Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process.

An increasing number of non-mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log10 reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.

Structure-function relationships of the soluble form of the antiaging protein Klotho have therapeutic implications for managing kidney disease.

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and ß-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

Evolution of a comprehensive, orthogonal approach to sequence variant analysis for biotherapeutics.

Amino acid sequence variation in protein therapeutics requires close monitoring during cell line and cell culture process development. A cross-functional team of Pfizer colleagues from the Analytical and Bioprocess Development departments worked closely together for over 6 years to formulate and communicate a practical, reliable sequence variant (SV) testing strategy with state-of-the-art techniques that did not necessitate more resources or lengthen project timelines. The final Pfizer SV screening strategy relies on next-generation sequencing (NGS) and amino acid analysis (AAA) as frontline techniques to identify mammalian cell clones with genetic mutations and recognize cell culture process media/feed conditions that induce misincorporations, respectively. Mass spectrometry (MS)-based techniques had previously been used to monitor secreted therapeutic products for SVs, but we found NGS and AAA to be equally informative, faster, less cumbersome screening approaches. MS resources could then be used for other purposes, such as the in-depth characterization of product quality in the final stages of commercial-ready cell line and culture process development. Once an industry-wide challenge, sequence variation is now routinely monitored and controlled at Pfizer (and other biopharmaceutical companies) through increased awareness, dedicated cross-line efforts, smart comprehensive strategies, and advances in instrumentation/software, resulting in even higher product quality standards for biopharmaceutical products.

Ultrasensitive detection and quantification of acidic disaccharides using capillary electrophoresis and quantum dot-based fluorescence resonance energy transfer.

Rapid and highly sensitive detection of the carbohydrate components of glycoconjugates is critical for advancing glycobiology. Fluorescence (or Förster) resonance energy transfer (FRET) is commonly used in detection of DNA, in protein structural biology, and in protease assays but is less frequently applied to glycan analysis due to difficulties in inserting two fluorescent tags into small glycan structures. We report an ultrasensitive method for the detection and quantification of a chondroitin sulfate disaccharide based on FRET, involving a CdSe-ZnS core-shell nanocrystal quantum dot (QD) streptavidin conjugate donor and a Cy5 acceptor. The disaccharide was doubly labeled with biotin and Cy5. QDs then served to concentrate the target disaccharide, enhancing the overall energy transfer efficiency, with unlinked QDs and Cy5 hydrazide producing nearly zero background signal in capillary electrophoresis using laser-induced fluorescence detection with two different band-pass filters. This method is generally applicable to the ultrasensitive analysis of acidic glycans and offers promise for the high-throughput disaccharide analysis of glycosaminoglycans.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Hexuronic acid stereochemistry determination in chondroitin sulfate glycosaminoglycan oligosaccharides by electron detachment dissociation.

Electron detachment dissociation (EDD) has previously provided stereo-specific product ions that allow for the assignment of the acidic C-5stereochemistry in heparan sulfate glycosaminoglycans (GAGs), but application of the same methodology to an epimer pair in the chondroitin sulfate glycoform class does not provide the same result. A series of experiments have been conducted in which glycosaminoglycan precursor ions are independently activated by electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD) to assign the stereochemistry in chondroitin sulfate (CS) epimers and investigate the mechanisms for product ion formation during EDD in CS glycoforms. This approach allows for the assignment of electronic excitation products formed by EID and detachment products to radical pathways in NETD, both of which occur simultaneously during EDD. The uronic acid stereochemistry in electron detachment spectra produces intensity differences when assigned glycosidic and cross-ring cleavages are compared. The variations in the intensities of the doubly deprotonated (0,2)X(3) and Y(3) ions have been shown to be indicative of CS-A/DS composition during the CID of binary mixtures. These ions can provide insight into the uronic acid composition of binary mixtures in EDD, but the relative abundances, although reproducible, are low compared with those in a CID spectrum acquired on an ion trap. The application of principal component analysis (PCA) presents a multivariate approach to determining the uronic acid stereochemistry spectra of these GAGs by taking advantage of the reproducible peak distributions produced by electron detachment.

Complete mass spectral characterization of a synthetic ultralow-molecular-weight heparin using collision-induced dissociation.

Glycosaminoglycans (GAGs) are a class of biologically important molecules, and their structural analysis is the target of considerable research effort. Advances in tandem mass spectrometry (MS/MS) have recently enabled the structural characterization of several classes of GAGs; however, the highly sulfated GAGs, such as heparins, have remained a relatively intractable class due their tendency to lose SO(3) during MS/MS, producing few sequence-informative fragment ions. The present work demonstrates for the first time the complete structural characterization of the highly sulfated heparin-based drug Arixtra. This was achieved by Na(+)/H(+) exchange to create a more ionized species that was stable against SO(3) loss, and that produced complete sets of both glycosidic and cross-ring fragment ions. MS/MS enables the complete structural determination of Arixtra, including the stereochemistry of its uronic acid residues, and suggests an approach for solving the structure of more complex, highly sulfated heparin-based drugs.

Proteoglycan sequence.

Proteoglycans (PGs) are among the most structurally complex biomacromolecules in nature. They are present in all animal cells and frequently exert their critical biological functions through interactions with protein ligands and receptors. PGs are comprised of a core protein to which one or multiple, heterogeneous, and polydisperse glycosaminoglycan (GAG) chains are attached. Proteins, including the protein core of PGs, are now routinely sequenced either directly using proteomics or indirectly using molecular biology through their encoding DNA. The sequencing of the GAG component of PGs poses a considerably more difficult challenge because of the relatively underdeveloped state of glycomics and because the control of their biosynthesis in the endoplasmic reticulum and the Golgi is poorly understood and not believed to be template driven. Recently, the GAG chain of the simplest PG has been suggested to have a defined sequence based on its top-down Fourier transform mass spectral sequencing. This review examines the advances made over the past decade in the sequencing of GAG chains and the challenges the field face in sequencing complex PGs having critical biological functions in developmental biology and pathogenesis.

The proteoglycan bikunin has a defined sequence.

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan, with a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike nucleic acids and proteins, whose synthesis is template driven, Golgi-synthesized glycosaminoglycans are not believed to have predictable or deterministic sequences. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size-similar and charge-similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular ions corresponding to monocompositional peptidoglycosaminoglycans. Fragmentation using collision-induced dissociation unexpectedly afforded a single sequence for each monocompositional parent ion, unequivocally demonstrating the presence of a defined sequence. The biosynthetic pathway common to all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment in the understanding of information storage in complex glycans.

Structural characterization of heparins from different commercial sources.

Seven commercial heparin active pharmaceutical ingredients and one commercial low molecular weight from different manufacturers were characterized with a view profiling their physicochemical properties. All heparins had similar molecular weight properties as determined by polyacrylamide gel electrophoresis (M(N), 10-11 kDa; M(W), 13-14 kDa; polydispersity (PD), 1.3-1.4) and by size exclusion chromatography (M(N), 14-16 kDa; M (W), 21-25 kDa; PD, 1.4-1.6). one-dimensional (1)H- and (13)C-nuclear magnetic resonance (NMR) evaluation of the heparin samples was performed, and peaks were fully assigned using two-dimensional NMR. The percentage of glucosamine residues with 3-O-sulfo groups and the percentage of N-sulfo groups and N-acetyl groups ranged from 5.8-7.9%, 78-82%, to 13-14%, respectively. There was substantial variability observed in the disaccharide composition, as determined by high performance liquid chromatography (HPLC)-mass spectral analysis of heparin lyase I-III digested heparins. Heparin oligosaccharide mapping was performed using HPLC following separate treatments with heparin lyase I, II, and III. These maps were useful in qualitatively and quantitatively identifying structural differences between these heparins. The binding affinities of these heparins to antithrombin III and thrombin were evaluated by using a surface plasmon resonance competitive binding assay. This study provides the physicochemical and activity characterization necessary for the appropriate design and synthesis of a generic bioengineered heparin.

Controlled Photochemical Depolymerization of K5 Heparosan, a Bioengineered Heparin Precursor.

Heparosan is a polysaccharide, which serves as the critical precursor in heparin biosynthesis and chemoenzymatic synthesis of bioengineered heparin. Because the molecular weight of microbial heparosan is considerably larger than heparin, the controlled depolymerization of microbial heparosan is necessary prior to its conversion to bioengineered heparin. We have previously reported that other acidic polysaccharides could be partially depolymerized with maintenance of their internal structure using a titanium dioxide-catalyzed photochemical reaction. This photolytic process is characterized by the generation of reactive oxygen species that oxidize individual saccharide residues within the polysaccharide chain. Using a similar approach, a microbial heparosan from Escherichia coli K5 of molecular weight >15,000 was depolymerized to a heparosan of molecular weight 8,000. The (1)H-NMR spectra obtained showed that the photolyzed heparosan maintained the same structure as the starting heparosan. The polysaccharide chains of the photochemically depolymerized heparosan were also characterized by electrospray ionization-Fourier-transform mass spectrometry. While the chain of K5 heparosan starting material contained primarily an even number of saccharide residues, as a result of coliphage K5 lyase processing, both odd and even chain numbers were detected in the photochemically-depolymerized heparosan. These results suggest that the photochemical depolymerization of heparosan was a random process that can take place at either the glucuronic acid or the N-acetylglucosamine residue within the heparosan polysaccharide.

Control of the heparosan N-deacetylation leads to an improved bioengineered heparin.

The production of the anticoagulant drug heparin from non-animal sources has a number of advantages over the current commercial production of heparin. These advantages include better source material availability, improved quality control, and reduced concerns about animal virus or prion impurities. A bioengineered heparin would have to be chemically and biologically equivalent to be substituted for animal-sourced heparin as a pharmaceutical. In an effort to produce bioengineered heparin that more closely resembles pharmaceutical heparin, we have investigated a key step in the process involving the N-deacetylation of heparosan. The extent of N-deacetylation directly affects the N-acetyl/N-sulfo ratio in bioengineered heparin and also impacts its molecular weight. Previous studies have demonstrated that the presence and quantity of N-acetylglucosamine in the nascent glycosaminoglycan chain, serving as the substrate for the subsequent enzymatic modifications (C5 epimerization and O-sulfonation), can impact the action of these enzymes and, thus, the content and distribution of iduronic acid and O-sulfo groups. In this study, we control the N-deacetylation of heparosan to produce a bioengineered heparin with an N-acetyl/N-sulfo ratio and molecular weight that is similar to animal-sourced pharmaceutical heparin. The structural composition and anticoagulant activity of the resultant bioengineered heparin was extensively characterized and compared to pharmaceutical heparin obtained from porcine intestinal mucosa.

Heparin mapping using heparin lyases and the generation of a novel low molecular weight heparin.

Seven pharmaceutical heparins were investigated by oligosaccharide mapping by digestion with heparin lyase 1, 2, or 3, followed by high performance liquid chromatography analysis. The structure of one of the prepared mapping standards, ΔUA-Gal-Gal-Xyl-O-CH(2)CONHCH(2)COOH (where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, Gal is ß-d-galactpyranose, and Xyl is ß-d-xylopyranose) released from the linkage region using either heparin lyase 2 or heparin lyase 3 digestion, is reported for the first time. A size-dependent susceptibility of site cleaved by heparin lyase 3 was also observed. Heparin lyase 3 acts on the undersulfated domains of the heparin chain and does not cleave the linkages within heparin's antithrombin III binding site. Thus, a novel low molecular weight heparin (LMWH) is afforded on heparin lyase 3 digestion of heparin due to this unique substrate specificity, which has anticoagulant activity comparable to that of currently available LMWH.

Analysis of E. coli K5 capsular polysaccharide heparosan.

Heparosan is the key precursor for the preparation of bioengineered heparin, a potential replacement for porcine intestinal heparin, an important anticoagulant drug. The molecular weight (MW) distribution of heparosan produced by the fermentation of E. coli K5 was investigated. Large-slab isocratic and mini-slab gradient polyacrylamide gel electrophoresis (PAGE) were used to analyze the MW and polydispersity of heparosan. A preparative method that allowed fractionation by continuous-elution PAGE was used to obtain heparosan MW standards. The MWs of the heparosan standards were determined by electrospray ionization Fourier-transform mass spectrometry (ESI-FT-MS). A ladder of the standards was then used to determine the MW properties of polydisperse heparosan samples. Unbleached and bleached heparosan produced by fermentation of E. coli K5 had similar number-averaged MWs (M(N)), weight-averaged MWs (M(W)), and MW ranges of 3,000 to 150,000 Da.

E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor.

Heparosan is an acidic polysaccharide natural product, which serves as the critical precursor in heparin biosynthesis and in the chemoenzymatic synthesis of bioengineered heparin. Heparosan is also the capsular polysaccharide of Escherichia coli K5 strain. The current study was focused on the examination of the fermentation of E. coli K5 with the goal of producing heparosan in high yield and volumetric productivity. The structure and molecular weight properties of this bacterial heparosan were determined using polyacrylamide gel electrophoresis (PAGE) and Fourier transform mass spectrometry. Fermentation of E. coli K5 in a defined medium using exponential fed-batch glucose addition with oxygen enrichment afforded heparosan at 15 g/L having a number average molecular weight of 58,000 Da and a weight average molecular weight of 84,000 Da.

Domain structure elucidation of human decorin glycosaminoglycans.

The structure of the GAG (glycosaminoglycan) chain of recombinantly expressed decorin proteoglycan was examined using a combination of intact-chain analysis and domain compositional analysis. The GAG had a number-average molecular mass of 22 kDa as determined by PAGE. NMR spectroscopic analysis using two-dimensional correlation spectroscopy indicated that the ratio of glucuronic acid to iduronic acid in decorin peptidoglycan was 5 to 1. GAG domains terminated with a specific disaccharide obtained by enzymatic degradation of decorin GAG with highly specific endolytic and exolytic lyases were analysed by PAGE and further depolymerized with the enzymes. The disaccharide compositional profiles of the resulting domains were obtained using LC with mass spectrometric and photometric detection and compared with that of the polysaccharide. The information obtained through the disaccharide compositional profiling was combined with the NMR and PAGE data to construct a map of the decorin GAG sequence motifs.

Proteoglycomics: recent progress and future challenges.

Proteoglycomics is a systematic study of structure, expression, and function of proteoglycans, a posttranslationally modified subset of a proteome. Although relying on the established technologies of proteomics and glycomics, proteoglycomics research requires unique approaches for elucidating structure-function relationships of both proteoglycan components, glycosaminoglycan chain, and core protein. This review discusses our current understanding of structure and function of proteoglycans, major players in the development, normal physiology, and disease. A brief outline of the proteoglycomic sample preparation and analysis is provided along with examples of several recent proteoglycomic studies. Unique challenges in the characterization of glycosaminoglycan component of proteoglycans are discussed, with emphasis on the many analytical tools used and the types of information they provide.

High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis.

Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6h using continuous elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies.

Stereoselective synthesis of a C-linked neuraminic acid disaccharide: potential building block for the synthesis of C-analogues of polysialic acids.

C-linked neuraminic acid disaccharide was synthesized in a diastereoselective manner from a sulfone donor and aldehyde acceptor, which was protected as a propargyl ether, through a samarium-mediated coupling reaction. The resulting disaccharide has acetal and phenyl sulfide functional groups that can be easily converted into aldehyde and phenyl sulfone groups by photolysis and oxidation reactions to serve as disaccharide acceptor and donor, respectively.