19 LC/MS ANALYSIS OF PROTEINS AND PEPTIDES IN DRUG DISCOVERY Guodong Chen, Yan-Hui Liu, and Birendra N. Pramanik 19.1 INTRODUCTION The modern drug discovery process, in general, involves the identification of a biochemical target (usually protein target), screening of synthetic com- pounds or compound libraries from combinatorial chemistry/natural sources for a lead compound, and optimization of the lead compound (activity, selec- tivity, pharmacokinetics, etc.) for recommending a potential clinical candidate. The ultimate goal is to develop highly potent compounds (small molecules) that bind noncovalently with target proteins and produce the desired thera- peutic response with minimal side effects [1]. In addition, the discovery of DNA structures by Francis Crick and James Watson laid a foundation for the $30 billion-a-year biotechnology industry that has produced some 160 drugs and vaccines, treating everything from breast cancer to diabetes. Recent advances in recombinant DNA technology have provided means to produce and develop protein products as novel drugs, vac- cines, and diagnostic agents. For example, INTRON A (interferon α-2b) is one of the first recombinant protein drugs introduced on the market.This synthetic E. coli recombinant DNA-derived protein functions as a natural interferon produced by the human body as part of the immune system in response to the presence of enemy cells. It not only interferes with foreign invaders that may cause infections, but also prevents the growth and spread of other diseased 837 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. cells in the body. This protein drug is effective in treating hepatitis C virus and a variety of tumors . ENBREL (etanercept) is another protein drug used for treatment of rheumatoid arthritis.It is produced from a Chinese hamster ovary mammalian cell expression system. This protein drug is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75-kilodalton (kDa) tumor necrosis factor receptor (TNF). TNF is one of the chemical messengers that are involved in the inflammatory process.Too much TNF produced in the human body overwhelms the human immune system’s ability to control inflammation in the joints. ENBREL binds to and inactivates some TNF molecules before they can trigger inflammation, thus reducing inflammatory symptoms [2, 3]. One of difficulties encountered in producing large quantities of biologically active proteins is the elimination of microheterogeneity related to these pro- teins. The therapeutic proteins and the drug target proteins are usually asso- ciated with post-translational modifications, such as phosphorylation [4], glycosylation [5], aggregation, and disulfide bond formation [6], with all contributing to the heterogeneity of the proteins. These post-translational modifications control many biological activities/processes. Therefore, charac- terization of proteins with respect to assessment of purity and structure is an integral part of the overall efforts toward drug development, including sub- mission of the analytical data to the regulatory agencies. Furthermore, progress in genomics and proteomics research has generated new proteins that require rapid characterization by analytical methods [7]. 19.2 GENERAL STRATEGIES FOR ANALYSIS OF PROTEINS/PEPTIDES The analytical strategies for protein characterization rely heavily on high- performance liquid chromatography (HPLC) and/or electrophoretic separa- tion of proteins/peptides, followed by other detection methods [e.g., mass spectrometry (MS)]. 19.2.1 HPLC Methods in Proteins/Peptides Achieving good separation of proteins/peptides is always one of many challenges in chromatographic separations. Proteins are highly complex mole- cules with enormous amount of structural diversity, including hydrophobic/ hydrophilic and anionic/cationic interactions. The differences in physical, chemical, and functional properties of proteins/peptides provide the molecu- lar basis for their separations.There are five basic chromatographic separation methods, including size-exclusion chromatography, ion-exchange chromatog- raphy, reversed-phase chromatography, hydrophobic interaction chromatog- raphy (HIC), and affinity chromatography (detailed discussions on the first three techniques are provided in Part I of this book) [8, 9]. 838 LC/MS ANALYSIS OF PROTEINS AND PEPTIDES IN DRUG DISCOVERY Size-exclusion chromatography (often referred to as gel filtration or gel per- meation chromatography) is a chromatographic process involving separation of proteins on the basis of their differential apparent molecular sizes [10]. The column packing materials usually consist of particles with well-controlled pore size. When mobile-phase liquid flows through these particles, the proteins (solutes) with different size can get into and out of the pores with different accessibility. For a specific size-exclusion column with a specific pore size, pro- teins with molecular weights above the exclusion limit (in daltons) of the column are too large to enter the pores and are excluded from the column. Proteins with molecular weights less than the exclusion limit can have differ- ent access to pores of particles and elute after the void volume, depending on their size and shape. In theory, there is a linear relationship between the log- arithm of protein molecular size (molecular weight) and the elution volume of the protein.A calibration curve based on this linear relationship can be used to determine the molecular weight of proteins, assuming that the protein is globular and symmetrical in shape, and there is no other interaction between the protein and column. In practice, denaturants (e.g., 0.1% SDS) are some- times used in the mobile phase to disrupt possible formation of undesired protein aggregates in solution and promote uniformity in conformations of proteins. Thus, the separation can be performed in near-ideal situations to obtain more accurate molecular weight determination of proteins using this approach. Several parameters should be given special consideration in method devel- opment of size-exclusion chromatography. Although its nature of separation requires no interactions between the proteins and stationary phase, the column packing material often exhibits anionic and hydrophobic characters. The addition of salts to the mobile phase can suppress these column effects. However, a higher concentration of salts (>0.5 M) might promote hydropho- bic interactions between proteins and the column. Amount of salts added to the mobile phase should be carefully adjusted. Another factor is pH value. The formation of silanolate anions from column can be minimized by carrying out experiments at pH values less than 7. Typical experimental conditions include mobile phases with low ionic strength buffers (<0.1M) in near- physiological pH ranges—that is, 50mM phosphate buffer with 100mM KCl (pH 6.8). Flow rates can vary from 0.5mL/min to 1.0mL/min,although a better resolution can be achieved with slower flow rates.The sample injection volume and analyte concentration is also critical for optimum performance. The loading capacity is very low for size-exclusion chromatography. Generally, the sample injection volume should not exceed 5% of the column bed volume in order to maintain good resolution. Protein samples should be concentrated without causing precipitation prior to analysis. Once an appropriate method is developed, size-exclusion chromatography can be an excellent method for separation of protein complexes. It is also suitable for buffer exchange as a desalting procedure in protein purifications (salts can be easily separated from proteins by size-exclusion chromatography) and estimation of the molecular GENERAL STRATEGIES FOR ANALYSIS OF PROTEINS/PEPTIDES 839 weight of proteins. A key advantage of this technique is that the biological activity of proteins is maintained during the separation. Ion-exchange chromatography relies on reversible , electrostatic (or ionic) interactions between charged proteins/peptides in the mobile phase and charged ion-exchange group on the stationary phase [11]. Proteins/peptides normally possess either net positive or negative charges depending on pH. They are positively charged at pH values below their pI (isoelectric point) and negatively charged at pH values above their pI. For acidic proteins and pep- tides (pI < 6), they are normally separated using anion-exchange columns because they are negatively charged. Basic proteins and peptides (pI > 8) are usually chromatographed on cation-exchange column because they are posi- tively charged. The choice of pH is important for optimum separation results. The pH of the mobile phase is typically set at least one pH unit away from the pK a of its ion-exchange resin in order to keep 90% of the full charge on the column. For anion-exchange column, the pH is chosen to be lower than the pK a . For cation exchangers, the pH is set to be higher than the pK a . Other key parameters include the ionic strength of the mobile phase. The salts used in the buffer solution are the counterions that might bind to the ion-exchange column in competition with proteins/peptides. Thus, if a protein/peptide is strongly bound to the ion-exchange column, a stronger counterion can be used to improve the elution. Some common counterions with their relative strength include Cs + > K + > NH + 4 > Na + and PO 4 3− > CN − > HCOO − > CH 3 COO − .The unique feature of ion-exchange chromatography is that the biological activity of proteins is almost always preserved, and this separation method can also be used to concentrate dilute protein samples. More recently, another related technique — chromatofocusing — has emerged as a chromatographic technique complementary to electrophoretic methods for pI determination. Chromatofocusing is an ion-exchange tech- nique in which a pH gradient is established across the column, allowing for the eventual separation of amphoteric substances (i.e.,proteins) based on their pI. The main advantages of chromatofocusing are high loadability of the column, high resolution power allowing separation of two proteins (i.e., protein and a degradation product variant) differing less than 0.05pI units, and the high efficiency due to both gradient elution mode and special focusing effect of the polyampholytes. Furthermore, peptides and proteins are less likely to precipitate in chromatofocusing than in isoelectrical focusing. Reversed-phase (RP) chromatography is a hydrophobic separation tech- nique based on the interaction between the nonpolar regions of proteins/ peptides and the stationary phase [12]. It typically utilizes volatile organic sol- vents (acetonitrile, etc.) as mobile phases under acidic pH conditions. It pro- vides high speed and high efficiency and is compatible with MS detection.This technique is the most widely used HPLC method in the separation of peptides and proteins. There are a number of factors to be considered in method development of RPLC for separation of proteins and peptides. Appropriate pore size is one 840 LC/MS ANALYSIS OF PROTEINS AND PEPTIDES IN DRUG DISCOVERY of primary considerations in selecting a column. For proteins greater than 10 kDa, large pore size (300Å) is necessary to reduce restriction of the protein into the stationary phase and avoid poor recoveries and decreased efficien- cies. Polypeptides (<10 kDa) can be effectively separated using a column with a small pore size (<150Å). The hydrophobicity of the protein is also impor- tant when choosing a column. In general, C18 column is used for hydrophilic proteins/small peptides, and C4 or C5 bonded phase is used for hydrophobic proteins/large polypeptides. The use of C4/C5 column for hydrophobic pro- teins may reduce undesired protein absorption on the column because more retentive C18 column for hydrophobic proteins can lead to irreversible binding of the protein to the column.The most commonly used mobile phase in RPLC involves acetonitrile solution with 0.1% trifluoroacetic acid (TFA). In addition, alcohols such as isopropanol are sometimes used for large and more hydrophobic proteins to enhance the elution and improve recovery. Note that all mobile phase reagents should be of the highest quality to avoid the appearance of ghost peaks from solvent impurities. Some ion-pairing reagents are often used to optimize resolution and retention. For example, hydropho- bic, anionic ion-pairing reagents (i.e.,TFA and pentafluoropropionic acid) can complex with positively charged basic residues and influence the chromatog- raphy. On the other hand, hydrophobic, cationic ion-pairing reagents (i.e., tri- ethylamine acetate) interact with negatively charged groups (i.e., carboxylic acid, free carboxyl terminus at pH > pK a ) and effect their retention. Thus, manipulation of ion-pairing reagent and pH value provides alternative approaches in optimizing RPLC. Variation of flow rate and gradient rate can have an impact on the chromatography as well. An increase in flow rate or a decrease in gradient rate improves resolution, although it may result in a loss of sensitivity.Typically, a shallower gradient is employed to maintain good res- olution—that is, 0.25% to 4% per minute. Column temperature also affects the separation. Higher column temperature usually improves column effi- ciency, peak shape, and resolution. However, it may lead to the loss of bio- logical activity of the protein. Hydrophobic interaction chromatography involves weak interactions of hydrophobic patches on the surface of the intact protein and nonpolar groups on the stationary phase [13]. This technique uses aqueous mobile phases of high ionic strength and neutral pH. It does not denature or unfold proteins and can be used to detect protein conformational changes. Key factors affect- ing protein separations include column, salt, mobile-phase pH, and tempera- ture. Most columns used in HIC are made of silica-based stationary phases with modified aryl groups, diol derivatives, and short alkyl chains. The overall hydrophobicity of the stationary phase is determined by both the nonpolar character of the bonded ligands and their density. Strong column-solute inter- actions should be avoided to reduce denaturation.The type and concentration of salt are critical in HIC. One of considerations in choosing a salt is its surface tension. Salts with higher surface tension values may lead to the increase in solute retention. The amount of proteins bound to the column also increases GENERAL STRATEGIES FOR ANALYSIS OF PROTEINS/PEPTIDES 841 with increasing of salt concentration. More hydrophobic proteins should be separated using salts with higher surface tensions . Commonly used salts with relative surface tension include KCl < NaCl < Na 2 HPO 4 < (NH 4 ) 2 SO 4 < Na 3 PO 4 , with typical concentrations ranging from 1M to 3M in order to max- imize selectivity or column capacity. The pH value in HIC is usually main- tained in the neutral range (pH 5–8). Appropriate pH for the optimization of resolution/selectivity in HIC can only be made empirically since proteins differ significantly in their susceptibility to denaturation with changing of pH. Another important parameter in developing HIC method is temperature. In general, proteins tend to be more stable at lower temperatures. To maintain the conformations of proteins,the lowest temperature sufficient for separation should be used in the HIC technique. As an illustration of HIC technique, the recombinant human growth hormone (hGH) and methionyl hGH (met-hGH) were well-separated by the HIC technique [14]. The optimized conditions were found to be 1M ammonium phosphate dibasic, pH 8.0/propanol (99.5:0.5) and 0.1M sodium phosphate dibasic, pH 8.0/propanol (97.5:2.5) for mobile phase A and B, respectively, with a descending gradient from 100% A to 100% B in 30 minutes at a column (TSK-phenyl 5PW, 75 × 7.5mm) temperature of 30°C. Note that the addition of a small amount of propanol as organic modifiers significantly decreases elution time while maintaining resolution and efficiency. This HIC method allowed separation of several hGH variants from the main hGH peak while retaining their native structures. Affinity chromatography is based on reversible, specific binding of one biomolecule to another [15]. The analyte to be purified is specifically and reversibly adsorbed to a ligand (binding substance) that is immobilized by a covalent bond to a chromatographic bed material (matrix). The choice of ligand is a critical factor in affinity chromatography, because it determines the interaction mode between the solute and the ligand. There are two types of ligands: specific ones and multifunctional ones. Specific ligands include potent binders of single classes of peptides or proteins, such as enzyme substrates/ inhibitors and antigens/antibodies. Examples of multifunctional ligands include (a) concanavalin A that binds to some specific carbohydrate residues and (b) nucleotides that bind to enzymes. The chromatography steps involve sample loading in which samples are applied under favorable conditions for their specific binding to the ligand. Analytes of interest are consequently bound to the ligand while unbound substances are washed away. Recovery of molecules of interest can be achieved by changing experimental conditions to favor desorption (elution). Various elution techniques used include changes in mobile-phase composition (e.g., ionic strength, pH) and disruption of ligand/solute complex using competitive ligands in the mobile phase. The sep- aration of analytes depends on their native conformations (for proteins) and relative binding affinities for the immobilized ligand on the column.The affin- ity interactions can be extremely specific, an antibody binding to its antigen, and so on. This technique is a powerful tool in investigating protein–protein, 842 LC/MS ANALYSIS OF PROTEINS AND PEPTIDES IN DRUG DISCOVERY protein–peptide, and drug–protein interactions. Its applications in inhibitor screening using affinity chromatography–MS methods in drug discovery will be discussed later in this chapter . 19.2.2 MS Methods for Protein Characterization MS is another powerful analytical technique for protein characterization.This technique measures mass-to-charge ratios of ions in the gas phase, providing both molecular weight (MW) information and structural information [16]. The introduction of electrospray ionization (ESI) [17, 18] and matrix-assisted laser desorption/ionization (MALDI) [19] or soft ionization [20] has revolu- tionized applications of MS in protein characterization, making it quite straightforward to analyze proteins with molecular weight of over 1 million daltons (Da). ESI forms multiple-charged ions for proteins/peptides by spray- ing the sample solution through a nozzle under a strong electrical field. The molecular weight of a protein can be calculated from a group of [M + nH] n+ ions in the ESI spectrum with a better precision. Also, multiple-charge ions appear at m/z values which are only fractions of the actual molecular weight of the analyte. This allows one to observe high-molecular-weight proteins beyond the normal mass range of a mass spectrometer. In addition, ESI oper- ates at atmospheric pressure, which allows the direct on-line analysis by inter- facing HPLC with MS.The MALDI technique has high ionization efficiencies for proteins and can achieve a mass range of over 500kDa when coupled with a time-of-flight (TOF) mass analyzer. In this technique, proteins are mixed with an IR or UV absorbing matrix in large excess and the mixed sample is deposited on a sample target, dried, and inserted into the mass spectrometer for laser irradiation. In contrast to multiple-charge ions in ESI, the singly charged ions are the most abundant species in the MALDI-MS spectrum. Higher sensitivity (lower femtomole) can be achieved with MALDI-MS analysis. The very first step in protein characterization is the molecular weight deter- mination.With multiple-charge ions formed in ESI, a deconvoluted mass spec- trum can be generated to give an average molecular weight of the protein by calculating from successive multiple-charged ions. For example, Figure 19-1 shows an ESI mass spectrum of a recombinant interferon α-2b (antiviral protein drug) with a charge distribution of +9 to +13. The deconvoluted spec- trum (Figure 19-1, insert) gives a molecular weight of 19,266.3Da for this protein. The mass measurement precision and accuracy are enhanced by the use of all the observed multiple-charged ions (typically better than 0.01% for masses up to 100kDa) [21]. The MALDI-MS technique can also be employed to analyze intact proteins with high tolerance of impurities (salts, etc.). Figure 19-2 illustrates a MALDI-TOF mass spectrum of 1pmol of anti-IL-5 MAB protein with an average molecular weight of 146.5kDa [1]. The singly charged molecular ion [M + H] + is observed at m/z 146,485, along with a doubly charged molecular ion. GENERAL STRATEGIES FOR ANALYSIS OF PROTEINS/PEPTIDES 843 The protein identification or sequence determination of a protein can be achieved using two different approaches: “top-down” [22, 23] and “bottom- up” [24].A top-down experiment involves high-resolution measurement of an intact molecular weight and direct fragmentation of protein ions by tandem mass spectrometry (MS/MS) [25]. This approach surveys an entire protein sequence with 100% coverage. Post-translational modifications such as glyco- 844 LC/MS ANALYSIS OF PROTEINS AND PEPTIDES IN DRUG DISCOVERY Figure 19-1. Positive ion ESI mass spectrum of rh-IFN-α-2b.The insert shows a decon- voluted spectrum. Figure 19-2. MALDI-TOF mass spectrum of 1pmol of anti-IL-5 MAB protein. (Reprinted from reference 1, with permission of the Thomson Corporation.) sylation and phosphorylation tend to remain intact during MS/MS fragmen- tation at the protein level. The fragment ions obtained allow the protein iden- tification by database retrieval, quick positioning of the N- and C-termini, confirmation of large sections of sequences, and partial or exact localization of modifications. This is a preferred method for protein identifications. However, there are some obstacles that need to be overcome before this approach can be widely accepted as a standard in protein identifications.These challenges include accessibility of expensive MS instrumentation for accurate mass measurements of large proteins, development of suitable MS instru- mentation for efficient MS/MS data acquisition in automatic fashion, and appropriate database search algorithm. In contrast to the top-down method- ology, the bottom-up experiment refers to the process in which proteins are digested into smaller peptides under enzymatic cleavages without measuring the accurate mass value of the intact protein. These enzymatic digested pep- tides (tryptic peptides, etc.) often can be unique in terms of their mass, amino acid composition/sequence, and separation characteristics. They can be sepa- rated/detected and either (a) directly searched against a genome or protein database for protein identification (peptide mass mapping) or (b) further dis- sociated in a tandem mass spectrometric experiment to generate fragment ions for database search (sequence tagging) [26, 27]. The principal fragment ions in polypeptide ions are b ions (N-terminus) and y ions (C-terminus) resulted from cleavages of amide bonds under collision-induced dissociations [28]. These are amino acid-specific fragment ions and can be used to derive sequences of polypeptides. Further database search based on the MS/MS information can lead to identification of proteins. The general sequence cov- erage from this approach (5–70%) is far less than 100% from top-down approach. Post-translational modifications are likely to be lost during MS/MS fragmentation at the peptide level. In spite of these limitations, the bottom-up approach has become a current standard method in protein identifications because of its high-throughput format and well-refined methodology—for example, mature instrumentation and excellent software development [29]. Some specific examples using this approach will be described in the following sections. 19.3 APPLICATIONS FOR BIOTECHNOLOGY PRODUCTS AND DRUG TARGETS 19.3.1 Biotechnology Products Development The production of biologically important proteins by recombinant DNA tech- niques and development of modified counterparts is a very challenging field. Certain criteria of safety, quality, and efficacy are required for the develop- ment and approval of these protein products as therapeutic agents. The presence of structural variations during the different steps in the protein APPLICATIONS FOR BIOTECHNOLOGY PRODUCTS AND DRUG TARGETS 845 production process could affect the protein’s biological properties and alter the safety , potency, and stability of the protein product. The development of sensitive analytical techniques for the analysis of therapeutic proteins is essen- tial for the quality control and structural characterization of recombinant protein products. Two examples are illustrated below, including recombinant human granulocyte-macrophage colony stimulating factor (rh-GM-CSF) and interferon alpha-2b (rh-IFN-α-2b). 19.3.1.1 rh-GM-CSF. GM-CSF belongs to a group of interacting glycopro- teins that regulate the differentiation, activation, and proliferation of multiple blood-cell types from progenitor stem cells. This particular glycoprotein is essential for the proliferation and differentiation of progenitor cells into mature granulocytes and macrophages [30]. It enhances the production and function of white blood cells with its potential clinical applications for follow- up treatment for patients who have gone through chemo or radiation therapy for tumors, as well as bone marrow transplantation. GM-CSF has been cloned and expressed in various cell lines that include yeast, Chinese hamster ovary, and E. coli.The E. coli derived GM-CSF used in this study contains 127 amino acid and has a molecular weight of ∼14,477.6Da. One of the first measurements performed to characterize a protein is deter- mination of the molecular weight. It is an important physical parameter that can be used to confirm primary structure and identity of the protein, charac- terize post-translational modifications, and determine batch-to-batch repro- ducibility in the production of recombinant proteins. The mature protein sequence for human GM-CSF with four cysteine residues is shown in Table 19-1 [31]. Figure 19-3A displays the ESI-MS spectrum of rh-GM-CSF, con- taining a series of multiply-charged ions ranging from the 7+ to the 16+ charge state that correspond to molecular ions of the protein. The measured average molecular weight (14,472Da, as shown in the insert) suggests the presence of two disulfide bonds in the rh-GM-CSF because the calculated averaged molecular weight of rh-GM-CSF derived from the sequence is 14,477.6Da 846 LC/MS ANALYSIS OF PROTEINS AND PEPTIDES IN DRUG DISCOVERY TABLE 19-1. Amino Acid Sequence of rh-GM-CSF from E. Coli APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVI -T 1 -→ T 2 → T 3 → V 1 → V 2 → V 3 →-V 4 ->-V 5 -> SEMFDLQEPTC 54 LQTRLELYKQGLRGSLTKLKGPLTMMASHYK T 4 → T 5 → T 6- → T 7 →T 8 > T 9 → V 6 > V 7 → V 8 → V 9 QHC 88 PPTPETSC 96 ATQIITFESFKENLKDFLLVIPFDC 121 WEPVQE T 10 →-T 11 -> T 12 → → V 10 →-V 11 -> V 12 →-V 13 → a T he T n and V n indicate expected tryptic and S. aureus V8 protease peptides, respectively. [...]... also shifted to higher charge states (17+, 18+, 19+, 20+) for the reduced form, indicating a more open form of protein structure for protonations upon disulfide-bonds reduction Furthermore, the molecular weight information obtained from ESI-MS spectrum has a higher accuracy of mass measurement (generally better than 0.01%) The primary structural information of the protein can be obtained by enzymatic cleavage... earlier than the parent protein It is well known APPLICATIONS FOR BIOTECHNOLOGY PRODUCTS AND DRUG TARGETS 853 Figure 19-6 RP -HPLC chromatographic profile of an “in-process” sample from E coli recombinant DNA derived IFN-α-2b Peak 1 is IFN-α-2b Isoform peak 2 and 3 are putative scrambled disulfides Isoform peak 4 is a putative open disulfide The HPLC was run under a linear gradient of 49–65% B (10 : 90 H2O... permission of John Wiley & Sons, Ltd.) TABLE 19-5 ESI-MS Analysis of the Tryptic Digest of CHO IL-4 for HPLC Peak 11 & 14 Sequence Position Expected Mr Observed Mr for CHO IL-4 11 38–47 and 89–102 22–42 and 65–75 2698.2 3516.0 14 38–47 and 89–115 4230.8 — 5286, 5577, and 5868 7682 and 7976 HPLC Peak Observed Mr for Deglyc IL-4 Tryptic Peptide 2698.5 — T5,6-S-S-T16 T4,5*-S-S-T10 4231.6 T5,6-S-S-T16,17 Source:... individual peak for signal patterns characteristic of glycopeptides This HPLC/ ESI-MS approach proved to be useful in detecting several glycoforms in CHO IL-4 Not only were the main asialo and sialylated biantennary glycoforms detected, but also additional signals indicative of higher branching were well-separated This rapid assessment of glycosylation at the molecular level is invaluable for an initial... the NADH study, the amount of NAD+ APPLICATIONS FOR BIOTECHNOLOGY PRODUCTS AND DRUG TARGETS 859 Figure 19-10 The pyruvate formation with the N-terminal cysteine The C-2 carbonyl in pyruvic acid initially forms a ketimine intermediate (A) The sulfhydryl (SH) group of Cys-1 generated from the reduced cysteine 1–98 disulfide bond in Iso-4 tends to favor the formation of the more thermodynamically stable... isoforms, Iso-2, Iso-3, and Iso-4 The solid line indicates the disulfide bond formation, while the dashed line indicates the reduced disulfide bond or partial disulfide bond formation The pyruvic modification of the N-terminal cysteine of E coli derived recombinant IFN-α-2b via a ketimine linkage has not been reported previously There were only two cases in the literature that involved the ketimine formation... glycoprotein is essential for the secretion of soluble rabies virus glycoprotein [41] Changes in levels and types of glycosylation can be associated with disease It has been illustrated that detecting changes in glycan structure may be used as a diagnostic for aggressive breast cancer [42] Glycan profiling of normal and diseased forms of a glycoprotein has provided new insights for future research in rheumatoid... to these isoforms, a fourth component, a variant of IFN-α-2b, was detected either co-eluting with or as a small shoulder eluting in front of the target protein peak (peak 1) The separation of this shoulder peak from IFN-α-2b depended on the HPLC column load; for example, better separation was obtained with lower column loads as illustrated in Figure 19-7 The exact structures of these isoforms and the... Two of the three isoforms, Iso-2 and Iso3, were predicted to be incorrectly folded forms of the target protein with scrambled disulfides The third isoform, Iso-4, was thought to be reduced IFNα-2b containing four free cysteine sulfhydryls (SH) The level of Iso-4 was observed to decrease during the purification process, suggesting that Iso-4 may refold back to IFN-α-2b Earlier RP -HPLC data provided experimental... an “affinity handle” for subsequent affinity or covalent chemistry-based purification [89–91] Carbodiimidecatalyzed condensation of cysteamine with a phosphate group formed phosphoramidate with a free sulfhydryl group [89] β-elimination of phosphoserine or phosphothreonine followed by Michael addition also generates a free sulhydryl group [90] The free sulfhydryl moiety formed bases for affinity enrichments . interferes with foreign invaders that may cause infections, but also prevents the growth and spread of other diseased 837 HPLC for Pharmaceutical Scientists, . STRATEGIES FOR ANALYSIS OF PROTEINS/PEPTIDES The analytical strategies for protein characterization rely heavily on high- performance liquid chromatography (HPLC)