like cells or similar supportive in vitro cell models
  • Cytotoxicity, cell proliferation tests (MTT, direct contact, etc.)
  • Freezing/thawing and cell banking services (liquid nitrogen)

Molecular biology

  • DNA, RNA and protein extractions from cell tissue, cell suspension, blood, bone marrow, etc.
  • Western blotting
  • PCR (ARMS, cDNA retrotranscription, etc.)
  • ELISA
  • Generation of lentiviral vectors
  • Transient and stable transfections
  • CRISPR/CAS9 genome editing system

Cell Bioreactors
Production of drug products, cytokines, growth hormones, hematopoietic growth factor, antibodies, etc. on large scale (bioreactors BIOSTAT Qplus 12 fold 0.5L CC, BIOSTAT B Twin 2L CC, BIOSTAT Cplus 30L CC)

BIOCentre’s Downstream processing lab provides purification services of small molecules or proteins and training services.

Protein purification encompasses numerous methods for the isolation of a native or recombinant protein from cells or biological material.

HOMOGENIZATION
Isolation of proteins and metabolites through cell disruption by bead method (glass beads), high pressure homogenizing or ultrasonic homogenizing method.

LIQUID CHROMATOGRAPHY AND FRACTION COLLECTION
Method and process development using preparative liquid chromatographic systems with automated fraction collectors. Separation and purification of proteins and small molecules in highly selective column media at high resolution (SEC, IEX, RP and affinity chromatography). Fraction collection in time or volume mode; collection of peaks by peak detection, time windows or a combination of both.

FILTRATION
Tangential flow filtration (TFF) method for separation and purification of small molecules, proteins or cell suspensions. TFF can be used to concentrate sample or for diafiltration (desalting and buffer exchange) ranging in volume from 25 mL to dozens of liters. As well, it can be used to fractionate large from small molecules, for harvesting cell suspensions and clarifying fermentation broths and cell lysates.

PROCESS OPTIMISATION
Flexible instrument setup offers analysis in wide range of analyte amount and is ideal for fast and secure development of scalable chromatographic processes for biopharmaceutical production. Media screening and method optimization is done at small laboratory scale and can be scaled up to pilot production scale. Fine-tuning and robustness testing of the optimized process is done at larger scale with larger columns.

EQUIPMENT AND INSTRUMENTATION
High pressure homogenizer Panda Plus, Geo Niro Savi; ultrasonic homogenizer Bandelin, Sonoplus; GE Healthcare Life Sciences ÄKTA avant 25, ÄKTA Avant 150, and ÄKTA Pilot; AKTA Crossflow and Sartorius SARTOFLOW Advanced.

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.

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.

BIOCentre’s Bioanalytical Laboratory I (chemistry) provides services of developing, optimization, transfer and validation of various analytical methods (UV/VIS and fluorescence spectroscopy, liquid chromatography UHPLC-UV/VIS, HPLC-RID, HPLC-FLD, LC-(QQQ)-MS/MS and gas chromatography GC-(Ion-trap)-MS/MS).

Using HPLC equipped with UV/VIS (DAD) detector, we can develop quantitative methods for the measurement of a wide range of compounds in the field of biopharmaceuticals, food technology, agrochemicals, cosmetics or environmental science. Some examples include biologically active ingredients, degradation products (impurities), preservatives, antioxidants, additives, water-soluble vitamins, organic acids, biomolecules (hormones, proteins, peptides, etc.).

Assay determination of sugars, alcohols and complex carbohydrates can be provided using HPLC – REFRACTIVE INDEX detector.

Compounds with natural fluorescence (for example, polycyclic aromatic hydrocarbons) as well as fluorescent compounds produced by pre-column derivatization (bioactive peptides, traces of drugs, etc.) can be analyzed by HPLC – FLUORESCENCE detector.

Aroma profiling of food, drinks, beverages, flavor and fragrance can be done using manual Head-Space–Solid Phase Micro Extraction fiber and GC-MS/MS instrument.

In addition, GC-MS/MS qualitative (semi quantitative) and / or quantitative analysis of numerous compounds can be done using automatic split/splitless injection for example volatile organic compounds, trace levels of contaminants, esters, fatty acids, alcohols, aldehydes, terpenes, contaminants in food, essential oils (lavender, lemon oil), perfumes, menthol.

GC-MS/MS analysis can be used for metabolite identification and profiling, fatty acid profiling in microbes, detection of metabolites in serum, as well detection of volatile lipophilic compounds in plant species.

For targeted analysis, we can developed LC-MS/MS methods for quantification of trace organic compounds in complex matrices: food safety studies, environmental studies, drug discovery, toxicology, forensics and bioanalysis.

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.