A novel twin‐column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants

Downstream processing of mAb charge variants is difficult owing to their similar molecular structures and surface charge properties. This study aimed to apply a novel twin‐column continuous chromatography (called N‐rich mode) to separate and enrich acidic variants of an IgG1 mAb. Besides, a comparison study with traditional scaled‐up batch‐mode cation exchange (CEX) chromatography was conducted. For the N‐rich process, two 3.93 mL columns were used, and the buffer system, flow rate and elution gradient slope were optimized. The results showed that 1.33 mg acidic variants with nearly 100% purity could be attained after a 22‐cycle accumulation. The yield was 86.21% with the productivity of 7.82 mg/L/h. On the other hand, for the batch CEX process, 4.15 mL column was first used to optimize the separation conditions, and then a scaled‐up column of 88.20 mL was used to separate 1.19 mg acidic variants with the purity of nearly 100%. The yield was 59.18% with the productivity of 7.78 mg/L/h. By comparing between the N‐rich and scaled‐up CEX processes, the results indicated that the N‐rich method displays a remarkable advantage on the product yield, i.e. 1.46‐fold increment without the loss of productivity and purity. Generally, twin‐column N‐rich continuous chromatography displays a high potential to enrich minor compounds with a higher yield, more flexibility and lower resin cost.     

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%–17%), main (18%–23%), and basic (59%–68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.     

Displacement to separate host‐cell proteins and aggregates in cation‐exchange chromatography of monoclonal antibodies

An efficient and consistent method of monoclonal antibody (mAb) purification can improve process productivity and product consistency. Although protein A chromatography removes most host‐cell proteins (HCPs), mAb aggregates and the remaining HCPs are challenging to remove in a typical bind‐and‐elute cation‐exchange chromatography (CEX) polishing step. A variant of the bind‐and‐elute mode is the displacement mode, which allows strongly binding impurities to be preferentially retained and significantly improves resin utilization. Improved resin utilization renders displacement chromatography particularly suitable in continuous chromatography operations. In this study we demonstrate and exploit sample displacement between a mAb and impurities present at low prevalence (0.002%–1.4%) using different multicolumn designs and recycling. Aggregate displacement depends on the residence time, sample concentration, and solution environment, the latter by enhancing the differences between the binding affinities of the product and the impurities. Displacement among the mAb and low‐prevalence HCPs resulted in an effectively bimodal‐like distribution of HCPs along the length of a multi‐column system, with the mAb separating the relatively more basic group of HCPs from those that are more acidic. Our findings demonstrate that displacement of low‐prevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through protein A chromatography.     

Evaluation of differences between dual salt‐pH gradient elution and mono gradient elution using a thermodynamic model: Simultaneous separation of six monoclonal antibody charge and size variants on preparative‐scale ion exchange chromatographic resin

The efficiencies of mono gradient elution and dual salt‐pH gradient elution for separation of six mAb charge and size variants on a preparative‐scale ion exchange chromatographic resin are compared in this study. Results showed that opposite dual salt‐pH gradient elution with increasing pH gradient and simultaneously decreasing salt gradient is best suited for the separation of these mAb charge and size variants on Eshmuno^® CPX. Besides giving high binding capacity, this type of opposite dual salt‐pH gradient also provides better resolved mAb variant peaks and lower conductivity in the elution pools compared to single pH or salt gradients. To have a mechanistic understanding of the differences in mAb variants retention behaviors of mono pH gradient, parallel dual salt‐pH gradient, and opposite dual salt‐pH gradient, a linear gradient elution model was used. After determining the model parameters using the linear gradient elution model, 2D plots were used to show the pH and salt dependencies of the reciprocals of distribution coefficient, equilibrium constant, and effective ionic capacity of the mAb variants in these gradient elution systems. Comparison of the 2D plots indicated that the advantage of opposite dual salt‐pH gradient system with increasing pH gradient and simultaneously decreasing salt gradient is the noncontinuous increased acceleration of protein migration. Furthermore, the fitted model parameters can be used for the prediction and optimization of mAb variants separation in dual salt‐pH gradient and step elution. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:973–986, 2018     

Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design

Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.     

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform-Like Purification Process

Single chain variable fragment-IgGs (scFv-IgG) are a class of bispecific antibodies consisting of two single chain variable fragments (scFv) that are fused to an intact IgG molecule. A common trend observed for expression of scFv-IgGs in mammalian cell culture is a higher level of aggregates (10%–30%) compared to mAbs, which results in lower purification yields in order to meet product quality targets. Furthermore, the high aggregate levels also pose robustness risks to a conventional mAb three column platform purification process which uses only the polishing steps (e.g., cation exchange chromatography [CEX]) for aggregate removal. Protein A chromatography with pH gradient elution, high performance tangential flow filtration (HP-TFF) and calcium phosphate precipitation were evaluated at the bench scale as means of introducing orthogonal aggregate removal capabilities into other aspects of the purification process. The two most promising process variants, namely Protein A pH gradient elution followed by calcium phosphate precipitation were evaluated at pilot scale, demonstrating comparable performance. Implementing Protein A chromatography with gradient elution and/or calcium phosphate precipitation removed a sufficient portion of the aggregate burden prior to the CEX polishing step, enabling CEX to be operated robustly under conditions favoring higher monomer yield. From starting aggregate levels ranging from 15% to 23% in the condition media, levels were reduced to between 2% and 3% at the end of the CEX step. The overall yield for the optimal process was 71%. Results of this work suggest an improved three-column mAb platform-like purification process for purification of high aggregate scFv-IgG bispecific antibodies is feasible. © 2018 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers. Biotechnol. Prog., 35: e2720, 2019     

Application of displacement chromatography for the analysis of a lipid raft proteome

Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC–MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC–MS approach.     

Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography

This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E. coli. protein (ECP) impurities. The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem. Several operational parameters were investigated to study their effects on the yield of the displacement process. The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification. Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step. The process was successfully scaled-up with respect to particle and column diameter. The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.     

Continuous separation of multicomponent protein mixtures by annular displacement chromatography

Displacement chromatography is a predominantly nonlinear mode of chromatography, which has certain advantages over the elution mode for preparative bioseparations. Whereas continuous production (and separation) processes have their theoretical benefits in this context, protein displacement chromatography has up to now only been performed in the batch mode. In this contribution, we demonstrate that the principle of continuous annular chromatography can be adapted to displacement chromatography. Separations of up to three standard proteins (two whey proteins, soybean trypsin inhibitor) were developed and optimized using a small (4 x 250 mm) batch column. These separations were subsequently transferred directly to the continuous system (500-mL column). Separations of similar quality in terms of final product purity and recovery yield were obtained using the continuous system.     

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) analyses. First, the salt gradient (using K⁺ as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC–MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.     

Comparison of linear gradient and displacement separations in ion-exchange systems

The linear gradient mode of chromatography is the most widely employed mode of operation in ion-exchange chromatographic separations. However, in recent years, the displacement mode has received considerable attention because of its promise of high throughput and high resolution. To enable a comparison of these two modes of chromatography, it is essential to identify the optimum operating conditions for each. We employed an iterative algorithm to carry out the necessary optimization. The Steric Mass Action model of ion-exchange chromatography is used in concert with the solid-film linear-driving force model to describe the chromatographic behavior of the solutes in these systems. The performances of displacement and gradient modes of chromatography are compared for different types of separation problems. It turns out that for "easy" separations, both the modes are equally effective. However, for challenging separations, the displacement mode is superior to the gradient mode. Our results shed significant light on the performance of gradient and displacement modes in protein ion-exchange systems.     

Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow

Analysis of glycans via a porous graphitized carbon liquid chromatography (PGC-LC) coupled with electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) is a powerful analytical method in the field of glycomics. Isobaric glycan structures can be identified reliably with the help of PGC-LC separation and subsequent identification by ESI-MS(/MS) in negative ion mode. In an effort to adapt PGC-LC-ESI-MS(/MS) to the nano-scale operation, spray instability along the nano-PGC-LC gradient was repeatedly observed on an LTQ Orbitrap Elite mass spectrometer equipped with a standard nano-electrospray ionization source. A stable electrospray was achieved with the implementation of a post-column make-up flow (PCMF). Thereby, acetonitrile was used to supplement the eluate from the nano-PGC-LC column. The improved spray stability enhanced detection and resolution of glycans during the analysis. This was in particular the case for smaller O-glycans which elute early in the high aqueous content regime of the nano-PGC-LC elution gradient. This study introduces PCMF as an easy-to-use instrumental adaptation to significantly improve spray stability in negative ion mode nano-PGC-LC-ESI-MS(/MS)-based analysis of glycans.     

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.     

Cross-scale quality assessment of a mechanistic cation exchange chromatography model

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing-scale can be affected by variability in feed composition, mobile phase and resin properties, or dispersion effects depending on the chromatography system at hand. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. In this study, a multicomponent steric mass-action (SMA) adsorption model was applied to the scale-up of a CEX polishing step. Based on chromatograms and elution pool data ranging from laboratory- to manufacturing-scale, the proposed modeling workflow enabled early identification of differences between scales, for example, system dispersion effects or ionic capacity variability. A multistage model qualification approach was introduced to measure the model quality and to understand the model's limitations across scales. The experimental SDM and the in silico model were qualified against large-scale data using the identical state of the art equivalence testing procedure. The mechanistic chromatography model avoided limitations of the SDM by capturing effects of bed height, loading density, feed composition, and mobile phase properties. The results demonstrate the applicability of mechanistic chromatography models as a possible alternative to conventional SDM approaches.     

Characterization of a unique IgG1 mAb CEX profile by limited Lys-C proteolysis/CEX separation coupled with mass spectrometry and structural analysis

The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.     

Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non‐infectious retrovirus‐like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO^? and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV. Biotechnol. Bioeng. 2012;109: 157–165. © 2011 Wiley Periodicals, Inc.     

Cathepsin L Causes Proteolytic Cleavage of Chinese-Hamster-Ovary Cell Expressed Proteins During Processing and Storage: Identification,Characterization, and Mitigation

A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019     

Shotgun proteome analysis utilising mixed mode (reversed phase‐anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis

The 2‐D peptide separations employing mixed mode reversed phase anion exchange (MM (RP‐AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP‐AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP‐AX) compared with the traditional SCX hyphenated to RP‐LC‐MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP‐AX) for 2‐D LC proteomic studies was also extended in the analysis of iTRAQ‐labelled HeLa and cyanobacterial proteomes using nano‐flow chromatography interfaced to the MS/MS. We demonstrate MM (RP‐AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.