33 research outputs found
HPLC enrichment/isolation of proteins for post-translational modification studies from complex mixtures
A pancreas tumor miatt végzett pancreatico-duodenectomiák korai eredményei
ABSZTRAKT
Bevezetés:
Napjaink egyik legrosszabb prognózisú daganata a pancreascarcinoma. Az évente felfedezett új esetek száma és az 5 éves halálozás gyakorlatilag megegyezik.
Mai tudásunk szerint a hasnyálmirigy daganat egyetlen kuratív kezelése a radikális (R0) resectio. A pancreasfejben lévő daganat sebészi terápiája a pancreatico-duodenectomia.
Munkámban a pancreatico-duodenectomia korai eredményeit elemzem különös tekintettel a rekonstrukciós módszerekre.
Betegek és módszerek:
A DEOEC Sebészeti intézetben 2000. szeptember és 2011. december között 54 betegnél végeztek pancreatico-duodenectomiát hasnyálmirigy fej daganat miatt. A betegek közül 25 férfi (46,2%) és 29 nő (53,8%) volt. Átlagéletkoruk 52,2±12,3 év. A pancreatico-duodenectomia során 27 betegnél pancreatico-gastrostomiával (PG), 27 betegnél pancreatico-jejunostomiával (PJ) történt a rekonstrukció.
Eredmények:
A pancreatico-duodenectomia műtét során intraoperatív halálozás nem volt. Az 54 esetből 53 alkalommal (98,2%) zavartalanul folyt a műtét. Egy esetben (1,8%) a műtét során intraoperatív komplikáció miatt splenectomiát végeztek.
A 27 pancreatico-jejunostomiával rekonstruált beteg közül 18 esetben (66,7%) zavartalanul telt a posztoperatív szakasz. 9 esetben (33,3%) volt posztoperatív szövődmény. A 27 pancreatico-gastrostomiás betegből 19 esetben (70,4%) zavartalan volt a posztoperatív időszak. 8 esetben (29,6%) fordult elő posztoperatív szövődmény.
A posztoperatív szövődményeket tekintve a szignifikáns különbséget nem találtam a két eljárás között. (Khí-négyzet próba= 0,086 a P=0,769)
A posztoperatív szakaszban 3 beteg halt meg, varratelégtelenség talaján kialakult sepsis tüneteiben egy, vagy többszörös reoperáció után. A PG csoportban egy, a PJ csoportban két beteg halt meg. Az összmortalitás az 54 betegre vonatkoztatva 5,5% volt.
A posztoperatív mortalitásban szignifikáns különbség nem volt a két eljárás között, ennek valószínűleg az alacsony esetszám is lehet az oka. (Khí-négyzet próba=0,353 P= 0,5525)
Következtetés:
Az irodalom és az eredmények alapján nincs szignifikáns különbség a posztoperatív mortalitást tekintve a két rekonstrukciós eljárás között. A posztoperatív morbiditást tekintve sem sikerült szignifikáns különbséget találni az eljárások között, ezért minden esetben individuálisan kell kiválasztani a rekonstrukciós műtéti eljárást. Ebben a döntésben többek között a hasnyálmirigy állománya, annak zsírossága, a hasnyálmirigy kivezető csövének átmérője és a sebész jártassága fő faktorok.egységes, osztatlanáltalános orvostudományimagyarnappaliP.J
Systematic comparison of a new generation of columns packed with sub-2 μm superficially porous particles
The aim of this study was to evaluate the possibilities/limitations of recent RP-LC columns packed with 1.6 m superficially porous particles (Waters Cortecs) and to compare its potential to other existing sub-2 mcore–shell packings. The kinetic performance of Kinetex 1.3 m, Kinetex 1.7 mand Cortecs 1.6 mstationary phases was assessed. It was found that the Kinetex 1.3 mphase outperforms its counterparts for ultra-fast separations.Conversely, the Cortecs 1.6 m packing seemed to be the best stationary phase for assays with longer analysis time in isocratic and gradient modes, considering small molecules and peptides as test probes. This exceptional behaviour was attributed to its favourable permeability and somewhat higher mechanical stability ( Pmax of 1200 bar). The loading capacity of these three columns was also investigated with basic and neutral drugs analysed under acidic conditions. It appears that the loading capacities of Cortecs 1.6 m and Kinetex 1.7 m were very close, while it was reduced by 2–7-fold on the Kinetex 1.3 m packing. However, this observation is dependent on the nature of the compound and certainly also on mobile phase conditions
New developments and possibilities of wide-pore superficially porous particle technology applied for the liquid chromatographic analysis of therapeutic proteins
This review paper discusses the success of columns packed with superficially porous particles (SPP) in liquid chromatography for the analysis of peptides and proteins. First, it summarizes the history of SPP, including the development of different SPP generations from particles of 50 um to sub-2 um. It also critically discusses the improved kinetic performance of SPP particles in comparison to fully porous particles. The current trends and applications of columns packed with SPPs for the analysis of peptides and proteins (including mAbs and ADC at the intact and sub-unit levels) are shown, as well. Finally, some of the potential perspectives for this technology are also described, including the radially oriented mesopores or the applicability of the technology for chiral separations
Superheated reversed phase chromatography with ultrashort columns for the analysis of therapeutic proteins
Analysis of recombinant monoclonal antibodies in hydrophilicinteraction chromatography: A generic method developmentapproach
Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separa-tion and analysis of small polar compounds. A recently introduced widepore stationary phase expandedHILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present somegeneric HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonalantibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessingvarious isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in thisstudy. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduc-tion. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation ofpolar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible withother chromatographic modes. Three samples were selected and chromatographic conditions were fur-ther optimized to maximize resolution. A commercial software was used to build up retention models.Experimental and predicted chromatograms showed good agreement and the average error of retentiontime prediction was less than 2%. Recovery of various species and sample stability under the appliedconditions were also discusse
Evaluation of strategies for overcoming trifluoroacetic acid ionization suppression resulted in single-column intact level, middle-up, and bottom-up reversed-phase LC-MS analyses of antibody biopharmaceuticals
Utility of a high coverage phenyl-bonding and wide-pore superficially porous particle for the analysis of monoclonal antibodies and relatedproducts
A wide-pore silica-based superficially porous material with a high coverage phenyl bonding was evalu-ated for the analysis of monoclonal antibodies and antibody-drug conjugates. This new material is basedon 2.7 um particles having a shell thickness of 0.40 um and average pore size of approximately 450 Å.Various important features of this reversed phase column technology were explored, including kinetic performance for large biomolecules (i.e. speed of analysis, efficiency and peak capacity), recovery ofproteins, selectivity for resolving modifications, and the possibility to reduce the amount of trifluoroacetic acid in the mobile phase. A systematic comparison was also performed with other existing modernwide-pore phases possessing differences in structure/morphology and chemistry.If all these figures of merit are considered, it is clear that this phenyl bonded wide-pore superficially porous stationary phase is one of the most promising materials to have been developed in recent years.Indeed, it offers kinetic performance comparable to the most efficient wide-pore SPP column on themarket. In terms of protein recovery, this new phase was found to be superior to silica-based and silica-hybrid C4 bonded materials, particularly with separations performed at sub-80◦C temperature. Under such conditions, it in fact shows recoveries that are quite similar to a divinyl benzene (DVB) polymer-based material. More importantly, due to its unique, high coverage phenyl bonding, it offers additionalsteric effects and potentially even t-t interactions that yield advantageous selectivity for mAb sub-unit peaks and ADC species as compared to commonly used C4 or C18 bonded phases. Last but notleast, mobile phases consisting of only 0.02–0.05% trifluoroacetic acid can be successfully used with thiscolumn, without significant loss in recovery and peak capacity
Protocols for the analytical characterization of therapeutic monoclonal antibodies. III – Denaturing chromatographic techniques hyphenated to mass spectrometry
The full analytical characterization of therapeutic monoclonal antibodies (mAbs) requires a large variety of complementary information that can be obtained by chromatographic methods. A series of protocols papers has been proposed to cover the chromatographic techniques and the enzymatic and chemical sample preparation procedures generally applied for the analytical characterization of therapeutic mAbs. The present protocol paper focuses on denaturing chromatographic techniques hyphenated to mass spectrometry, namely reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC), to assess the subtle mAbs structural heterogeneity resulting from glycosylation patterns and post-transcriptional modifications (PTMs). In this paper, some generic protocols are provided, using a wide range of therapeutic mAbs approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA), to illustrate the possibilities offered by the last generation of RPLC and HILIC columns when performing LC-MS analysis at the middle-up level
Impact of organic modifier and temperature on protein denaturationin hydrophobic interaction chromatography
The goal of this study was to better understand the chromatographic conditions in which monoclonalantibodies (mAbs) of broad hydrophobicity scale and a cysteine conjugated antibody-drug conjugate(ADCs), namely brentuximab-vedotin, could denaturate. For this purpose, some experiments were car-ried out in HIC conditions using various organic modifier in natures and proportions, different mobilephase temperatures and also different pHs. Indeed, improper analytical conditions in hydrophobic inter-action chromatography (HIC) may create reversed-phase (RP) like harsh conditions and therefore proteindenaturation. In terms of organic solvents, acetonitrile (ACN) and isopropanol (IPA) were tested with pro-portions ranging from 0 to 40%. It appeared that IPA was a less denaturating solvent than ACN, but shouldbe used in a reasonable range (10–15%). Temperature should also be kept reasonable (below 40◦C), tolimit denaturation under HIC conditions. However, the combined increase of temperature and organiccontent induced denaturation of protein biopharmaceuticals in all cases. Indeed, above 30–40◦C and10–15% organic modifier in mobile phase B, heavy chain (HC) and light chain (LC) fragments dissociated.Mobile phase pH was also particularly critical and denaturation was significant even under moderatelyacidic conditions (pH of 5.4).Today, HIC is widely used for measuring drug-to-antibody ratio (DAR) of ADCs, which is a critical qualityattribute of such samples. Here, we demonstrated that the estimation of average DAR can be dependenton the amount of organic modifier in the mobile phase under HIC conditions, due to the better recoveryof the most hydrophobic proteins in presence of organic solvent (IPA). So, special care should be takenwhen measuring the average DAR of ADCs in HIC