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From this they concluded that both carbon atoms in acetic acid were used for the synthesis of fatty acids and cholesterol symptoms of the flu order discount prothiaden on line. Eventually Rittenberg became director of the isotope laboratory at P & S in 1941 and remained there until he retired treatment zit buy prothiaden 75 mg otc. In addition to using isotopes to study the biosynthesis of cholesterol treatment gout order prothiaden with a visa, Bloch also used the tracers to examine the precursor role of cholesterol in bile acids and steroid hormones treatment plan goals and objectives purchase prothiaden 75 mg with mastercard. However symptoms dizziness nausea cheap prothiaden 75 mg mastercard, Bloch encountered several logistical problems when starting this experiment treatment zap buy prothiaden american express. First, labeled cholesterol was unavailable commercially so he had to spend much of his time introducing deuterium into cholesterol by platinum-catalyzed exchange in heavy wateracetic acid mixtures. Second, the only practical source for isolating the progesterone metabolite pregnanediol in sufficient quantity was from human pregnancy urine, and his request to the P & S department of obstetrics and gynecology for permission to administer labeled cholesterol to one of its patients was denied. In 1946 Bloch moved to the Department of Biochemistry at the University of Chicago and then to Harvard University in 1954. He continued to study fatty acids and cholesterol as well as the enzymatic synthesis of the tripeptide glutathione. Eventually, through the combined 2 All biographical information on David Rittenberg was taken from Ref. H4 Classics efforts of Bloch, John Cornforth, and George Popjafik, the origin of each of the 27 individual carbon atoms of cholesterol (from the methyl or carboxyl group of acetate) was established. Bloch also aided in the identification of several important landmarks in the series of more than 30 reactions in the biosynthesis of cholesterol, including the cyclization of squalene to lanosterol. Knowledge of the biosynthetic pathway for cholesterol eventually aided in the discovery of statins, drugs that interfere with cholesterol synthesis, which are now widely used to treat high cholesterol. Further studies on the biological uptake of deuterium into organic substances, with special reference to fat and cholesterol formation. Lehninger the Relationship of the Adenosine Polyphosphates to Fatty Acid Oxidation in Homogenized Liver Preparations (Lehninger, A. Although his interests soon changed to chemistry, Lehninger would later make use of his writing talents to author three classic textbooks: Biochemistry, the Mitochondrion, and Bioenergetics. Inspired by the work of Otto Warburg and Hans Krebs, Lehninger went on to graduate school at the University of Wisconsin and received his Ph. Witzemann was on the metabolism of acetoacetate and the oxidation of fatty acids by disrupted liver preparations. At the time, much of what was known about glycolysis and the citric acid cycle had been elucidated from minced tissue and tissue extracts. However, similar studies on fatty acid oxidation had been hampered by the fact that ruptured liver cells lost their ability to oxidize fatty acids. Munfioz had some success with liver homogenates at low temperatures in the presence of oxygen, inorganic phosphate, fumarate, cytochrome c, adenylic acid, and magnesium ions (1), but these experiments were not always reproducible. Subsequent work would show that fatty acids are activated by the formation of a thioester linkage between the carboxyl group of the fatty acid and the sulfhydryl group of coenzyme A. During this time, Lehninger discovered several papers on oxidative phosphorylation, and from then on the mechanisms of energy capture and transduction in cells became the central focus of his research. During his 6 years in Chicago, Lehninger and two of his students would make two significant discoveries that would contribute greatly to the study of metabolism. Friedkin would show that electron transport this paper is available on line at. He enrolled at De Paul University in 1937 as a chemistry major and then went to the University of Chicago in 1941 for graduate training in organic chemistry. To pay his tuition, Kennedy also got a job in the chemical research department of Armour and Company, one of the large meat packers in Chicago. As part of the war effort, his job at Armour was to assist in the large scale fractionation of bovine blood to obtain pure bovine serum albumin. It was believed that the bovine serum albumin might be useful for treating shock in soldiers on the battlefield. However, by the end of 1942, hope had faded that bovine serum albumin would be an effective treatment, and the Red Cross started to collect blood from volunteers instead. Armour opened a new facility in Fort Worth, Texas for the fractionation of human blood from donors, and Kennedy was sent to Fort Worth to assist in this effort. He remained in Texas until 1945, when the war was clearly nearing its end and large amounts of human plasma proteins had been stockpiled. Returning to the University of Chicago, Kennedy immediately transferred from the Department of Chemistry to the Department of Biochemistry. His experience on the plasma project had led to a new appreciation of biochemistry. At that time, Lehninger was studying oxidative phosphorylation and fatty acid oxidation. He agreed that this would be desirable, but went on to point out rather gently that fatty acid oxidation had not yet been demonstrated in a soluble extract from which individual enzymes might be isolated. Despite this initial incident, Lehninger agreed to take Kennedy on as a graduate student, and he began to work on the problem of fatty acid oxidation in 1947. Lehninger had observed that both fatty acid oxidation and oxidative phosphorylation were inhibited in a strikingly parallel fashion when particulate enzyme preparations of homogenized rat livers were exposed to hypotonic buffers. The activity could be preserved by adding either salts or iso-osmotic amounts of sucrose to the buffers. These studies led Lehninger and Kennedy to surmise that fatty acid oxidation, oxidative phosphorylation, and the Krebs cycle must all be taking place in one organelle, bounded by a membrane impermeable to certain solutes. Although their enzyme preparations were quite crude, they were convinced that the organelle was the mitochondrion, even though functionally and morphologically intact mitochondria had not yet been isolated. Kennedy immediately tested mitochondria isolated by this method and obtained convincing evidence that oxidative phosphorylation, fatty acid oxidation, and the reactions of the Krebs cycle did indeed occur in the mitochondria. After finishing graduate school, Kennedy went to the University of California, Berkeley, to work with Horace A. Although the initial discovery had already been made, there was much to be learned about these extracts and Kennedy aided in this effort. He then returned to the University of Chicago in 1951, after being given a joint appointment in the Department of Biochemistry and the newly organized Ben May Laboratory for Cancer Research. In Chicago, Kennedy started to study the origins of the phosphodiester bond of phosphatidylcholine using labeled choline. Determined to understand why he and Kornberg had obtained contradicting results, Kennedy, along with his graduate student Samuel Weiss, undertook a detailed examination of the differences between the two studies. They also showed that the two cytidine coenzymes were present in high quantities in liver and yeast. In 1959, Kennedy was invited to become a Hamilton Kuhn Professor and head of the Department of Biological Chemistry at the Harvard Medical School. He continued his research on phospholipid biosynthesis and was able to formulate a detailed picture of the pathways of biosynthesis of the principal glycerophosphatides and of triacylglycerol by 1961. Kennedy is currently at Harvard as the Hamilton Kuhn Professor of Biological 2 Chemistry and Molecular Pharmacology, Emeritus. Preparation of Cell-free Extracts That Catalyze the Conversion of Ethanol and Acetate to Butyrate and Caproate (Stadtman, E. Studies of Monocyclic and Bicyclic Interconvertible Enzyme Cascades, in Situ (Mura, U. When he was 10, his family moved to San Bernardino, California, where he attended high school. After graduating from high school in 1937, Stadtman enrolled in several science courses at San Bernardino Valley College, hoping to eventually set up a soil-testing laboratory. However, he soon realized that he needed a more rigorous education and enrolled at the University of California, Berkeley. After spending a year in Alaska, involved in a wartime project of mapping the AlaskanCanadian (Al-Can) Highway, Stadtman returned to Berkeley looking for work. Around this time, Stadtman also met his future wife, Thressa Campbell, who was working as a laboratory assistant in the food technology department. Since then, Barker had been searching for an explanation for the observation that C. He made a break14 through when he obtained some C and used the isotope to label acetate and demonstrate that fatty acid synthesis is accomplished by the multiple condensation of 2-carbon molecules (1). Barker surmised that acetyl phosphate might be the active acetate formed in the above reaction. Initially, like Barker, Stadtman used C to trace the metabolic pathways in whole cell preparations of C. Gunsalus showed Stadtman how to dry bacterial cells and grind the dried preparations to break open cell walls, producing a cell-free extract. This discovery was especially significant because up until that time most biochemists believed that the capacity to make fatty acids was a unique property of specialized cellular systems or particulate organelles. Unfortunately, at that time, most universities had antinepotism rules that did not allow more than one family member to work in the same department. Intended to protect universities from charges of favoritism, the rules often had the effect of discriminating against married women. No one seriously challenged the rules until the 1960s, when the American Association of University Women began to protest their unfairness. In 1952, he successfully carried out the first in vitro net synthesis of acetyl-CoA using only basic materials (acetyl phosphate, CoA, and phosphotransacetylase). Roy Vagelos also demonstrated that long-chain fatty acid synthesis is catalyzed by an enzyme complex in which malonyl-CoA is the source of active acetate. The activity of glutamine synthetase is subject to feedback inhibition by 7 different end products of glutamine metabolism. In view of these results, Stadtman surmised that glutamine synthetase activity was controlled by a cascade system in which two systems of reversible covalent modification were tightly linked. Each system was composed of two reversible reactions, or two interconvertible enzyme cycles, the linkage of which resulted in the formation of a bicyclic cascade system. This cascade system allowed enzyme activity to be shifted gradually in response to metabolite availability. Boon Chock carried out a theoretical analysis of this bicyclic cascade system to understand its implications in enzyme regulation. Stadtman had discovered that after a freeze-thaw cycle, treatment of Escherichia coli cells with a nonionic detergent rendered them permeable to small metabolites but allowed the cells to retain the protein components of the cascade system. Using these cells, they were able to study the effects of different substrates and allosteric effects on the cascade system and to confirm the previous theoretical and in vitro studies. Stadtman was also President of the American Society for Biological Chemists (now American Society for Biochemistry and Molecular Biology) from 1 1982 to 1983 and has been a member of the National Academy of Sciences since 1969. The aerobic oxidation of ethanol and butyrate with the formation of acetyl phosphate. The activation of molecular hydrogen and the conversion of acetyl phosphate and acetate to butyrate. The phosphoroclastic decomposition of acetoacetate to acetyl phosphate and acetate. Hokin 32 Enzyme Secretion and the Incorporation of P into Phospholipides of Pancreas Slices (Hokin, M. Investigating this phenomenon further, they found that most of the radioactivity was incorporated into the phospholipid fraction. This was a surprising discovery as up until then phospholipids were regarded as inert structural components of membranes. The 32 Hokins incubated pigeon pancreas slices with various compounds along with P to see the effects on phosphate incorporation into phospholipids. They found that when enzyme secretion was stimulated by acetylcholine or carbamylcholine, both of which induce amylase secretion, the 32 incorporation of P into phospholipids was on average 7. Separating individual phospholipids for analysis was difficult at that time, but fortunately Rex Dawson devised a method that permitted the analysis of diacylglycerophospholipids by deacylation and two-dimensional separation of the water-soluble backbone (1). The Hokins used this 32 method to show that hormone stimulation of pancreatic slices mainly increased the rate of P incorporation into phosphoinositide but that phosphatidylcholine, phosphatidylserine, and phosphatidic acid also contained radiolabeled phosphate (2).

Yellowing of the sclerae medications 3 times a day 75 mg prothiaden fast delivery, skin medications that cause high blood pressure order prothiaden on line amex, and oral mucosa are all characteristic of jaundice medicine overdose order prothiaden without prescription, the accumulation of bilirubin treatment neuroleptic malignant syndrome 75 mg prothiaden with amex, the catabolic product of the heme moiety of hemoglobin medicine assistance programs buy 75 mg prothiaden amex. The figure illustrates general preservation of myocardial architecture with some fragmentation symptoms after embryo transfer discount 75 mg prothiaden with mastercard, more intense cytoplasmic staining corresponding to increased cellular eosinophilia, and loss of nuclei, all of which are characteristics of coagulative necrosis. The figure illustrates fatty change of the liver, which is characterized by the accumulation of intracellular parenchymal triglycerides. It is seen most frequently in the liver, heart, and kidney and is commonly secondary to alcoholism. Fatty change results from an imbalance between the uptake, utilization, and mobilization of fat from liver cells. In advanced form, primary (hereditary) hemochromatosis is characterized by the triad of cirrhosis, diabetes, and hyperpigmentation, or so-called bronze diabetes. The disease is most often caused by a mutation in the Hfe gene on chromosome 6 and is characteristically familial rather than sporadic. The skin hyperpigmentation is due largely to increases in melanin and to lesser accumulations of hemosiderin. Metastatic calcification, or deposition of calcium in previously normal tissue, is caused by hypercalcemia. In this patient, tumor metastases to the bone with increased osteolytic activity caused mobilization of calcium and phosphate, resulting in hypercalcemia. Metastatic calcification should be contrasted with dystrophic calcification, in which the serum calcium concentration is normal and previously damaged tissues are the sites of deposition. Myelin figures, cell blebs, mitochondrial swelling, and glycogen depletion are all signs of reversible injury. Nuclear changes such as pyknosis, karyorrhexis, and karyolysis are signs of cell death and are, of course, irreversible. Leukocytes engulf and destroy bacteria, tissue debris, and other particulate material. This is limited by the extent of tissue destruction and by the regenerative capacity of the specific tissue. They are divided into three families: selectins, immunoglobulin (Ig)-family adhesion proteins, and integrins. P-selectins, stored in endothelial Weibel-Palade bodies and platelet alpha granules, relocate to the plasma membrane after stimulation by mediators such as histamine and thrombin. Intercellular adhesion molecules 1 and 2 are expressed on endothelial cells and bind to integrin molecules on leukocytes. Vascular cell adhesion molecules are similarly expressed on endothelial cells and bind to integrin molecules on leukocytes. These changes begin with a brief period of vasoconstriction, followed shortly by dilation of arterioles, capillaries, and postcapillary venules. The resultant marked increase in blood flow to the affected area is clinically manifest by redness and increased warmth of the affected area. Causes include endothelial changes that vary from contraction of endothelial cells in postcapillary venules, with widening of interendothelial gaps, to major endothelial damage involving arterioles, capillaries, and venules. Important causes of neutrophilia (increased neutrophils in the peripheral blood) include bacterial infections and other causes of acute inflammation, such as infarction. The early release of neutrophils into the peripheral blood in acute inflammation is from the bone marrow postmitotic reserve pool. There is often an increase in the proportion of less mature cells such as band neutrophils (Figure 2-1). In older individuals, chronic lymphocytic leukemia is a common cause of lymphocytosis. The most important causes of eosinophilia include allergies such as asthma, hay fever, and hives and also parasitic infections. Important causes of basophilia include chronic myelogenous leukemia and other myeloproliferative diseases. Adhesion occurs as leukocytes adhere to the endothelial surface and is mediated by the interaction of integrins on leukocytes binding to Ig-family adhesion proteins on endothelium. This is the process by which leukocytes are attracted to and move toward an injury. Chemotaxis and other forms of cellular migration are measured in an in vitro system (Boyden chamber technique) that assesses the migration of cells from an upper chamber through a microporous membrane to a lower chamber filled with a chemoattractant. This process is mediated by diffusible chemical agents (Table 2-1); movement of leukocytes occurs along a chemical gradient. It is the coating of particulate material by substances referred to as opsonins, which immobilize the particles on the surface of the phagocyte. Vasoactive amines (1) Histamine mediates the increase in capillary permeability associated with the contraction of endothelial cells in postcapillary venules that occurs with mild injuries. Binding of specific antigen to basophil and mast cell membrane-bound Ige (complement is not involved) 2. Binding of complement fragments C3a and C5a, anaphylatoxins, to specific cell-surface receptors on basophils and mast cells (specific antigen and IgE antibodies are not involved) 3. The complement system consists of a group of plasma proteins that participate in immune lysis of cells and play a significant role in inflammation. C5a is chemotactic, mediates the release of histamine from platelet-dense granules, induces the expression of leukocyte adhesion molecules, and activates the lipoxygenase pathway of arachidonic acid metabolism. This is a cavity filled with pus (neutrophils, monocytes, and liquefied cellular debris). This is the final result of tissue destruction, with resultant distortion of structure and, in some cases, altered function. This change is marked by the replacement of neutrophils and monocytes with lymphocytes, plasma cells, and macrophages. It often includes proliferation of fibroblasts and new vessels, with resultant scarring and distortion of architecture. Because enzyme-deficient neutrophils cannot produce H2O2 and bacterial H2O2 is destroyed by bacterial catalase, H2O2 is not available as a substrate for myeloperoxidase. In effect, the substrate for myeloperoxidase is produced by the bacteria, and the bacteria in a sense kill themselves. Chronic inflammation can occur when the inciting injury is persistent or recurrent or when the inflammatory reaction is insufficient to completely degrade the agent. It occurs in two major patterns: chronic nonspecific inflammation and granulomatous inflammation. A cellular reaction with a preponderance of mononuclear (round) cells (macrophages, lymphocytes, and plasma cells), often with a proliferation of fibroblasts and new vessels. Note the presence of lymphocytes, macrophages, and plasma cells (marked by arrows). Note the absence of caseation in this granuloma taken from a lymph node from a patient with sarcoidosis. The lesion consists of focal accumulations of altered macrophages referred to as epithelioid cells. B lymphocyte activation by macrophage-presented antigen results in the formation of antibody-producing plasma cells. This type of inflammation is characterized by granulomas, which are nodular collections of specialized macrophages referred to as epithelioid cells. Interaction with the antigen-specific T-cell receptor of these cells triggers the release of cytokines (especially, interferon-), which mediate the transformation of monocytes and macrophages to epithelioid cells and giant cells. The presence of multinucleated giant cells derived from macrophages is also characteristic. The langhans giant cell has nuclei arranged in a horseshoe-shaped pattern about the periphery of the cell and is particularly characteristic of, but not specific for, the granulomatous inflammation of tuberculosis. Giant cells, derived from macrophages, are a frequent component of granulomatous inflammation. This typical example with the nuclei arranged in the periphery in a horseshoe pattern is referred to as a Langhans giant cell. Granulomatous inflammation is the characteristic form of inflammation associated with a number of diverse etiologic agents, including: a. It requires that the surviving affected parenchymal cells have the capacity to regenerate. They include cells of the epidermis and gastrointestinal mucosa, cells lining the surface of the genitourinary tract, and hematopoietic cells of the bone marrow. Characteristically, these cells undergo few divisions but are capable of division when activated; that is, they can regenerate from G0 cells when needed. These cells have been considered to be incapable of division and regeneration (a view challenged by recent provocative new evidence involving stem cells). They are replaced by scar tissue (typically fibrosis; gliosis in the central nervous system) after irreversible injury and cell loss. Generally, growth factor receptors are transmembrane proteins that respond to ligand interaction by conformational changes that induce tyrosine kinase activity in their intracellular domains. Fibroblast growth factors promote the synthesis of extracellular matrix protein (including fibronectin) by fibroblasts, endothelial cells, monocytes, and other cells. As the amount of collagen in granulation tissue progressively increases, the tissue becomes gradually less vascular and less cellular. Progressive contraction of the wound also occurs, often resulting in a deformity of the original structure. Metabolic disorders, such as diabetes mellitus (associated with both susceptibility to infection and impaired circulation) 5. Dietary deficiency of ascorbic acid or protein, both of which are required for collagen formation Review Test directions: Each of the numbered items or incomplete statements in this section is followed by answers or by completions of the statement. The leukocyte count is history of progressively worsening produc14,000/mm3, with 74% segmented neutrotive cough, fever, chills, and signs of toxicity. Prominent physical findings include signs Which of the following describes the expectof consolidation and rales over the right ed findings at the affected sitefi Sputum culture is positive for (A) Fistula (abnormal duct or passage) conStreptococcus pneumoniae. An intra-alveolar necting to the abdominal wall exudate filling the alveoli of the involved (B) Granulation tissue (new vessels and portion of the lung is present. Which of the young fibroblasts) with a prominent following types of inflammatory cells is most infiltrate of eosinophils likely a prominent feature of this exudatefi A routine complete blood count perareas of abscess formation formed on a 22-year-old medical student reveals an abnormality in the differential 4. Extensive investigation results in a during the past several weeks and reports diagnosis of chronic granulomatous disease that she frequently had such episodes in the of childhood. Which of the following closely characterizes the abnormality in this cell types is most likely to be increasedfi A 16-year-old boy presents with a 24-hour formation history of severe abdominal pain, nausea, (d) Inability to kill streptococci vomiting, and low-grade fever. The accompanying figure is representative of the findings in a hilar lymph node from a 54-year-old man who sought medical care for low-grade fever, anorexia, fatigue, night sweats, weight loss, and persistent cough with bouts of hemoptysis. A chest x-ray had revealed a right apical infiltrate with beginning cavitation, and examination of the sputum had revealed acid-fast bacilli. This condition is typified by a form of inflammation that invariably includes which of the followingfi In the laboratory, the students (c) fi2-Integrin demonstrate that the compound is a potent (d) P-selectin vasodilator and platelet antiaggregant. A 70-year-old man presents with the sud(A) Labile cells den onset of left-sided weakness, spasticity, (B) Multipotent adult progenitor cells and hyperactive and pathologic reflexes. The (c) Permanent cells most serious consequences of this disorder (d) Stable cells are the result of damage to which of the following cell typesfi In the early stages of acute inflammation, the neutrophil is the most prominent inflammatory cell. It is noteworthy that, in many instances, bacterial infections are characterized by neutrophilic infiltrates. This type of reaction is primarily mediated by the release of histamine from tissue mast cells, and the associated cellular infiltrate and peripheral blood findings represent mobilization and increased numbers of eosinophils. The clinical findings are typical of acute appendicitis, another example of severe acute inflammation. Because the danger of perforation is great, early appendectomy is the treatment of choice. Suppurative or purulent inflammation is characterized by the prominent areas of edema resulting from increased vascular permeability, congestion, and a purulent (pus-containing) exudate consisting of necrotic cells and large numbers of neutrophils. In addition, other signs of acute inflammation, such as congestion, are prominent.

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The research organizations at the time at which the experiments flew are preserved in the appendix of this document. Of the investigations summarized here, some are completed with results released, some are completed with preliminary results, and some remain ongoing. Through predominantly used for the production of therapeutic glycoour own process development and characterization efforts with proteins. Obtaining a consistent glycoform profile in over the past, present, and future state of the control of this production is desired due to regulatory concerns because a very important metabolic pathway. An therapeutics currently on the market, which is the impetus optimal profile may involve a spectrum of product glycans for understanding their metabolic control. Glycosylation optimization will improve culture process, and culture media toward the resulting glycotherapeutic efficacy and is an ongoing goal for researchers form profile. This review will focus on as new attempts toward achieving uniform, consistent, and/or the advancements made in glycosylation control in a manuoptimal glycosylation. These efforts share a common need for facturing process, as well as the next steps in understanding a more fundamental understanding of the metabolic pathway, and controlling protein glycosylation. The ultimate goal would be to Keywords: bioprocessing/mammalian cell culture/protein take the lessons learned from previous studies, combined with glycosylation/protein therapeutics/protein quality new technologies, to transform a well-characterized cell line toward one that can control the glycosylation profile. In order to truly incorporate process quality by design (QbD) into the final Introduction glycoprotein product (Rathore and Winkle 2009), more fundaProtein glycosylation is of paramount importance to the efficacy mental studies on the dependence of glycosylation on process and manufacturing of therapeutic glycoproteins. Mammalian conditions and cellular physiology will be required to gain physcell expression systems are the preferred method for the comiological insight. The information gathered from such research mercial production of these glycoproteins because their innate together with clinical studies will continue to enhance our capaprotein processing machinery, including that of protein glycobility toward generating highly efficacious glycoprotein drugs. Glycosylation of proteins takes on the form of oligosaccharides attached to either the side chain of asparagine (N-linked) Protein glycosylation and product quality or serine/threonine (O-linked) with the former being the most Metabolic pathway 1 the addition of oligosaccharides onto a glycoprotein is a comTo whom correspondence should be addressed: Tel: +1-315-432-2131; e-mail: plex metabolic pathway, characterized by the en bloc transfer zhengjian. In most cosidases and sialyltransferases to facilitate better end-capping industrial bioprocesses, this large repertoire of glycans is typiwith sialic acid to maximize residence time (Kronman et al. Control of this metabolic pathway can that is not typically found in adult humans because it is an onbe manifested through a variety of different conditions, includcofetal antigen (Muchmore et al. The importance of galactose (Gal) monosaccharides attached N-Linked Glycosylation. The workhorses most often used in have been shown with monoclonal IgG1 antibodies, where there mammalian cell culture processes. In contrast to N-glycans, which have a common core pentasaccharide structure, O-glycans have up to eight different core structures that make their study and experimental measurement more difficult. The final glycoprotein forms can be variable depending on the level of sialylation and terminal sugars. The level of antennarity and sialylation will be dependent on cell culture conditions and expression levels of each enzyme. Both molecular controls (mutants, knockouts, loss or gain-of-function) and cell culture parameters can modulate the final form for a glycoprotein. The following are known to modulate the glycoform: (1) Gnt Ifi/ Lec1 Mutant, (2) fi(1. In some cases, this immune recognition is desired the reticulo-endothelial system, which relies on several recepfor targeted drug delivery. Corresponding enzyme molecules without this modiN-Acetylglucosamine also plays a principal role in determinfication are quickly removed from circulation by hepatocytes ing N-glycan antennarity and the resulting level of terminal gly(Furbish et al. This is especially true in IgG, the backbone molecule predict due to lack of consensus recognition sequences; howof most therapeutic monoclonal antibodies (Rademacher 1993). Most ular glycoprotein, matching those attributes on the recombinant reports to date document the role of O-glycans in the glycobiologic is not trivial. A universal set of acceptance limits does not exist, tion on the properties of recombinant glycoproteins is the culbut what is universal is the idea of comparability, and the fact mination of each of its glycosylation sites, which may have that the process must provide for it for batch release of the mavarying, or even opposing infiuences depending on its glycoterial. It has been classified into two types as a result: the molecule remains the same (Woodcock et al. In industry, predicting the manufacfacturing process on the protein product (Schenerman et al. Overall sialylation was increased in the perfusion mode cultures compared There have been numerous strides toward understanding and imto fed-batch mode, the slower growing cells in perfusion mode proving protein quality control, including a better understanding facilitated a more fully glycosylated protein compared to the of molecular engineering and cell culture processes on the refed-batch mode where cells grew faster. Documented reports have shown both direct and inverse relationships between that of protein synthesis rates and the reProcess strategies sulting protein glycosylation. This the Golgi facilitated a more fully glycosylated structure (Wang process mapping is a fine example of range finding efforts that et al. Dissolved oxygen levels are monitored critical in designing new protein therapeutics. Bioreactor pH has been found to affect protein glycosylation (see #11 in Figure 3). The culture pH of a hybridoma cell Manufacturing mode line has been shown to affect the resulting galactosylation and the three most common cell culture production modes are sialylation of the monoclonal antibody (Muthing et al. The production method can the highest levels of agalacto and monogalacto complex Nhave a pronounced effect on the resulting glycoform profile glycans were measured at pH 7. Effect of cell culture process variables and media on glycosylation Figure 3 legend Variable Effect on glycosylation References 9 Low glucose/glutamine Glycosylation of monoclonal antibodies by human (Hayter et al. Cellular and protein engineering effects on glycosylation Figure 3 legend Variable Effect on glycosylation References 1 GnT I Knockout creates high-mannose-type IgG1 antibody (Kanda et al. Cell viability is critpool in glucoseand glutamine-limited cultures (Nyberg et al. Galactose feeding can help facilitate a more fully galacmedium and step-wise remove monosaccharides from the glytosylated N-glycan profile (Andersen 2004). Temperature shifts in a production bioreactor can dia to induce a desired cellular behavior, such as high speincrease volumetric product titers while maintaining glycoform cific productivity. It is had an overall decrease in product quality through increased still unknown whether higher productivity correlates to the exmicroheterogeneity, reduced sialylation, and decreased in vivo pression rate, thereby, reducing the intracellular processing time activity (Sung et al. Indeed, the pleiotropic effects of for glycosylation and causing an increase in less sialylated prosodium butyrate on the gene and protein expression level of tein population. High-cell viability (less sialidase activity) in mammalian cells have been well documented (Yee et al. Supplementan important engineering variable for the resulting glycoform ing additional amino acids (cysteine, isoleucine, leucine, trypprofile (see #20 in Figure 3). These undefined components include peptones, sialylation on tetrasialylated glycans by 41%, and the proyeast extracts, plant hydrolysates, and serum. The literature has portion of tetraantennary glycans by 37%, and with less than reported the effects of some of these undefined components on 30 mM ammonia decreased tetrasialylated glycans by 73%, protein glycosylation (Gawlitzek et al. A and the proportion of tetraantennary glycans by 57% (Yang monoclonal IgG1 produced by mouse hybridoma in serum-free and Butler 2002). Protein engineering strategies Cellular engineering strategies Inserting additional N-linked sites has been proven to be clinA number of strategies have been attempted to modify the ically beneficial for recombinant glycoproteins. Understanding these the above studies all attempted to introduce more of a pardifferences may require a more in-depth physiological underticular enzyme or eliminate enzyme function through cellular standing of the mammalian cell responses to a particular set engineering; however, these studies merely controlled absoof conditions, as well as the detailed glycoprotein characterizalute expression levels. Achieving uniform and/or consistent protein glycosylation views highlighting various structural requirements for Golgimay be characterized by a certain set of gene/protein expresspecific enzyme localization (Grabenhorst and Conradt 1999). There may be a set of physiologizymes to enhance the glycosylation machinery at key protein cal requirements that need to be met within a particular clone processing organelles. Once this could be identified, it would serve as bisected hybrid-type glycans (Ferrara et al. The optimal glycoform at each glycosylation site of a protein sugars, but more such studies are necessary. We would also like to thank species, and its overall intrinsic contribution toward efficacy or Dr. Such an analysis would also be All authors were employed by Bristol-Myers Squibb & Co. The scientific studies reviewed in the previous section all attempted to analyze only one component of the protein glycosylation pathway, which is more likely simultaneously dependent Abbreviations on the interplay of many components toward the determination of the final glycoform profile (see Figure 3). The effect of ammonia on the O-linked for early-stage processes that are amenable to parental cell line glycosylation of granulocyte colony-stimulating factor produced by Chinese modifications. Biotechnol Cell-free synthesis of enzymically active tissue-type plasminogen activator. Optimisation of the cellular metabolism of glycosylation for reoligosaccharide content with in vivo clearance profile. The macroheterogeneity of erythropoietin in the circulation: the role of carbohydrates. Temperature effects on productIgG antibody with anti-alpha-galactosyl specificity. Glycosylation of recombinant protein therapeutics: Contion and stability in the Golgi. Improvement of interferon-gamma sialylation in Chinese nant adeno-associated virus type 2 by transfection of 293 suspension cells. Large-scale purification of human by Chinese hamster ovary cell culture using serum-free media. Oligosaccharide specific endocytosis by isoN-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids. GlycoVis: Visualizing glycan Effect of constant and variable domain glycosylation on pharmacokinetics distribution in the protein N-glycosylation pathway in mammalian cells. Mori K, Kuni-Kamochi R, Yamane-Ohnuki N, Wakitani M, Yamano K, Jenkins N, Castro P, Menon S, Ison A, Bull A. Getting the glycosylation right: ImpliMori K, Iida S, Yamane-Ohnuki N, Kanda Y, Kuni-Kamochi R, Nakano R, cations for the biotechnology industry. The primary site of hydroxylation of Jones D, Kroos N, Anema R, van Montfort B, Vooys A, Van Der Kraats S, N-acetylneuraminic acid is the cytosolic sugar nucleotide pool. Effects of buffering conditions and culture pH on Julenius K, Molgaard A, Gupta R, Brunak S. Prediction, conservaproduction rates and glycosylation of clinical phase I anti-melanoma mouse tion analysis, and structural characterization of mammalian mucin-type IgG3 monoclonal antibody R24. Nat Rev Drug Kanda Y, Yamada T, Mori K, Okazaki A, Inoue M, Kitajima-Miyama K, Discov. Immunogenicity of biological activity among nonfucosylated therapeutic IgG1 antibodies with N-glycolylneuraminic acid-containing carbohydrate chains of recombinant three different N-linked Fc oligosaccharides: the high-mannose, hybrid, human erythropoietin expressed in Chinese hamster ovary cells. Metabolic effects on recombinant interferon-gamma glycosylation in conBiochemistry. Infiuence of intracellular nucleotide and nucleotide sugar contents anN-terminalextensionprolongsthehalf-lifeandincreasestheinvivoactivon recombinant interferon-gamma glycosylation during batch and fed-batch ityoffolliclestimulatinghormone. Hierarhuman alpha-(1,3)fucosyltransferase gene into Chinese hamster ovary chy of post-translational modifications involved in the circulatory longevity cells. Species-specific variation sylation of a monoclonal antibody produced under nominally identical cell in glycosylation of IgG: Evidence for the species-specific sialylation and culture conditions in two different bioreactors. Glycoengitration in serum-free continuous culture affects N-linked glycosylation of a neering of therapeutic glycoproteins: In vitro galactosylation and sialylation monoclonal antibody. Conceptsandprinexpressed in Chinese hamster ovary cells in high productivity cultures. Biologacetyllactosamines attached to lysosomal membrane glycoproteins are inicals. Challenges in therapeutic glycoprotein activity of Bowes melanoma and human colon fibroblast derived tissue production. Impactofdynamic thesis inhibitors on the glycosylation site occupancy of recombinant human online fed-batch strategies on metabolism, productivity and N-glycosylation prolactin. Glycoengineering: the effect of glycosylation on antibody-dependent cellular cytotoxicity.

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