Bioprocessing and protein glycosylation analysis
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| Anzahl der Teile | 163 | |
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| Lizenz | CC-Namensnennung - keine Bearbeitung 4.0 International: Sie dürfen das Werk in unveränderter Form zu jedem legalen Zweck nutzen, vervielfältigen, verbreiten und öffentlich zugänglich machen, sofern Sie den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen. | |
| Identifikatoren | 10.5446/50400 (DOI) | |
| Herausgeber | 05jdrrw50 (ROR) | |
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00:00
Alterung
00:21
SchönenVisualisierungChemisches Experiment
00:29
ProteineArzneimittelforschungKohlenhydratchemieRauschgiftChemische StrukturMolekülChemisches Experiment
00:38
RauschgiftBesprechung/Interview
00:47
RauschgiftKohlenhydratchemieErythrozytOberflächenchemieErythropoietinChemische StrukturProteineMeeresspiegelBesprechung/Interview
01:09
AntikörperMolekülEukaryontische ZelleMeeresspiegelTumorAbschreckenBindungsenergieMonoklonaler AntikörperBesprechung/Interview
01:21
BindungsenergieTumorAbschreckenZeichnung
01:28
TumorStammzelleRauschgiftKohlenhydratchemieAntikörperBesprechung/Interview
01:46
TumorStammzelleKohlenhydratchemieAntikörperStoffwechselwegRauschgiftBesprechung/Interview
02:01
RauschgiftBesprechung/Interview
02:07
RauschgiftChemisches Experiment
02:16
RauschgiftChemisches Experiment
02:22
ProteineEukaryontische ZelleSüßkraftChemisches Experiment
02:31
Eukaryontische ZelleProteineChemisches Experiment
02:42
ChromstahlProteineEukaryontische ZelleChemisches ExperimentFlussdiagramm
02:50
ProteineEukaryontische ZelleChemisches Experiment
02:57
ProteineAlkoholische LösungEukaryontische ZelleVollernterChemisches Experiment
03:07
ProteineAlkoholische LösungEukaryontische ZelleChemisches Experiment
03:15
ChromatographieChemischer ProzessSystemische Therapie <Pharmakologie>FiltrationAdvanced glycosylation end productsGangart <Erzlagerstätte>Elektronische ZigaretteKonzentratChemisches Experiment
03:21
FiltrationKonzentratGangart <Erzlagerstätte>
03:27
Chemischer ProzessElektronische ZigaretteSystemische Therapie <Pharmakologie>FiltrationChromatographieAdvanced glycosylation end productsGangart <Erzlagerstätte>Fülle <Speise>Chemisches Experiment
03:38
Gangart <Erzlagerstätte>IonenaustauschChemisches Experiment
03:47
Chemischer ProzessWasserfall
03:54
GradingChemisches Experiment
04:01
Chemischer ProzessReglersubstanzProteineKohlenhydratchemieChemisches Experiment
04:11
Chemisches Experiment
04:16
Chemischer ProzessProteineKohlenhydratchemieSystemische Therapie <Pharmakologie>Chemisches Experiment
04:26
GalactoseKohlenhydratchemieChemisches ExperimentComputeranimationBesprechung/Interview
04:33
MannaneKomplikationMonosaccharideChemisches Experiment
04:41
Chemische StrukturKettenlänge <Makromolekül>MonosaccharideSymptomatologieKohlenhydratchemieTechnische ZeichnungVorlesung/Konferenz
04:49
ProteineKohlenhydratchemieEnzymChemisches Experiment
04:58
KohlenhydratchemieMolekülProteineBesprechung/Interview
05:07
ChromatographieMischenFluoreszenzfarbstoffChemisches Experiment
05:14
PeriodateBiologisches MaterialChemische StrukturIsotopenmarkierungKlinische ChemieCarbonatplattformChemisches Experiment
05:22
Biologisches MaterialTechnische Zeichnung
05:28
KapillarelektrophoreseChemisches Experiment
05:35
ChromatographieWasserMassenspektrometerHPLCChemisches Experiment
05:44
Chemisches Experiment
05:50
Biologisches MaterialMischenPhasengleichgewichtCHARGE-AssoziationAktives ZentrumChemisches Experiment
05:58
FluoreszenzfarbstoffChemisches Experiment
06:05
EmissionsspektrumMündungCHARGE-AssoziationChemischer ProzessBiologisches MaterialChemisches ExperimentComputeranimation
06:12
CHARGE-AssoziationMassenspektrometerChemisches ExperimentBesprechung/Interview
06:22
MassenspektrometerLab on a ChipChemische StrukturChemisches Experiment
06:30
Lab on a ChipKartoffelchipsDiagrammFlussdiagrammChemisches Experiment
06:38
Biologisches MaterialInterkristalline KorrosionKartoffelchipsChemischer Prozess
06:47
Chemischer ProzessChemisches ExperimentBesprechung/Interview
06:54
GlykolsäureRauschgiftOrganische ChemieGlykobiologieSetzen <Verfahrenstechnik>Eukaryontische ZelleChemische StrukturChemisches Experiment
07:03
GlykobiologieRauschgiftChemisches Experiment
07:16
Computeranimation
Transkript: English(automatisch erzeugt)
00:11
Hello everybody, I'm Professor Pauline Rudd, I'm standing here in the National Institute for Bioprocessing Research and Training in Ireland.
00:22
So in this building we have a very fine suite of bioprocessing equipment and we also have very extensive analytical facilities to do research and to train people in the modern technologies. Now the new drugs that are being made are really quite different from the drugs
00:40
that have been used until now. Most of the earlier drugs were small molecules, the ones that are being made today are large protein structures which have sugars attached to the surface and the function of these sugars is very important. For example if you have a drug like erythropoietin which is used to treat anaemia, if the
01:00
sugars are not correct, they're not the right structures, the drug will be cleared so quickly from the patient it won't have time to be effective in raising the red blood cell level. A huge class of molecules that is now being made are known as monoclonal antibodies. Antibodies are Y-shaped molecules and in the tips of the Y there are many loops
01:23
and these loops are designed to bind to specific targets, for example on tumour cells. And we now know that when these drugs hit their target on tumour cells, the sugars that are attached to the stem of the Y are very important in deciding what will happen
01:41
next. And normally when you're treating tumours you want the tumour cell to be killed, so in other words you want the stem of the antibody to initiate an event which will kill the tumour cell. And we also found out in the last 10 years or so that the sugars actually determine which
02:00
kind of pathway is opened up in order to kill the tumour cell. So we have technologies here where we can understand how these drugs are made, we can teach people how to make them and we can then take the product and we can do very careful analysis to make sure that these drugs are going to work properly in the patient.
02:28
Hello my name is Jared, welcome to Nybrids Power Plant Facility. It consists of an upstream suite, a downstream suite and a fill-finish suite. Upstream is mainly concerned with replicating the cells and producing our protein of interest.
02:41
We produce them in bioreactors such as the one beside me. We've got a 150 litre stainless steel production bioreactor. Here in Nybrids we use Chinese hamster ovary cells, or CHO cells, such as in the flask beside me. These are the cells that produce our protein of interest. After we produce our product, we then need to remove the cell waste.
03:02
We do this in microfiltration or centrifugation, otherwise known as the harvest. After we've harvested the cells from the liquid, we then can transfer the solution down to downstream, which is concerned with purifying out the protein of interest. Here we are in Nybrids downstream suite. The downstream processing involves chromatography steps and filtration steps.
03:25
The filtration step can actually achieve a concentration from our 100 litre volume down to 5 litres of product at the end of the downstream process. Here you can see our old filtration system behind me. Moving on, our capture chromatography and our second chromatography step, which is
03:42
anion exchange. These chromatography steps will remove any contaminants and impurities that may be present. Welcome to Nybrids fill-finish suite. This is where the final product is aseptically filled into a container ready for the patient. Here in Nybrids, we use RAV technology to aseptically fill the product in a grade A environment.
04:04
It utilizes laminar flow to protect the vials. The filling process itself consists of vials moving along the beam, being filled, stoppered, capped, and finally crimped. Once this process has taken place, they're now ready for quality controls, such as
04:21
characterizing the sugars of the protein. Hi, my name is Rebecca and I'm a postdoctoral researcher at Nybrids. Glycans consist of single sugar units called monosaccharides, such as galactose and mannose. These monosaccharides are linked together in different ways, which results in complicated
04:42
branched glycan structures. Often we use symbols to represent the monosaccharides of the glycan chain. To start our glycan analysis, we first have to remove the sugars from the protein backbone. To do this, we use a bacterial enzyme. This cleaves between the protein and the sugar backbone here.
05:03
In order to be able to see the glycans, we label them with a fluorescent molecule. This attaches here. Then we can separate the mixture of glycans using liquid chromatography combined with fluorescence detection. The glycan release and labeling can be automated using our novel robotic platform.
05:20
This is important for high-throughput screening so that we can analyze thousands of clinical samples in a short period of time. The released and labeled glycans are then ready for analysis. As glycans are very complex, we often use more than one analytical technique to determine their structures, mainly liquid chromatography, capillary electrophoresis and mass spectrometry.
05:40
For example, this is a Waters UPLC instrument. The glycans are dissolved in a liquid phase, placed in a vial, and then into the autosampler of the instrument. The sample is then injected onto the column, and the mixture of glycans are separated according to size and charge as they interact with the solid phase on the inside of the column.
06:03
As the glycans elute from the column, a signal is recorded by the fluorescence detector. This gives a glycan profile. In mass spectrometry, the sample is transformed from the liquid to the gas phase. During this process, the glycans are ionized, which imparts them with a charge. The mass-to-charge ratio is then determined by the mass analyzer.
06:23
The mass spectrometer can also select an ion of interest and fragment it to gain more structural information. This is very useful, as some glycans have the same mass, but different structures, and we can differentiate them in this way. This is the Agilent HPLC chip cube mass spectrometer. The microfluidic chip is designed for ease of use in a plug-and-play manner.
06:45
The nano column of the chip separates the glycans, and then the glycans are ionized on the integrated nanospray tip. The miniaturization and integration of these processes results in very high sensitivity, which can be useful when there are limited sample amounts.
07:01
Glycomics is the study of all glycan structures of a given cell type or organism. The glycome may in fact be one of the most complex entities in nature. The advanced technology is helping to unlock the structural complexity of the glycome and to help the design and manufacture of safer drugs. Thanks for watching and goodbye.