Publications
2016
U. Cernigoj, U. Vidic, B. Nemec, J. Gaspersic, J. Vidic,N. L. Krajnc, A. Strancar, A. Podgornik. Journal of Chromatography A, 1464 (2016) 72–78
We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and variousaffinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant non linear effect ofligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12 mg/mL exceeding theoretical monolayer adsorption value of 7.8 mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11 nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4 nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacitylinearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher.
2015
L. Hernandez, D. Stewart, L. Zumalacarregui, D. Amaro
Chinese Journal of Chromatography A, 1000-8713 (2015) 642-646
Affinity and ion exchange conventional chromatography have been used to capture erythropoietin (EPO) from mammalian cell culture supernatant. Currently, chromatographic adsorbent perfusion is available, however a limited number of applications have been found in the literature. In this work, three anion exchange chromatographic supports (gel, membrane and monolithic) were evaluated in the capture step of the recombinant erythropoietin purification process. The influences of load and flow rate on each support performance were analyzed. Also the purity of the EPO molecules was determined. A productivity analysis, as a decision tool for larger scale implementation, was done. As a conclusion, the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity. However, the monolithic column admits high operating velocity, showing the highest adsorption capacity and productivity.
2014
P. Leblebici, M. E. Leblebici, F. Ferreira-da-Silva, A. E.Rodrigues, L. S. Pais
Journal of Chromatography B, 962 (2014) 89-93
Monolithic columns have attracted significant attention for the purification of large biomolecules. In the present study, a step gradient elution method was evaluated for the separation of human immunoglobulinG (hIgG) into its subclasses on CIM (convective interaction media) r-protein A (recombinant protein A)monolithic column. hIgG was loaded onto the column and bound protein was eluted with a pH gra-dient. The subclass content of the eluted fractions was analyzed by enzyme-linked immunosorbentassay (ELISA). Results showed that separation of IgG3 from the other three subclasses can be success-fully achieved with high selectivity (100%) and throughput on monolithic media. It was also revealedthat enriched fractions of IgG1 and IgG2 could be obtained from purified hIgG in a 28 min long chro-matographic run. Three fractions with high IgG1 content (89.1%, 94.3% and 88.8%) were recovered. Furthermore, IgG2 was enriched to 64% successfully. A rapid step gradient elution scheme without any additives in buffers was proven to obtain enriched preparations of the two important subclasses with high throughput. The separation time can be reduced even more by increasing the flow rate without anyloss in selectivity, which will be beneficial in industrial scale applications.
M. M. St. Amand, B. A. Ogunnaike, A.S. Robinson
Published online in Wiley Online Library, 2013
One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired posttranslational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2DHPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using
a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered
to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions.
2013
A. Martinčič, M. Cemazar, G. Sersa, V. Kovač, R. Milačič, J. Ščančar
Talanta 116 (2013)141-148
Conjoint liquid chromatography (CLC) on monolithic convective interaction media (CIM) disks coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP-MS) detectors was used for the first time in speciation analysis of Pt in human serum spiked with Pt-based chemotherapeutics. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. Such a set-up allows rapid two-dimensional separation by affinity and ion-exchange (IE) modes to be carried out in a single chromatographic run. By applying isocratic elution with Tris–HCl–NaHCO3 buffer (pH 7.4) in the first minute, followed by gradient elution with 1 mol L-1 NH4Cl (pH 7.4) in the next 9 min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Pt from Pt bound to transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Further elution with acetic acid (AcOH) in the next 3 min allowed separation of Pt associated with IgG. Separated Pt species were quantified by post-column isotope dilution—ICP-MS. Pt recovery on the CLC column was close to 100%. In comparison to commonly applied procedures that involve separation of protein peaks by size-exclusion chromatography (SEC) followed by IE separation of metal-based chemotherapeutic fractions bound to serum proteins, the CLC method developed is much faster and simpler. Its sensitivity (LOQs adequate for quantification of all separated Pt species, lower than 2.4 ng Pt mL-1), good selectivity and method repeatability (RSD±3%) enabled investigation of the kinetics of interaction of Pt-based chemotherapeutics with serum proteins and the distribution of Pt species in spiked human serum. Pt species present in spiked serum were bound preferentially to HSA. The proportion of Pt associated with IgG and Tf was lower than 13%. Cisplatin and especially oxaliplatin react rapidly with serum proteins, while carboplatin much less. The method developed may be reliably applied in preclinical and clinical studies of the kinetics of the interaction and distribution of different metallodrugs with proteins in blood serum.
H. G. Schwelberger, J. Feurle, F. Ahrens
Journal of Neural Transmission 120 (2013) 983-986
Diamine oxidase (DAO) was purified to homogeneity from human seminal plasma by consecutive chromatographic fractionation on heparin-sepharose, phenyl-sepharose, CIM-QA, and Superdex 200. Human seminal plasma DAO behaves electrophoretically similar to DAO proteins from other human tissues and has very similar enzymatic properties with histamine and aliphatic diamines being the preferred substrates as well as significant conversion of polyamines. The cellular source and functional importance of DAO in human semen remain to be determined.
O. Zitka, S. Skalickova, S. Krizkova, M. Vlkova, V. Adam, R. Kizek
Chromatographia (2013) 76:611-619
In this study, we optimized method for the isolation and detection of lactoferrin from human saliva using 3 mm short monolithic disc. We optimized the conditions for separation as flow rate 4 mL min-1 and ionic strength of effluent as 2 M·NaCl. We estimated limit of detection of whole method, which was hyphenated to the Bradford’s assay, down to 100 ng mL-1. The purity of the isolated fractions was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and recovery of isolation was found to be 51 % using minimally processed sample of saliva. Further, we tested the optimized method on group of healthy volunteers (n = 7). We were able to distinguish between the healthy subjects and subject suffering from celiac disease, which reported at least 2.5× higher level of lactoferrin in comparison to healthy ones. The results were correlated with standard enzyme-linked immunosorbent assay (ELISA) kit with obtained correlation coefficient R2 = 0.8446. Analysis of lactoferrin in saliva by monolithic disc and subsequent offline photometric detection is faster and cheaper method compared to ELISA commercial kit. The total analysis of one sample takes.
D. A. Ribeiro, D. F. Passos, H. C. Ferraz, L. R. Castilho
Journal of Chromatography B, 938 (2013) 111-118
Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose® FF and Fractogel® TMAE, the monolith CIM® QA and the membrane adsorber Sartobind® Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca2+ ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45 mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step.
J. A. Martin, P. Parekh, Y. Kim, T. E. Morey, K. Sefah, N. Gravenstein, D. M. Dennis, W. Tan
PLOS ONE, March 2013, Volume 8, Issue 3, e57341
Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.
P. Fagan, C. Wijesundera
Journal of Separation Science, 10.1002/jssc.201201156
Eicosapentaenoic and docosahexaenoic acids are important bio-active fatty acids in fish oils. Monolithic HPLC columns both in the polymeric cation exchange (silver-ion) and RP formats were compared with corresponding packed columns for the isolation of these acids from tuna oil ethyl esters. Monolithic columns in both formats enabled rapid (typically 5–10 min) separations compared with packed columns (30 min). Polymeric monolithic silver-ion disc column rapidly furnished mixtures of eicosapentaenoic and docosahexaenoic esters (90% purity) within 5–10 min, but was unable to resolve individual esters. A preparative version of the same column (80 mL bed volume) enabled isolation (>88% purity) of 100 mg quantities of eicosapentaenoic and docosahexaenoic esters from esterified tuna oil within 6 min. Baseline separation of eicosapentaenoic and docosahexaenoic esters was achieved on all RP columns. The results show that there is potential to use polymeric monolithic cation exchange columns for scaled-up preparation of eicosapentaenoic and docosahexaenoic ester concentrates from fish oils.
J. A. Martin, P. Parekh, Y. Kim, T. E. Morey, K. Sefah, N. Gravenstein, D. M. Dennis, W. Tan
PLOS ONE, March 2013, Volume 8, Issue 3, e57341
Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.
2012
C. Scott
BioProcess International, November 2012, pg. 31-42
Monoclonal antibodies (MAbs) remain the largest segment of the biopharmaceutical market, but they are not the only recombinant proteins in development. Remember that the first biopharmaceutical approved for sale was recombinant insulin — a hormone — back in the 1980s. And proteins aren't the only recombinant biologics. The sector has expanded since then to include gene therapies and viral vectors, vaccines, and even cells and tissues. Companies around the world are developing such products for cancer, neurological, infectious disease, metabolic, autoimmune, and cardiovascular disorders, to name just the most prominent. And although MAbs are finally fulfilling their “magic bullet” promise, many other approaches are becoming available to drug developers targeting those markets — and others.
Meanwhile, funding challenges are increasing emphasis on manufacturing and development efficiencies. Even though total funding of the biotechnology industry has rebounded since the 2008 recession — from about US$13 billion for the United States industry in 2008 to about $21 billion in 2010, for example — a growing share of that money is going to the less risky investments. According to Ernst & Young's 2011 Beyond Borders report, that means mature and already-profitable companies are taking a larger portion of the financial pie.
At the same time, the average number of drug approvals per year has decreased: from about three dozen in the United States from 1996 to 2004 to under two dozen for the years since. And even though markets are opening up in China, India, and other countries, the cost of doing business on a global scale makes it no easy task to reach them. So biopharmaceutical companies need to curb the rise of development and manufacturing costs. Single-use technologies are helping with the latter in large part. And platform technologies have helped antibody makers shorten development times by starting out with certain rules of thumb — rather than trying out hundreds of available purification technologies, for example, in many different combinations to find what works best for every new product candidate.
Do nonantibody makers have similar options when it comes to their own process development work? As is so often the case in bioprocessing, the answer to that question is “It depends. ..” on the product class; on the expression system; and on the regulatory history of the company, process, and type of molecule.
K. Sushma, C. J. Bilgimol, M. A. Vijayalakshmi, P. K. Satheeshkumar
Journal of Chromatography B, 891 - 892 (2012) 90 - 93(2012) 90 - 93
Anti TNF-α molecules are important as therapeutic agents for many of the autoimmune diseases in chronic stage. Here we report the expression and purification of a recombinant single chain variable fragment (ScFv) specific to TNF-α from inclusion bodies. In contrast to the conventional on column refolding using the soft gel supports, an efficient methodology using monolithic matrix has been employed. Nickel (II) coupled to convective interaction media (CIM) support was utilized for this purpose with 6 M guanidine hydrochloride (GuHCl) as the chaotropic agent. The protein purified after solubilization and refolding proved to be biologically active with an IC50.
M. Srajer Gajdosik, J. Clifton, D. Josić,
Journal of Chromatography A, 1239 (2012) 1- 9
Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed.
A. Albreht, I. Vovk
Journal of Chromatography A, 1227 (2012) 210-218
The separation and isolation of major whey proteins is already extensively covered in the literature although no study has been published in which monolithic columns were used. In our research we present, for the first time, the use of short convective interaction media (CIM) monolithic columns for the separation of all major whey proteins and isolation of β-lactoglobulin variant A and B (β-LgA and β-LgB) from a commercial product whey isolate (WI). Although our primary interest was directed towards finding a proper monolithic column and chromatographic conditions for the purification and isolation of β-LgA and β-LgB, three additional analytical LC methods, each having its own potential application target, were also developed in the course of our research. On the monolithic diethylaminoethyl convective interaction media analytical column (CIMac DEAE), the separation of major whey proteins was achieved by gradually lowering the pH of the mobile phase. The ever-so-hard obtainable linear external pH gradient was very linear in the range of pH 5.5–3 and the developed ion-exchange (IE) high-performance liquid chromatographic (HPLC) method was amenable to mass spectrometry (MS). A very fast baseline separation, with UV detection, of all major whey proteins was achieved on a prototype CIMac reversed-phase styrene-divinylbenzene (RP-SDVB) monolithic column in only 4 min and the performance of this column proved superior in comparison with the packed particle POROS perfusion column. The developed RP-HPLC–MS method is fast and, due to the MS detector, can offer low limits of detection and quantitation. Finally, in order to fulfill our primary interest, a scale-up method was developed, using a prototype 8 mL analogue of the CIMac RP-SDVB column, for the isolation of native and chemically unmodified β-LgA and β-LgB from WI with purities higher than 90% and 81%, respectively. The proteins were to be used in further protein–ligand binding studies. The developed methods excel in speed of the analysis, sensitivity, resolution, and simplicity. Thus, it is shown for the first time that short monolithic columns are applicable to the separation and isolation of major whey proteins and that their use has some obvious benefits.
P. Gagnon
Journal of Chromatography A, 1221 (2012) 57-70(2012) 57-70
This article reviews technology trends in antibody purification. Section 1 discusses non-chromatography methods, including precipitation, liquid–liquid extraction, and high performance tangential flow filtration. The second addresses chromatography methods. It begins with discussion of fluidized and fixed bed formats. It continues with stationary phase architecture: diffusive particles, perfusive particles, membranes and monoliths. The remainder of the section reviews recent innovations in size exclusion, anion exchange, cation exchange, hydrophobic interaction, immobilized metal affinity, mixed-mode, and bioaffinity chromatography. Section 3 addresses an emerging trend of formulating process buffers to prevent or correct anomalies in the antibodies being purified. Methods are discussed for preventing aggregate formation, dissociating antibody-contaminant complexes, restoring native antibody from aggregates, and conserving or restoring native disulfide pairing.
E. S. Sinitsyna, J. G. Walter, E. G. Vlakh, F. Stahl, C. Kasper, T. B. Tennikova
Talanta 93 (2012) 139-146
Macroporous monoliths with different surface functionalization (reactive groups) were utilized as platforms for DNA analysis in microarray format. The slides based on a copolymer glycidyl methacrylate-co- ethylene dimethacrylate (GMA-EDMA) have been chosen as well known and thoroughly studied standard. In particular, this material has been used at optimization of DNA microanalytical procedure.
The concentration and pH of spotting solution, immobilization temperature and time, blocking agent and coupling reaction duration were selected as varied parameters. The efficiency of analysis performed on 3-D monolithic platforms was compared to that established for commercially available glass slides. As a practical example, a diagnostic test for detection of CFTR gene mutation was carried out. Additionally, the part of presented work was devoted to preparation of aptamer-based test-system that allowed successful and highly sensitive detection both of DNA and protein.
2011
M. Pucic, A. Knezevic, J. Vidic, B. Adamczyk, M. Novokmet, O. Polasek, O. Gornik, S. Supraha-Goreta, M. R. Wormald, I. Redzic, H. Campbell, A. Wright, N. D. Hastie, J. F. Wilson, I. Rudan, M. Wuhrer, P. M. Rudd, Dj. Josic, and G. Lauc
Mol Cell Proteomics. Oct 2011; published online Jun 8, 2011
All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously.
Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.
E. F. Maksimova, E. G. Vlakh, T. B. Tennikova
Journal of Chromatography A, 1218 (2011) 2425-2431
A series of macroporous monolithic methacrylate-based materials was synthesized by in situ free radical UV-initiated copolymerization of functional monomers, such as glycidyl methacrylate (GMA), butyl methacrylate (BuMA), 2-aminoethyl methacrylate (AEMA), 2-hydroxyethyl methacrylate (HEMA) and 2-cyanoethyl methacrylate (CEMA), with crosslinking agent, namely, ethylene glycol dimethacrylate (EDMA). The materials obtained were applied as the stationary phases in simple and robust technique – planar chromatography (PLC). The method of separation layer fabrication representing macroporous polymer monolith bound to the specially prepared glass surface was developed and optimized. The GMA–EDMA and BuMA–EDMA matrixes were successfully applied for the separation of low molecular weight compounds (the mixture of several dies), as well as poly(vinylpyrrolidone) and polystyrene homopolymers of different molecular weights using reversed-phase mechanism. The materials based on copolymers AEMA–HEMA–EDMA and CEMA–HEMA–EDMA were used for normal-phase PLC separation of 2,4-dinitrophenyl amino acids and polystyrene standards.
M. R. Etzel, T. Bund
Journal of Chromatography A, 1218 (2011) 2445-2450
Proteins conjugated to neutral biopolymers are of keen interest to the food and pharmaceutical industries. Conjugated proteins are larger and more charge shielded than un-reacted proteins, making purification difficult using conventional beaded chromatographic supports because of slow mass transfer rates, weak binding, and viscous solutions. Past methods developed for pharmaceuticals are unsuitable for foods. In this work, a food-grade whey protein–dextran conjugate was purified from a feed solution also containing un-reacted protein and dextran using either a column packed with 800 mL of a beaded support that was specifically designed for purification of conjugated proteins or an 8 mL tube monolith. The monolith gave a similar dynamic binding capacity as the beaded support (4–6 g/L), at a 42-fold greater mass productivity, and 48-fold higher flow rate, albeit at somewhat lower conjugate purity. Performance of the monolith did not depend on flow rate. In conclusion, monoliths were found to be well suited for the purification of whey protein–dextran conjugates.