Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica

Silica is widely used for chromatography resins due to its high mechanical strength, column efficiency, easy manufacturing (i.e. controlled size and porosity), and low‐cost. Despite these positive attributes to silica, it is currently used as a backbone for chromatographic resins in biotechnological downstream processing. The aim of this study is to show how the octapeptide (RH)4 can be used as peptide tag for high‐purity protein purification on bare silica. The tag possesses a high affinity to deprotonated silanol groups because the tag''s arginine groups interact with the surface via an ion pairing mechanism. A chromatographic workflow to purify GFP fused with (RH)4 could be implemented. Purities were determined by SDS‐PAGE and RP‐HPLC. The equilibrium binding capacity of the fusion protein GFP‐(RH)4 on silica is 450 mg/g and the dynamic binding capacity around 3 mg/mL. One‐step purification from clarified lysate achieved a purity of 93% and a recovery of 94%. Overloading the column enhances the purity to >95%. Static experiments with different buffers showed variability of the method making the system independent from buffer choice. Our designed peptide tag allows bare silica to be utilized in preparative chromatography for downstream bioprocessing; thus, providing a cost saving factor regarding expensive surface functionalization. Underivatized silica in combination with our (RH)4 peptide tag allows the purification of proteins, in all scales, without relying on complex resins.     

An immuno-chemo-proteomics method for drug target deconvolution

Chemical proteomics is an emerging technique for drug target deconvolution and profiling the toxicity of known drugs. With the use of this technique, the specificity of a small molecule inhibitor toward its potential targets can be characterized and information thus obtained can be used in optimizing lead compounds. Most commonly, small molecules are immobilized on solid supports and used as affinity chromatography resins to bind targets. However, it is difficult to evaluate the effect of immobilization on the affinity of the compounds to their targets. Here, we describe the development and application of a soluble probe where a small molecule was coupled with a peptide epitope which was used to affinity isolate binding proteins from cell lysate. The soluble probe allowed direct verification that the compound after coupling with peptide epitope retained its binding characteristics. The PKC-alpha inhibitor Bisindolylmaleimide-III was coupled with a peptide containing the FLAG epitope. Following incubation with cellular lysates, the compound and associated proteins were affinity isolated using anti-FLAG antibody beads. Using this approach, we identified the known Bisindolylmaleimide-III targets, PKC-alpha, GSK3-beta, CaMKII, adenosine kinase, CDK2, and quinine reductase type 2, as well as previously unidentified targets PKAC-alpha, prohibitin, VDAC and heme binding proteins. This method was directly compared to the solid-phase method (small molecule was immobilized to a solid support) providing an orthogonal strategy to aid in target deconvolution and help to eliminate false positives originating from nonspecific binding of the proteins to the matrix.     

Essential role of the concentration of immobilized ligands in affinity chromatography:: Purification of guanidinobenzoatase on an ionized ligand

Guanidinobenzoatase, a plasma protein with possible application as a ‘tumor marker’, has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by ‘controlled’ immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 μmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 μmol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, ×220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.     

The role of polymer nanolayer architecture on the separation performance of anion-exchange membrane adsorbers: I. Protein separations

This contribution describes the preparation of strong anion-exchange membranes with higher protein binding capacities than the best commercial resins. Quaternary amine (Q-type) anion-exchange membranes were prepared by grafting polyelectrolyte nanolayers from the surfaces of macroporous membrane supports. A focus of this study was to better understand the role of polymer nanolayer architecture on protein binding. Membranes were prepared with different polymer chain graft densities using a newly developed surface-initiated polymerization protocol designed to provide uniform and variable chain spacing. Bovine serum albumin and immunoglobulin G were used to measure binding capacities of proteins with different size. Dynamic binding capacities of IgG were measured to evaluate the impact of polymer chain density on the accessibility of large size protein to binding sites within the polyelectrolyte nanolayer under flow conditions. The dynamic binding capacity of IgG increased nearly linearly with increasing polymer chain density, which suggests that the spacing between polymer chains is sufficient for IgG to access binding sites all along the grafted polymer chains. Furthermore, the high dynamic binding capacity of IgG (>130 mg/mL) was independent of linear flow velocity, which suggests that the mass transfer of IgG molecules to the binding sites occurs primarily via convection. Overall, this research provides clear evidence that the dynamic binding capacities of large biologics can be higher for well-designed macroporous membrane adsorbers than commercial membrane or resin ion-exchange products. Specifically, using controlled polymerization leads to anion-exchange membrane adsorbers with high binding capacities that are independent of flow rate, enabling high throughput. Results of this work should help to accelerate the broader implementation of membrane adsorbers in bioprocess purification steps.     

Separation of human vitamin K-dependent coagulation proteins using hydrophobic interaction chromatography

A rapid and simple method was developed to separate human vitamin K-dependent plasma proteins from each other, yielding virtually homogeneous pools. The purification technique is based on the single use of hydrophobic interaction chromatography, starting from prothrombin concentrate (PC or DEFIX, also termed factor IX concentrate) as initial material. Phenyl-sepharose HP demonstrated optimal separation by comparing several hydrophobic resins as well as resins used in standard procedures like immobilised heparin and Cibacron blue. Under ideal conditions, factor X could be separated in a single step as well as prothrombin. Factor IX co-eluted with other minor proteins. Focus was given only on these three proteins due to their relative abundance. Complete separation of all proteins present in the starting material was achieved by MonoQ anion-exchange chromatography following the phenyl-sepharose run. The resulting purified material could be demonstrated to be of equal or higher purity than using described methods. This strategy employing hydrophobic interaction chromatography for blood macromolecules could be of immense value for purifying the human vitamin K-dependent proteins and represents a considerable simplification over other purification schemes. It not only involves minimal sample handling but also can be readily up-scaled and is a cost-efficient alternative.     

Glycosylation of α1-acid glycoprotein in inflammatory disease: analysis by high-pH anion-exchange chromatography and concanavalin A crossed affinity immunoelectrophoresis

High-pH anion-exchange chromatography with pulsed amperometric detection is a highly sensitive technique that can be used for detecting changes in sialylation and fucosylation, as well as different branching patterns of N-linked oligosaccharides in glycoproteins. We examined the N-glycans of α1-acid glycoprotein obtained from twelve patients with various inflammatory conditions with this technique, as well as traditional concanavalin A crossed affinity immunoelectrophoresis. We found the chromatographic profiles of N-glycans in all patients with rheumatoid arthritis to be very similar, but significantly different from normal controls. N-glycans from patients with ulcerative colitis also showed specific alterations in their chromatographic profiles. However, some heterogeneity was found between these patients, perhaps reflecting changes in glycosylation secondary to certain states of the disease, or to medical treatment. We conclude that this technique is useful for detailed mapping of glycosylation changes in α1-acid glycoprotein in clinical samples, and that it may be used to further increase our knowledge about glycosylation changes in response to inflammatory disease. Abbreviations: AC, acute cholangitis; AGP, α1-acid glycoprotein; CAIE, crossed affinity immunoelectrophoresis; Con A, concanavalin A; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; IEC, ion exchange chromatography; RA, rheumatoid arthritis; SLex, sialyl Lex; UC, ulcerative colitis This revised version was published online in November 2006 with corrections to the Cover Date.     

Preparation and properties of calcium-dependent resins with increased selectivity for calmodulin

Calmodulin from both animal and plant sources is known to bind a number of hydrophobic compounds with resultant inhibition of calmodulin function. Some of these compounds, including certain phenothiazine and naphthalene sulfonamide derivatives, have been previously shown to be useful in the chromatographic isolation of calmodulin, when covalently linked to a solid support. With the exception of fluphenazine linked to epoxide-activated Sepharose, these resins have the undesirable characteristics of requiring high salt concentrations in the elution buffer for efficient elution of calmodulin, thus decreasing the selectivity for this protein. The synthesis of nine Sepharose-ligand affinity resins is reported. Some of the ligands are newly synthesized naphthalene sulfonamide and phenothiazine derivatives. The synthetic ligands have been coupled to three types of Sepharose: epoxide-activated, CNBr-activated, and carbodiimide-activated. The properties of these resins are reported and their relative abilities to act selectively in the isolation of calmodulin are compared. 2-Trifluoromethyl-10-aminopropyl phenothiazine (TAPP), when linked to epoxide-activated Sepharose, was found to be the most useful for calmodulin isolation in terms of its combined stability, capacity, and ability to select for calmodulin. This resin was found to behave as a true affinity resin. A quantitative evaluation of its affinity behavior was consistent with the presence of two high-affinity Ca2+-dependent phenothiazine binding sites on calmodulin, in apparent agreement with previous reports which involved the use of different methods.     

Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides

This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient pressure strong anion-exchange resin Source 15Q combined with ammonium formate buffer (AF) was used for the separation of unsaturated and saturated pectic oligogalacturonides (OGAs) derived from enzymatic digestion of pectin. Routinely, multi-milligram quantities of defined sizes OGAs with DPs from 5 to 19 were produced in excellent purity (>95%). Elution of OGAs followed by direct analysis of the peak fractions by MALDI-TOF MS. Purified OGAs (DP 5-7) were chemoselectively immobilized onto aminooxy-terminated polyethylene glycol polyacrylamide (PEGA) supports. Solid-phase anchoring took place at the reducing end of the oligosaccharide and resulted in the formation of an oxime linkage. The very high coupling yields confirmed the general suitability of aminooxy-PEGA resins for the immobilization of OGAs of different lengths. The OGA-functionalized PEGA supports were subsequently treated with aq TFA at 40 or 60 degrees C, and the chemical degradation products released from the support were analyzed by ESIMS. In all cases, the original OGA was degraded into smaller oligomers of various sizes down to the monomer. This work illustrates some of the basic principles underlying a strategy ultimately aimed at solid-support assisted sequencing of oligosaccharides.     

Design, synthesis and screening of antisense peptide based combinatorial peptide libraries towards an aromatic region of SARS-CoV

A combination of high-performance affinity chromatography and antisense peptide based combinatorial peptide libraries was used to screen a potential inhibitor for SARS-CoV. An aromatic-amino acid-rich region within the transmembrane domain at the C terminal of spike (S) protein identified as a membrane-active region was chosen as the target sense peptide (SP) and immobilized as affinity ligand. Four antisense peptides were designed based on the degeneracy of genetic codes. One of them was screened as the lead peptide to construct the extended peptide libraries (EPL). The library screening was carried out at pH 5.5 so as to mimic the low-pH milieu required by virus fusion. After five cycles of screening, a dodecapeptide KKKKYRNIRRPG (DP) was identified to possess the highest binding affinity to the immobilized sense peptide. The dissociation constant of the complex between the DP and the SP was 5.64 x 10(-7) M in a physiological condition. The recognition between the DP and recombinant SARS S protein was demonstrated by ELISA assay to be in a saturable way. The competitive inhibition of the sense peptide in the competitive ELISA reveals the affinity binding between the DP and SARS S protein is specific and directed towards the target SP of the S protein. The results indicate this preferred polypeptide can be used as a lead compound of potent inhibitor of SARS-CoV. The mechanism study suggests the specific recognition between the DP and the target peptide was due to sequence-dependent and multi-modal affinity interaction.     

mRNA display selection and solid‐phase synthesis of Fc‐binding cyclic peptide affinity ligands

Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid‐phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on‐resin solid‐phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide—cyclo[Link‐M‐WFRHY‐K]—as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link‐M‐WFRHY‐K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link‐M‐WFRHY‐K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link‐M‐WFRHY‐K] is an attractive candidate for developing a cost‐effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest. Biotechnol. Bioeng. 2013; 110: 857–870. © 2012 Wiley Periodicals, Inc.     

Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography

Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and YfbG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties.     

Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L).     

Cd2+结合肽的筛选及与重金属离子的亲和作用

利用噬菌体随机十二肽库和亲和层析技术对重金属Cd进行亲和筛选,共获得两条Cd结合肽序列。将展示有Cd2 结合肽的噬菌体单克隆扩增物对不同重金属离子(Cd2 、Cr2 、Cu2 、Co2 、Zn2 、Ni2 )螯合的树脂进行亲和测定,结果表明Cu2 、Co2 、Zn2 、Ni2 对结合肽的亲和力高于Cd2 和Cr2 。抑菌解毒试验进一步确认了Cd2 结合肽对大肠杆菌重金属的解毒作用。显微观察可见金属结合肽与金属螯合树脂混合后分散度发生改变。     

Affinity Chromatography: An Enabling Technology for Large‐Scale Bioprocessing

Affinity chromatography (AC) has been used in large‐scale bioprocessing for almost 40 years and is considered the preferred method for primary capture in downstream processing of various types of biopharmaceuticals. The objective of this mini‐review is to provide an overview of a) the history of bioprocess AC, b) the current state of platform processes based on affinity capture steps, c) the maturing field of custom developed bioprocess affinity resins, d) the advantages of affinity capture‐based downstream processing in comparison to other forms of chromatography, and e) the future direction for bioprocess scale AC. The use of AC can result in economic advantages by enabling the standardization of process development and the manufacturing processes and the use of continuous operations in flexible multiproduct production suites. These concepts are discussed from a growing field of custom affinity bioprocess resin perspective. The custom affinity resins not only address the need for a capture resin for non‐platformable processes, but also can be employed in polishing applications, where they are used to define and control drug substance composition by separating specific product variants from the desired product form.     

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

In this work, two phage biopanning strategies were developed to identify affinity peptides for a single Fab and multiple kappa Fabs. For the biopanning rounds, protein L beads were employed to bind Fab targets in a fixed orientation, and NHS functionalized magnetic beads were used to facilitate evaluation of low pH elution conditions. The resulting peptide sequences were synthesized and the binding to different Fabs was evaluated using fluorescence polarization. The first biopanning approach yielded a peptide with similar affinities for two forms of the Fab (recombinantly expressed and post papain-digestion) as well as the intact antibody. While moderate affinity was observed toward a murine variant of the Fab with the same complementarity determining regions (CDR) region but different framework, minimal binding occurred to a Fab with high sequence homology but containing different CDR loops. The second biopanning strategy yielded a peptide with affinity for all three kappa Fabs indicating that it may be a good lead for the development of more general affinity reagents for recombinant kappa Fabs. Finally, an affinity peptide column was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The results presented in this article demonstrate that different peptide-based phage biopanning strategies can be effectively employed to identify affinity peptide leads for specific Fab and more general kappa Fab purifications.     

Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis

The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.     

Automated isocratic high-performance liquid chromatography of inositol phosphate isomers.

Isomers of inositol phosphates from biological samples can be analysed by anion-exchange h.p.l.c., by using isocratic elution with phosphate buffers. The method involves the preliminary processing of the extracted samples with conventional soft-gel anion-exchange resins, including the commonly used Dowex resins, followed by direct analysis with h.p.l.c. of a portion of relevant fractions. Run times (up to 20 min) and collected fraction numbers (up to 24) are minimal, so that if the method is used in conjunction with automated h.p.l.c. injection a high throughput of samples is achieved.     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

The clearance of host cell proteins (HCPs) is of crucial importance in biomanufacturing, given their diversity in composition, structure, abundance, and occasional structural homology with the product. The current approach to HCP clearance in the manufacturing of monoclonal antibodies (mAbs) relies on product capture with Protein A followed by removal of residual HCPs in flow-through mode using ion exchange or mixed-mode chromatography. Recent studies have highlighted the presence of “problematic HCP” species, which are either difficult to remove (Group I), can degrade the mAb product (Group II), or trigger immunogenic reactions (Group III). To improve the clearance of these species, we developed a family of synthetic peptides that target HCPs and exhibit low binding to IgG product. In this study, these peptides were conjugated onto chromatographic resins and evaluated in terms of HCP clearance and mAb yield, using an industrial mAb-producing CHO harvest as model supernatant. To gather detailed knowledge on the binding of individual HCPs, the unbound fractions were subjected to shotgun proteomic analysis by mass spectrometry. It was found that these peptide ligands exhibit superior HCP binding capability compared to those of the benchmark commercial resins commonly used in mAb purification. In addition, some peptide-based resins resulted in much lower losses of product yield compared to these commercial supports. The proteomic analysis showed effective capture of many “problematic HCPs” by the peptide ligands, especially some that are weakly bound by commercial media. Collectively, these results indicate that these peptides show great promise toward the development of next-generation adsorbents for safer and cost-effective manufacturing of biologics.     

Simple method to assess stability of immobilized peptide ligands against proteases

Although peptides are used as affinity chromatography ligands, they could be digested by proteases. Usually, peptide stability is evaluated in solution, which differs from the resin‐bounded peptide behavior. Furthermore, the study of the degradation products requires purification steps before analysis. Here, we describe an easy method to assess immobilized peptide stability. Sample peptides were synthesized on hydroxymethylbenzamide‐ChemMatrix resin. Peptidyl‐resin beads were then incubated with solutions containing proteases. Peptides were detached from the solid support with ammonia vapor and analyzed by matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry, allowing the detection of the whole peptides as well as their C‐terminal degradation products. The method allowed a fast evaluation of peptide ligand stability in solid phase towards proteases that may be present in the crude sample before their use as ligands in affinity chromatography. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.