Selection of pH-related parameters in ion-exchange chromatography using pH-gradient operations

This work demonstrates that the type of ion-exchanger (anion or cation), the mode of operation (bind-and-elute or flow-through), and the operational pH of ion-exchange chromatography (IEX) can be selected in a fast and rational way by analytical pH-gradient IEX operations, thereby eliminating the need for pH scouting or high-throughput screening. The developed approach was applied for the selection of an IEX process for the capture of a monoclonal antibody (MAb) from hybridoma cell culture supernatant (CCS). It was found within a day that MAb can optimally be captured by bind-and-elute mode cation-exchange chromatography (CEX) at pH 4.5 or anion-exchange chromatography (AEX) at pH 7.2 without lowering the salt concentration in the CCS. The performance of both CEX and AEX was predicted to be equal for this particular MAb capture.     

Assessing adsorbent-biomass interactions during expanded bed adsorption onto ion exchangers utilizing surface energetics

Biomass adhesion onto an adsorbent matrix or "interaction" as well as biological particle co-adhesion or "aggregation" can severely affect the overall performance of many direct-contact methods for downstream processing of bioproducts. Studies to quantitatively describe this biomass-adsorbent interaction were developed utilizing surface energetics. An indirect thermodynamic approach via contact angle and zeta potential measurements was utilized. Intact yeast cells, yeast homogenates, and disrupted bacterial paste were employed as model system. Various surfaces that are relevant to biochemical and environmental applications were characterized. The extended Derjaguin, Landau, Verwey, Overbeek (XDLVO) theory was found to appropriately predict biomass adhesion behaviour. It was observed that cell attachment onto anion-exchange supports is promoted by strong and close interaction within a secondary energy minimum followed by moderate multilayer cell aggregation. On the other hand, cell interaction with cation-exchange materials can take place within a reversible secondary energy minimum and at longer separation distance. The influence of particle charge and size, as well as the influence of the nature of the material under study were summarized in the form of energy vs. distance profiles. These investigations lead to many process-related conclusions: (a) process buffer conductivity windows can be recommended for anion-exchange chromatography (AEX) vs. cation-exchange chromatography (CEX) systems, (b) increased hydrodynamic shear is required to prevent biomass attachment onto AEX as compared to CEX, and (c) aggregation phenomena is a function of contact time and biomass concentration. Understanding biomass-adsorbent interaction at the particle (local) level is opening the pave for optimized operation of expanded bed adsorption methods at the process (macro) scale. A universal methodological approach is presented to guide both process and material design.     

Impact of multiple re-use of anion-exchange chromatography media on virus removal

We evaluated viral clearance in multiply-cycled anion-exchange media run in flow-through mode. We found that anion-exchange columns do not lose viral clearance capacity after extensive re-use, if they are cleaned with recommended buffers that do not chemically degrade the media. In contrast, anion-exchange (AEX) columns that are not cleaned or are cleaned with buffers that chemically degrade the media lost viral clearance capacity after extended use. In these cases, other performance attributes that changed at the same time were increased band spreading, decreased DNA clearance and accumulating backpressure that prevented re-use past 80-120 cycles. Thus, our data suggests that flow through mode anion-exchange columns that are cleaned with recommended cleaning buffers, and periodically monitored for band spreading, DNA clearance and/or backpressure need not be re-evaluated for viral clearance at the end of the validated media lifetime.     

Selective oxidation of polyfunctional 2-amino-1,3-propanediol derivatives

Oxidation of polyfunctional threo-(1S,2S)-2-amino-1,3-propanediol derivatives with a 717 anion-exchange resin-supported bromine has been investigated. The result showed that oxidized products were in close relationship with the substituents at nitrogen in the starting materials. Its primary and secondary amine derivatives were oxidized in the presence of Na(2)HPO(4) to give essentially a substituted chiral oxazoline or C(3)-O acylated product in high yield, while oxidation of its N,N-dimethyl derivative mainly gave a chiral N-methyl oxidation-formylation product. This selective oxidation was first observed in 2-amino-1,3-propanediol chemistry.     

Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized for the separation of recombinant human growth hormone (hGH) variants on a C18 silica column at 55 degrees C using an isocratic mobile phase which contained 27% 1-propanol in a 25 mM potassium phosphate buffer, pH 6.5. Three of the obtained peaks were characterized by tryptic mapping and mass spectrometry; two of the peaks were found to contain oxidized hGH (dioxy Met14/Met125 and Met125 sulfoxide) while the third contained a deamidated form (Asn149-->Asp149 or Asn152-->Asp152). Compared to the European Pharmacopoeia RP-HPLC method of hGH analysis, this new method gives two additional peaks and a 50% reduction in the analysis time.     

Evaluation of an icIEF-MS system for comparable charge variant analysis of biotherapeutics with rapid peak identification by mass spectrometry

Protein therapeutics are usually produced in heterogeneous forms during bioproduction and bioprocessing. Heterogeneity results from post-translational modifications that can yield charge variants and require characterization throughout product development and manufacturing. Isoelectric focusing (IEF) with UV detection is one of the most common methods to evaluate protein charge heterogeneity in the biopharmaceutical industry. To identify charge variant peaks, a new imaged microfluidic chip-based isoelectric focusing (icIEF) system coupled directly to mass spectrometry was recently reported. Bridging is required to demonstrate comparability between existing and new technology. As such, here we demonstrate the comparability of the pI value measurement and relative charge species distributions between the icIEF-MS system and the control data from a frequently utilized methodology in the biopharmaceutical industry for several blinded development-phase biopharmaceutical monoclonal antibodies across a wide pI range of 7.3–9.0. Hyphenation of the icIEF system with mass spectrometry enabled direct and detailed structural determination of a test molecule, with masses suggesting acidic and basic shifts are caused by sialic acid additions and the presence of unprocessed lysine residues. In addition, MS analysis further identified several low-abundance glycoforms. The icIEF-MS system provides sample quantification, characterization, and identification of mAb proteoforms without sacrificing icIEF quantification comparability or speed.     

Determination of protein oxidation by mass spectrometry and method transfer to quality control

Characterization and quantitative analysis of oxidation plays an important role in biopharmaceutical development. This study demonstrates an approach to the assessment of susceptible to oxidation methionine residues in monoclonal antibodies and recombinant proteins. A method for the determination of oxidation levels by peptide mapping with mass spectrometric (MS) detection is described and its advantages compared to the UV detection are presented. Good linearity and reproducibility for determination of oxidation with MS detection are demonstrated (R2 > 0.99; RSDs of 4-9%). Aspects of method transfer to quality control group (QC) are discussed. As well, a quick and easy flow injection/MS method is proposed to substitute for peptide map analysis. Peptide coverage, linearity, reproducibility, robustness, sensitivity and quantitative oxidation results are compared for the flow injection/MS and LC/MS approaches.     

Usage of nonporous polymeric particles for reversed-phase high-performance liquid chromatography of oxidized and deamidated forms of recombinant human growth hormone

In this work, a C(18) reversed-phase column with nonporous polymeric 2.5- micro m particles is utilized to initially test the analysis of oxidized and deamidated human growth hormone (hGH). Phosphate buffer (pH 7.5) with 24% 1-propanol was used for elution. This quick method (analysis time is 20 min) gave a selectivity, as judged by the number of detected peaks, and resolution of hGH variants that is better than many methods in which porous silica particle columns are used. Only mixtures of oxidized and deamidated hGH are analyzed, and no characterization of the peaks is performed. The results indicate that C(18) nonporous polymeric column material is a promising alternative for the chromatographic separation of several hGH variants.     

Isolation and partial characterization of angiotensinase A and aminopeptidase M from urine and human kidney by lectin affinity chromatography and high-performance liquid chromatography

Angiotensinase A (ATA) and aminopeptidase M (APM) were partially purified from human urine specimens and human kidney particles using wheat germ lectin affinity chromatography, anion-exchange Fast Protein Liquid Chromatography (FPLC) (Mono Q), chromatofocusing (Mono P, FPLC) and Superose 12 gel filtration. APM, a globular 5-nm glycoprotein, is localized in the brush border membrane of the proximal tubule; angiotensin II-degrading ATA is present on glomerular endothelia and podocytes and, to lesser extent, in the brush border. For the first time, both peaks of ATA and APM activity from urine samples were separated by the above-mentioned techniques with only slight overlap; ATP (146,000 dalton: pI4.8) was enriched more than 20-fold and APM (153,000 dalton, pI4.7) more than 50-fold compared with the activity of the starting material. Using similar separation steps, ATA and APM solubilized from kidney particles could not be resolved into two distinct peak fractions, however, except after hydrophobic interaction chromatography. Thus urine is a major source for the preparation of individual ATA and APM fractions, necessary to generate specific anti-enzyme antibodies for diagnostic purposes.     

Use of two-dimensional liquid fractionation for separation of proteins from cell lysates without the presence of methionine oxidation

A novel two-dimensional (2D) chromatographic method is developed to separate proteins from malignant breast cancer whole cell lysates. Protein mixtures are first separated according to their pIby chromatofocusing followed by an orthogonal non-porous reversed-phase separation. An important advantage of this 2D chromatographic method is that, unlike gel-based methods, it does not result in methionine oxidation. The lack of methionine oxidation during separation is demonstrated by the analysis of protein tryptic digests using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. Our novel 2D chromatographic method used in combination with on-target light-induced methionine oxidation provides a means for studying methionine-containing peptides. Methionine residues in peptide sequences are partially oxidized with light exposure. Neither the location nor the modification of methionine in the peptide sequence affects the oxidation. As a result, multiple peaks are observed in MALDI-TOF-MS spectra after light exposure. Sequence information derived from light-induced methionine can be applied to enhance the database search results obtained through peptide mass fingerprinting.     

Oriented immobilization of periodateoxidized monoclonal antibodies on amino and hydrazide derivatives of eupergit C

Amino and hydrazyno derivatives of Eupergit C were prepared by reaction of the beads with hexamethylene diamine (HMD) and adipic acid dihydrazide (ADH), respectively. Monoclonal antibodies (mAbs) against carboxypeptidase A (CPA) and horse radish peroxidase (HRP) were prepared, and those that did not inhibit the respective enzymatic activities were selected. The carbohydrate moieties of these antibodies were oxidized by reaction with sodium periodate and then coupled onto the modified beads. The oxidation and coupling reactions were optimized to achieve highly active matrix-conjugated antibodies. Thus, antibody-matrix conjugates that possessed antigen-binding activities close to the theoretical value of 2 mol antigen bound/mol immobilized antibody were obtained.     

Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8. The antibody gave a highly reproducible CIEF profile with three major peaks having average isoelectric point (pI) values of 6.83, 6.99, and 7.11. Intraday and interday reproducibility of pI values was found to be within RSD of 0.5%. The CIEF profile was also the same, with an alternate column cartridge and alternate batches of methyl cellulose. A plot of peak areas versus MU-B3 concentration was linear (R2 = 0.995) up to a concentration of 0.5 mg/mL in the sample solution. Peak area measurements were reproducible to within 7% RSD. The CIEF profiles of two other antibodies were distinctly different from the profile of MU-B3, showing that the assay is specific. After a sample of MU-B3 was subjected to heat stress by exposure to heat at 55 degrees C for 4 h, its CIEF profile was altered with extra peaks appearing at lower pI values, indicating that the assay could be used to monitor stability. The result of the heat stress experiment was also confirmed with a parallel slab-gel IEF analysis of the antibody sample before and after application of the heat stress. The results of this work suggest that imaging CIEF can be used for product testing under a quality control environment. The assay can be used for pI profiling of proteins and for monitoring structural changes (deamidation, glycosylation, etc.) during the manufacturing process and upon storage.     

Rapid analysis of titer,aggregate, and intact mass of antibody therapeutics using automated multi-dimensional liquid chromatography coupled with native mass spectroscopy

Monoclonal antibodies are tetrameric complex proteins, primarily produced using mammalian cell culture. Attributes such as titer, aggregates, and intact mass analysis are monitored during process development/optimization. In the present study, a novel workflow such that the Protein-A affinity chromatography is performed in the first dimension for purification and titer estimation, whereas size exclusion chromatography is employed in the second dimension to characterize size variants using native mass spectrometry. The present workflow offers a significant advantage over the traditionally used standalone Protein-A affinity chromatography followed by size exclusion chromatography analysis in that it can monitor these four attributes in 8 min while requiring a minimal sample size (10–15 μg) and not requiring any manual peak collection. In contrast, the traditional standalone approach requires manual collection of eluted peaks in Protein-A affinity chromatography followed by buffer exchange to a mass-compatible buffer, which can take up to 2–3 h with considerable risk of sample loss, degradation, and induced modifications. As the biopharma industry moves to make analytical testing efficient, we believe that the approach proposed here would be of significant interest due to its ability to monitor multiple process and product quality attributes in a single workflow and via rapid analysis.     

Rapid separation and determination of chromium

Chromium(III) is oxidized to chromium(VI) at room temperature or by conventional persulphate oxidation. The chromium(VI) is separated from other metal ions by retention on a small anion-exchange column, and then eluted with a perchlorate solution and measured spectrophotometrically with a flow-through cell. The method is rapid, selective and amenable to automation.     

Capillary electroendoosmotic chromatography of peptides

This review focuses on the current state of peptide separation by capillary electroendoosmotic chromatography (CEC). When carried out under optimised conditions, peptide separation by CEC methods represents an orthogonal and complementary technique to micro-HPLC (micro-HPLC) and high-performance capillary zone electrophoresis (HPCZE). The origin of the selectivity differences that can be achieved with these three separation techniques (CEC, micro-HPLC and HPCZE), respectively are discussed, and the current limits of performance with CEC methods documented. Peptide separations by CEC methods with n-alkyl bonded silicas or mixed-mode phases are also illustrated. The development of different variants of CEC and pressurised CEC (also commonly referred to in the literature as electrically-assisted micro-HPLC) are examined. The potential of coupling CEC systems to mass spectrometers for real-time analyses of peptides or protein digests has been examined. Several future directions for the application of this technique in phenotype/proteomic and zeomic mapping of naturally occurring peptides and proteins are highlighted.     

Thermal Stability of Oxidized Single‐Walled Carbon Nanotubes: Competitive Elimination and Decomposition Reaction Depending on the Degree of Functionalization

The thermal stability of oxidized single‐walled carbon nanotubes (SWNTs) with various degrees of oxidation was investigated. The oxidized SWNTs exhibited lower absorption and radial breathing mode (RBM) peaks and a higher intensity ratio of the D band to the G band (D/G) in their absorption and Raman spectra than those of the pristine SWNTs. After the thermal treatment, the D/G ratio of the oxidized SWNTs almost recovered its original intensity, regardless of the degree of oxidation. The absorption, photoluminescence (PL), and RBM peaks could not recover their original intensities when the oxidation degree was high. The results indicate that the elimination and decomposition reactions proceeded competitively depending on the degree of oxidation. In addition, a new PL peak was observed in the near‐infrared region, and the PL peak intensity increased with the subsequent thermal treatment. The theoretical calculations provided an insight into the possible pathways for the decomposition of oxidized SWNTs, showing that the O₂ elimination and CO/CO₂ evolution proceed competitively during thermal treatment.     

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.     

Membrane chromatography: Protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases

This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind^® D membrane adsorber and HiTrap™ DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind^® D membrane at equivalent volumetric throughput and higher capacities than the HiTrap™ DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap™ DEAE FF resin column and was the same for the new AEX membrane and Sartobind^® D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput.     

Mass spectrometry analysis of photo-induced methionine oxidation of a recombinant human monoclonal antibody

Oxidation of methionine (Met) residues of a recombinant fully human monoclonal antibody after exposure to light was investigated and compared with chemically induced oxidation using tert-butyl-hydroperoxide (tBHP). Met256 and Met432 in the Fc region in the samples exposed to light or incubated with tBHP were oxidized. The Fc mass spectra of the antibody exposed to light showed mainly peaks with a molecular weight (MW) increase of 32 Da, however the sample treated with tBHP showed peaks with increase of only 16 Da. These results suggested that either oxidation of one Met residue (either Met256 or Met432) catalyzed the oxidation of the second Met residue on the same heavy chain (HC) or Met residues of one HC were preferentially oxidized when the antibody was exposed to light, while Met256 and Met432 were randomly oxidized when the antibody was incubated with tBHP.     

Evaluation of the pattern and kinetics of degradation of adalimumab using a stability‐indicating orthogonal testing protocol

Forced degradation studies are crucial for the evaluation of the stability and biosimilarity. Here, adalimumab was subjected to oxidation, pH, temperature, agitation and repeated freeze–thaw in order to generate all possible degradation products. An orthogonal stability‐indicating testing protocol comprising SE‐HPLC, RP‐HPLC, TapeStation gel electrophoresis, dynamic light scattering (DLS), and functional receptor binding assay was developed and validated. The assay protocol was used for the assessment of the pattern and kinetics of aggregation/degradation of adalimumab. SE‐HPLC and DLS were used to show the formation of aggregates/fragments of adalimumab under nondenaturing conditions. TapeStation electrophoresis was performed under denaturing conditions to reveal the nature of aggregates. Results of the receptor binding assay agreed to those of SE‐HPLC and DLS which indicated that it can be used as an activity‐indicating assay for adalimumab. RP‐HPLC demonstrated excellent selectivity for adalimumab in the presence of its oxidized forms. The kinetics of degradation was studied in each case and the results showed that it followed the first‐order reaction kinetics. Correlation between the results supported the quality assessment of the tested product in industrial and clinical settings. This orthogonal protocol is a useful tool in stability assessment of monoclonal antibodies and a key criterion for the biosimilarity assessment.