PEG–protein conjugates

4 PEGeprotein conjugates: nonclinical and clinical toxicity considerations Peter L. Turecek1, Jürgen Siekmann2 1

BAXALTA INNOVATIONS G MB H, PART OF TAKEDA GROUP OF C OMPANIES, VIENNA, A US T R I A ; 2 FORMER EMPLOYEE OF BAXALTA INNOVATIONS GMB H, PART OF TAKEDA GROUP OF COMP ANIES, VIENNA, AUSTRIA

1. Introduction Polyethylene glycol (PEG) is a linear polyether of ethylene glycol usually synthesized by polymerization of ethylene oxide. It is a chemically inert, amphiphilic polymer of ethane-1,2-diol units that can be produced in various chain lengths and molecular weights. PEG has been used for decades as an excipient in cosmetics, consumer products, and also in pharmaceutical technology. There it serves not only as a formulation excipient for drugs but also as a drug itself in laxatives and, importantly, as a conjugate with drug molecules [1]. For modification of biopharmaceuticals including proteins such as enzymes, proenzymes, coenzymes and antibodies, peptides, and nucleic acids, PEG became particularly relevant in the development of so-called “next-generation” biologicals. In this context, PEG was and is still used to improve the pharmaceutical and medical use of biological drug molecules by covalent and noncovalent attachment often referred to as PEGylation. This technology primarily aims to prolong the drug’s half-life in circulation, to decrease plasma clearance, and/or to alter biodistribution, in comparison with non-PEGylated versions, thereby decreasing the dosing frequency required to maintain a therapeutic level [2]. Since 1990, more than 20 PEGylated biologicals have been approved with diverse clinical indications in the United States and Europe [3e6] and there are several more expected in the near future as there are approximately 20 other drug candidates in preclinical or clinical development (see for example Chapter 1 of this same book). Over the last 25 years, PEG has not been a subject of interest for intensive toxicological evaluation and analyses because of its nature as an inert molecule. This has recently changed with the advent of PEGylated proteins for chronic anddat least theoreticallydlifelong use in prophylactic therapies. Some of the newer developments in this field were primarily aiming to improve the quality of treatment by reducing the number of applications required in prophylactic therapy rather than improving Polymer-Protein Conjugates. https://doi.org/10.1016/B978-0-444-64081-9.00004-8 Copyright © 2020 Elsevier B.V. All rights reserved.

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pharmacodynamics. Many of these drug molecules are exceptionally safe and very well-tolerated. Therefore, an increase in convenience of treatment with such therapies may only be considered acceptable if the safety profiles would not be less favorable than before. Thus, clinicians, representatives of regulatory bodies, and industry scientists started to consider toxicity of PEG and PEG-drug conjugates. A review of publicly available toxicology information for approved PEGylated biopharmaceuticals indicated that their toxicological effects were derived from the active part of the drug substance rather than the PEG moiety [7]. Here we review the existing knowledge about safety and toxicity of PEG and its derivatives with focus on therapeutic PEGeprotein conjugates.

2. Toxicity of polyethylene glycol, its precursors, and impurities PEG, by the European Pharmacopoeia (EP) called “macrogols,” are mixtures of polymers with the general formula He(OCH2eCH2)neOH where n represents the average number of oxyethylene groups. The type of macrogol is defined by a number that indicates the average relative molecular mass [8]. As such, in the EP, PEGs in a molecular range from 300 to 35,000 Da are described, representing liquids in the low molecular mass range of up to 600 Da and solid substances with a waxy or paraffin-like appearance of above 1000 Da. The EP states that ethoxylated substances may contain varied amounts of ethylene glycol and diethylene glycol (DEG), as result of the manufacturing process. The reason why pharmacopoeias request quantitative determination of those substances and limit this impurity, e.g., for liquid PEGs at 0.4% [8] is that ethylene glycol and DEG are substances with relevant toxic effects where intoxications have been described as consequence of common technical and household use. Ethylene glycol, a common antifreeze, coolant, and industrial solvent, is responsible for many instances of accidental and intentional poisoning annually. Ethylene glycol, 1,2-ethanediol, has molecular formula C2H6O2 and a molecular mass of 62.07 g/mol. It is a sweet tasting, viscous, nonvolatile, colorless, and very hygroscopic liquid. Following ingestion, ethylene glycol is first hepatically metabolized to glycoaldehyde by alcohol dehydrogenase. Glycoaldehyde is then oxidized to glycolic acid, glyoxylic acid, and finally oxalic acid. While ethylene glycol itself causes intoxication, the accumulation of toxic metabolites is responsible for the potentially fatal acidosis and renal failure, which characterizes ethylene glycol poisoning [9]. Ethylene glycol is poorly absorbed by dermal and pulmonary routes but is readily absorbed from the gastrointestinal tract. Peak serum ethylene glycol concentrations occur 1e4 h after ingestion. Ethylene glycol is not protein bound and, because it is highly water-soluble, distributes evenly throughout body tissue with a volume of distribution of approximately 0.5e0.8 L/kg [10]. Approximately 80% of an absorbed dose of ethylene glycol is hepatically metabolized, with the remainder renally excreted unchanged. In the rhesus monkey, the kidney excretes 0.5%e10% of a dose of ethylene glycol as calcium oxalate [11]. The average elimination half-life of ethylene glycol is about 3 h. Typically symptoms of intoxications by ethylene glycol are

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caused by direct action on the central nervous system (CNS) and, predominantly, actions of substances generated by enzymatic metabolism such as the final oxidation product oxalic acid and its calcium salt which precipitates in various tissues including the brain, heart, lungs, and the kidneys, in which oxalate crystals are seen in the proximal renal tubules resulting in lethal kidney failure [12e14]. The estimated lethal dose of pure, undiluted ethylene glycol for an adult is approximately 1.4 mL/kg bodyweight (BW) [15]. Ethylene glycol poisoning is a medical emergency requiring immediate and aggressive treatment. Appropriate treatment includes aggressive supportive care, proper use of the antidotes fomepizole (4-methylpyrazole) or ethanol to inhibit the synthesis of toxic metabolites, hemodialysis when necessary to enhance the elimination of unmetabolized ethylene glycol and its toxic metabolites, and adjunctive therapy aimed at conversion of ethylene glycol to nontoxic metabolites. Intoxication by the other PEG impurity, DEG, is less common as this chemical is not in typical household use. DEG, 2-(2-hydroxyethoxy) ethanol or 3-oxapentan-1,5-diol, has molecular formula C4H10O3 and a molecular mass of 106.12 g/mol. DEG is a clear, colorless, practically odorless, viscous, hygroscopic liquid with a sweetish taste. As consequence of its use in a wide range of industrial products and as an impurity in poorly purified drug substances, it has also been involved in several fatal poisonings. Although the mechanism of toxicity is not clearly elucidated, it was suggested that the enzymatic metabolism of DEG to 2-hydroxyethoxyacetic acid (HEAA) is the major contribution to renal and neurological toxicities. The clinical effects of DEG poisoning are gastrointestinal symptoms with evidence of inebriation, metabolic acidosis, and evidence of emerging renal injury, like the symptoms seen by ethylene glycol toxicity which, in the absence of appropriate supportive care, can lead to death. If patients are stabilized, they may show various delayed neuropathies and other neurological effects, sometimes fatal [16]. Doses of DEG necessary to cause human morbidity and mortality are not well established. They are based predominantly on reports following some epidemics of mass poisonings, which may underestimate toxicity. The mean estimated fatal dose in an adult has been defined as approximately 1 mL/kg of pure DEG. Management of the DEG poisoning is to some extent also similar to treatment of patients after ethylene glycol exposure and requires attention to acidebase abnormalities. Prompt use of fomepizole or ethanol is important in preventing the formation of the toxic metabolite HEAA. Hemodialysis can also be critical, and assisted ventilation may be required [16]. PEG can be synthesized by polymerization of ethylene oxide (1,2-epoxyethane, oxirane) by alkaline catalysis. Reaction sometimes is initiated by ethylene glycol or ethylene glycol oligomers, which is then catalyzed by acidic or basic conditions [17]. Ethylene glycol and its oligomers may be preferred as starting materials because it allows us to obtain polymers with narrow molecular weight distribution and a low polydispersity. PEGs and PEG derivatives do not represent definite chemical entities but are mixtures of compounds with varying polymer chain lengths. Polydispersity of PEG should be as close as possible to 1 to yield an acceptable homogeneity of the final drug,

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which is particularly important when PEG is bound to heterogenic biological molecules [18]. Irrespective of its synthesis process, polymerization results in formation of repeated ether bonds in PEG. Ethers are characterized by high chemical stability where cleavage may only occur under strongly acidic or high basic conditions. Such conditions do not occur in biological systems, and particularly not in mammals. Ether bonds may be cleaved enzymatically; however, ether cleaving enzymes (etherases) have rarely been described in mammals. Typical ether cleaving organisms are bacteria, molds, or fungi [19,20]. Therefore, PEG polymers are considered noncleavable in mammals. Only recently it was found that cytochrome C, best known for its role in transferring electrons from complex III to complex IV while bound to the inner membrane of the mitochondrion, which is ubiquitously found in almost all species may, with help from an activating lipid and appropriate oxidants, cleave vinyl ether groups. This action of cytochrome C could explain the catabolic processing of plasmalogensdphospholipids containing a characteristic vinyl ether groupdwhich are precursors of lipids important for cellular signaling such as arachidonic acid [21,22]. If this activity would also work on ether bonds in PEG polymers has not been shown but there is not any observation pointing into this direction. Consequently, PEG could be considered an inert polymer not cleaved or metabolized in human cells, tissues, and organs, very much in contrast to monomeric ethylene glycol. As result of its inertness, PEG is described as nontoxic. PEG compounds are used in a great variety of cosmetic applications because of their solubility and viscosity properties and because of their low toxicity. Exposure of humans to PEG by cosmetics and health care products is massive. For example, cosmetic products contain PEG of various sizes in concentrations of about 3% in styling products, 5% in bath and shower products, 10% in toothpaste and shampoos, 25% in foundation and makeups, 30% skincare creams and lotions, and 90% in bath oils. The PEGs, their ethers, and their fatty acid esters produce little or no ocular or dermal irritation and have extremely low acute and chronic toxicities. They do not readily penetrate intact skin, and in view of the wide use of preparations containing PEG and PEG derivatives, only few case reports on sensitization reactions have been published, mainly involving patients with exposure to PEGs in medicines or following exposure to injured or chronically inflamed skin [23]. PEG is orally ingested when used in laxative therapy. Due to high prevalence of chronic constipation in individuals of all ages [24,25] with functional constipation having a prevalence of 9.5% (95% confidence interval (CI) 7.1e12.1) in children [26], and chronic idiopathic constipation having a median prevalence of 14% (95% CI 12e17) in adults and the associated impaired health-related quality of life, treatment of chronic idiopathic and functional constipation often includes adjunctive treatment with a laxative, generally an osmotic or bulk-forming agent [27,28]. Osmotic laxatives increase the volume of intestinal fluids to the stools, improving the progression of the stool in the colon, as well as improving defecation [29]. Such pharmaceutical preparations include PEG of molecular weights around 4000 Da. Most currently used products have PEG 3350 as active ingredient (average size 3350 Da). These PEG preparations are indicated as first-line osmotic treatments for chronic constipation in children and adults and for use

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for bowel cleansing before colonoscopy [29]. Typical standard doses of PEG range between 10 and 30 g/day. PEG was very well-tolerated as shown in many clinical trials in infants, preschool children, older children, adults, and elderly patients [30]. In children aged 0.5e15 years in clinical trials and postmarketing reports, most treatment-emergent adverse events (TEAEs) were minor, transient, and primarily involved the gastrointestinal system. The only common TEAEs were abdominal pain and diarrhea which may cause perianal soreness and uncommon TEAEs include vomiting, abdominal distension, and nausea. In adults, additional uncommon TEAEs include urgency to defecate and fecal incontinence [31]. All these effects do not represent any toxic reactions but can be explained by the osmotic mode of action of PEG. Long-term treatment with macrogol 4000 was also well-tolerated in open-label trials in children [32] or adults [33] with chronic constipation. Rare cases of anaphylaxis following the use of high molecular PEGs have been reported, with most being related to their use as lavage solutions for bowel preparation. The Allergy Vigilance Network recorded five cases of severe PEG-related anaphylaxis from 2010 to 2012 [34e36]. It is not known if PEG is absorbed from the gastrointestinal tract following oral ingestion. However, because of its amphiphilic nature, it may be expected that some small amounts of PEG would be found in the blood stream. Nevertheless, quantitative determination of PEG levels of humans exposed to oral PEG is missing. PEG is also directly injected into tissues and blood vessels upon use of PEG as an excipient in parenteral therapies. Small PEGs below 10,000 Da are selected as excipients because of their low toxicity, favorable solubility, and low viscosity [4]. Furthermore, high molecular weight PEGs (PEG 20,000 or 35,000 Da) are currently used as additives in liver preservation solutions before transplantation to limit the damage associated with cold ischemia reperfusion and prevent cell swelling and lipid oxidation [37e39]. Another important group of excipients used in pharmaceuticals are PEG derivatives used as detergents, primarily to enhance solubility and bioavailability of formulated drug substances. These derivatives of PEG include polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), and Poloxamer 188 (Pluronic F-68). PEG is a part of the chemical structure of these excipients and it was found to be released as a PEG derivative [40,41]. Some of these compounds are also used as emulsifiers in food preparations. Fig. 4.1 shows the structure of polysorbate 80, which is one of the most frequently used detergents as excipient for parenterally used drugs, primarily peptides and proteins. A comprehensive review investigated the occurrence of adverse events (AEs) that might be caused by PEG and PEG derivatives also including excipients [1]. Annual exposure to excipient PEG calculated for a 50 kg adolescent patient was between 100 and 1000 mg/year with most products where PEG was contained. Some drug products also used in children, intravenous (IV) immunoglobulins and plasma-derived coagulation factor VIII, formulated with PEG derivatives resulted in exposures of more than 10,000 mg/year. Because of the lack of clinical AEs that could be attributed to PEG, the authors looked for similarities between AEs to identify any trends. Safety outcomes were investigated by searching the medical literature, regulatory documents, and Internet sources for any report of clinical

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FIGURE 4.1 Chemical structure of detergent-bound polyethylene glycol (PEG) in polysorbate 80 (Tween 80). PEG has a formula of H(OeCH2eCH2)nOH. The PEG portions of the molecule are highlighted.

PEG-related AEs and by attempting to identify any salient pattern of AEs listed in the prescribing information or reported to the US Food and Drug Administration (FDA) or other reporting organization that might reflect PEG toxicity. Because to date there were no defined PEG-related AE, they looked to see if there were any similar AEs that can be attributed to PEG within the product prescribing informations, FDA website, general Internet searches, and websites that report AEs. After making extensive lists of the most common AEs, rare AEs, and postmarketing AEs, the authors conducted specific Internet searches for the therapy name along with renal, hepatic, CNS, or allergic symptoms. This comprehensive search of data in the public domain neither revealed any AEs specifically attributed to PEG exposure nor identified a pattern of events that may plausibly have been connected to PEG exposure. Despite paying attention to organ systems that appeared to be particularly affected by ultrahigh dose PEG exposure in preclinical toxicology studies (e.g., the CNS, kidney, liver), no pattern of AEs suggestive of possible PEG toxicity could be detected. PEG, as such, has two equivalent hydroxyl groups at each end of the polymeric chain. These hydroxyl groups remain reactive and can be oxidized and could also act as potential cross-linking agents. Therefore, reactivity resulting in toxic effects cannot entirely be excluded. In a few cases, PEG demonstrated oxidizing ability leading to incompatibility with other drug molecules such as the antibiotics bacitracin and benzylpenicillin [42]. All PEG polymers and derivatives for direct pharmaceutical use as laxatives or excipients contain PEG preparations with hydroxyl groups. Nevertheless, toxic effects related to potential reactivity have never been reported. By introduction of a terminal methoxy ether, undesired reactivity and cross-linking reactions in the PEGylation process can be omitted. Thus, nowadays as PEG reagents for the preparation of PEG protein conjugates exclusively “endcapped” methoxylated PEGs are used, which are also preferred to avoid cross-linking reaction during the preparation of the conjugates. In the literature these are referred to as “mPEG.”

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3. Polyethylene glycol clearance mechanism Absorption, distribution, metabolism, and excretion from the organism are important determinants for the safety of a compound. The pharmacokinetic, metabolism, and distribution of PEG after parenteral application were recently discussed in detail by Baumann et al. [43]. Briefly, PEG was shown to be readily distributed and excreted in several animal species. PEG size is an important determining factor for circulation time and excretion pathway [44]. Some reports referring to enzymatic metabolism of PEG molecules via alcohol (ADH) and aldehyde dehydrogenase might be misinterpreted as explained above [45,46]. Because PEG represents a family of compounds with large variations in terminal groups and size, it is important to distinguish literature covering PEGs with free hydroxy groups from references discussing “endcapped” PEGs used to modify pharmaceuticals. Still, PEG may be metabolized at its terminal ends, but the ratio is rendered negligible because of modification of the PEG chain ends with, for example, methoxy capping [43]. Ether linkages connecting ethylene glycol subunits in the PEG chain are highly stable in vivo because of the absence of etherases in eukaryotes as described. Nevertheless, formation of PEG peroxides in vitro is known to occur when PEG solutions are stored in the presence of oxygen [24]. PEG chains may be cleaved by free radicals in vivo after pino- or phagocytosis of PEG or PEG-containing particles when reactive oxygen species are present in the phagosome or lysosome, but at a minor rate, with no toxicological relevance [47]. For nonglycosylated globular proteins, the threshold for glomerular filtration is approximately 70 kDa. PEG molecules differ from globular proteins as PEG chains are highly hydrated, which amplifies the hydrodynamic radius of PEG in aqueous solutions. Furthermore, the flexible and linear PEG molecules can migrate through glomerular pores despite their large polymer size, a process referred to as “reptation” (lat. reptaredto creep) [47]. Therefore, no clear threshold for glomerular filtration can be determined for PEG. However, renal clearance is significantly slower for nonconjugated PEGs larger than 30 kDa [44]. A PEG size >30 kDa triggers increased halflife and favors other than renal clearance pathways, such as hepatic clearance or pinocytosis/phagocytosis by cells of the mononuclear phagocyte system (MPS), also known as the reticuloendothelial system (RES) or lymphoreticular system. MPS cells can engulf particles by a process called phagocytosis to form an intracellular phagosome, which fuses with the lysosome, the compartment involved in the breakdown of cellular components, thereby generating the phagolysosome. Inside the phagolysosome, contents are subsequently degraded and released intra- or extracellularly for further processing or elimination. PEG molecules with increasing molecular weight tend to have higher rates of cellular clearance; similarly, macrophage uptake of the inert polymer polyvinyl alcohol tends to increase with increasing molecular weight [48]. It has been established that the physicochemical properties of a particle’s surface, such as surface charge, size, functional groups, and hydrophobicity, affect the uptake of particles by phagocytic cells. In vitro experiments with nanoparticles coated with PEG of varying molecular weight showed

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that the lower negative charge of the surface after PEGylation reduced the uptake by macrophages [49]. PEG inside of phagocytic cells may be released into the circulation by decomposition of cell membranes after apoptotic decay of the phagocytic cell or by exocytosis [43], renal or biliary excretion follows. The vasculature of the liver is composed of discontinuous capillary walls that allow small and large polymeric molecules such as PEG to diffuse from the blood to the extravasal region. Elimination might also occur via paracellular pathways, such as transepithelial movement across intercellular junctional complexes [50]. Liver clearance via parenchymal cells leading to excretion via bile and feces increases with increasing molecular weight. Nonclinical studies for approved PEGylated proteins have demonstrated that PEG and PEGylated proteins distribute to various compartments [43]. Variability in the distribution pattern of PEGylated proteins, their metabolism, and excretion complicate our ability to clearly understand the specific effects of PEGylation. However, where proteolysis and kidney clearance are the primary degradation routes for the parent protein, increasing overall size of the PEGylated conjugate will limit renal clearance. When proteins themselves are too large for renal clearance, the protein will dominate the clearance mechanism. There appear to be no explicit rules with respect to PEG size in the case of clearance by receptor-mediated processes. Thus, the variation in nonclinical data from available distribution studies in FDA and EMA (European Medicines Agency) databases [51,52] is considered attributable to the different biological components of the conjugates, be it a protein or DNA of varying size, and to the size and quantity and dose of the PEG molecules used for chemical modification. Examples for PEG distribution and clearance studies are described further below for human coagulation factor VIII products modified with different PEG polymers.

4. Immunogenicity of polyethylene glycol In literature PEG sometimes is described as a nonantigenic and nonimmunogenic moiety and as being capable of reducing immunogenicity of immunogenic proteins by masking antigenic sites [17,53]. However, earlier immunization studies in animals demonstrated that PEGylated proteins induce anti-PEG antibodies [54]. In these studies, PEG was found to be nonimmunogenic when administered alone but acquired immunogenic properties when conjugated to an immunogenic protein. The immunogenic potential of PEG was found to be directly related to the immunogenic nature of the protein. For this reason, PEG was considered to be a polyvalent hapten, which is a small molecule that can induce an immune response only when attached to a large carrier molecule, for example, a protein. Review of newer literature tells that PEG and PEGylated proteins can be indeed immunogenic. Van Helden et al. presented evidence that PEGylation might even increase immunogenicity of proteins, depending on the amount of PEG bound to a protein [55]. The authors showed in a transgenic hemophilic mouse strain which expresses human coagulation factor VIII as a transgene and, therefore,

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recognizes the native human factor VIII as self-protein, that a high PEGylation degree (12 mol PEG per mol protein) induced break of immune tolerance by PEGylated human factor VIII. On the other hand, a low amount of bound PEG (2 mol PEG per mol protein) did not alter the immunogenicity of factor VIII and maintained immune tolerance against human factor VIII. In a similar study, the induction of PEG antibodies by PEGylated factor VIII was also investigated [56]. Two different mouse models were used to assess the immunogenicity of PEGylated factor VIII compounds, a transgenic hemophilic mouse model which expresses human factor VIII as a transgene and, therefore, tolerize human factor VIII as self-protein, and a conventional hemophilic mouse model that recognizes the human factor VIII as foreign protein. Only those mice that recognized factor VIII as a foreign protein developed PEG antibodies. The authors concluded that the development of PEG antibodies depends on the presentation of immunogenic CD4þ T cell epitopes provided by the protein. These data support the hapten concept for the immunogenicity of PEG. The immunological principles of antihapten antibody responses are that B cells recognizing the hapten in a hapten-carrier protein complex take up the whole complex, generate CD4þ T cell epitopes from the carrier protein, and present these epitopes to CD4þ T cells via MHC-class II expressed on their surface [57,58]. Another explanation for the development of PEG antibodies could be crosslinking of B cell receptors recognizing repetitive epitopes within the repetitive sequence of the PEG polymer. Antibody responses induced by strong cross-linking of specific B cell receptors can be generated without help of T cells. It requires multiple identical epitopes that are spaced regularly, as in PEG, to allow simultaneous interaction of multiple B cell receptors with each epitope. This signal is sufficiently strong to induce T celleindependent antibody responses [59,60]. IgM is the major isotype resulting from T celleindependent antibody responses as sometimes seen in immune responses to PEG. Additionally, antibody class switch can also occur under these conditions. For example, T celleindependent induction of PEG antibodies has been closely studied following administration of PEGylated liposomes [61e63], which was found being triggered by splenic B cells resulting predominantly in IgM-type anti-PEG antibodies [64]. These antibodies were transient and did not mature into a long-lived IgG response [65]. IgM immune complexes are efficient activators of the complement system and can not only clear antigen from the blood stream but also induce hypersensitivity through buildup of the complement anaphylatoxins. Several reports have documented the development of anti-PEG antibodies following treatment with PEGylated proteins or liposomes and have shown that the occurrence of anti-PEG antibodies results in reduced treatment effect and related hypersensitivity. The reduced efficacy is caused by accelerated clearance from the blood. It is seen after repeated exposure to the PEGylated drug molecule and is sometimes associated with the development of anti-PEG IgM antibodies and subsequent complement activation [62]. Formation of anti-PEG antibodies associated with rapid blood clearance and loss of therapeutic effects was, for example, observed in patients treated with PEG-uricase and PEG-asparaginase (PEG-ASN) [66,67].

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Formation of IgM anti-PEG antibodies in about 50% of patients undergoing hyposensitization with PEGylated allergens has been reported [68]. Recently, a similar phenomenon has also been seen when patients were treated with PEGylated B domainedeleted coagulation factor VIII, which is described separately in a section below. In contrast, other PEGylated biologics have proven to be safe and efficacious and have not been associated with the development of anti-PEG antibodies. Despite some hypotheses, it remains unclear why some PEGylated therapeutics induce a clinically relevant anti-PEG antibody response, whereas others do not. Some important factors may involve the molecular size of the PEG moiety, linker type, number of attached PEG polymer chains, and/or the overall immunogenicity of the protein itself [62,69]. Anti-PEG antibody formation has also been seen in healthy individuals and patients who have not received PEGylated drug molecules. This suggests that exposure to PEG polymers and PEG polymer containing molecules like detergents of the polysorbate and poloxamer types frequently contained in cosmetics and pharmaceuticals daily consumed by most people could also cause anti-PEG antibody formation [70e72]. The most systematic study so far available investigating the prevalence of anti-PEG antibodies in human subjects who have not been knowingly treated with PEGylated drug molecules was published by Lubich et al. [73]. The authors established validated robust antibody analytics and screened two cohorts of healthy individuals, more than 1000 healthy plasma donors, and one cohort of 110 subjects with congenital hemophilia A and hemophilia B patients for the expression of anti-PEG antibodies. Hemophilia patients were chosen because products of recombinant coagulation factors VIII and IX used for on-demand treatment or prophylaxis are frequently formulated with PEG containing excipients or, if derived from human plasma, may carry small but undefined amounts of PEG used in the manufacturing process. What the authors found was that IgM and/or IgG anti-PEG antibodies are expressed by some healthy individuals and by some patients with hemophilia A and B who have not received PEGylated biotherapeutics. Prevalence of anti-PEG antibody formation in healthy individuals was found at 23%e24%, which confirmed results obtained from a smaller group of blood donors published before [74]. The majority of positive individuals had antibody titers of <1:80, which were too low to be confirmed for specificity, and thus could represent either specific or multireactive antibodies. Nevertheless, a proportion of healthy individuals (4%e9%) showed anti-PEG antibodies of titers 1:80, with confirmed specificity for PEG tested with probes of differently sized linear or branched PEG derivatives. Prevalence in hemophilia patients was lower. Also, significant differences were found in the prevalence according to age with higher levels in younger patients. Relevant geographic differences were also found only in anti-PEG IgG but not in anti-PEG IgM levels (Table 4.1). While the reasons for these differences remained speculative, the major finding of the study was that persistent anti-PEG antibodies do not present a safety risk. Even high-affinity anti-PEG antibodies did not cross-react with human tissue when tested against a standard panel of different human tissues. None of these antibodies had a pathogenic character

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Table 4.1 Anti-PEG antibody formation in healthy individuals is common: prevalence of IgG and IgM anti-PEG antibodies in plasma donors in different plasma centers. Plasma center (state)

Anti-PEG IgG donors (%)

Anti-PEG IgM donors (%)

Ammon (ID) Austria (A) Elkhart (IN) Fargo (ND) Lakeland (FL) Laredo (TX)

w30 w8 w8 w22 w8 w0.5

w12 w12 w12 w10 w12 w10

The prevalence of IgG and IgM anti-PEG antibodies in plasma samples from healthy human subjects from different US locations and from Austria is shown. Plasma samples from 100 healthy individuals were analyzed from each center. From Lubich C, Allacher P, de la Rosa M, Bauer A, Prenninger T, Horling FM, et al. The mystery of antibodies against polyethylene glycol (PEG) - what do we know? Pharm Res 2016;33(9):2239e49.

even when anti-PEG responses were persistent. The authors therefore concluded that the occurrence of anti-PEG antibodies is not associated with any pathology.

5. Preclinical safety evaluation of PEGylated biologicalsdchemistry and toxicological assessment of approved PEGylated drugs Nonclinical development of new drug molecules includes both general toxicity studies and specific safety and efficacy studies which are highly dependent on the substance and its indication. Modification of biologicals, primarily proteins, by PEG is most often used to improve bioavailability and pharmacokinetics of otherwise short-lived drug molecules. Therefore, preclinical evaluation of such enhanced drug substances is guided by the knowledge obtained on safety and toxicity of the parent molecule without modification. Thus, no general guidance can be given on preclinical safety evaluation of PEGylated biologicals as the studies are driven by each molecule. Over decades of use, PEG has been recognized as safe without leaving specific toxicological concerns. Consequently, only a few protocols exist which particularly address PEG toxicity and are also in the public domain. Here some examples of different therapeutic proteins modified with different PEG derivatives using different coupling chemistries are described as also outlined in a recent comprehensive review paper [3]. PEG-INTRON is a mono-PEGylated interferon a2b synthesized using an acylating PEG reagent, which is a 12 kDa succinimidyl carbonate PEG (mPEG-SC). The mPEG-SC reagent forms a covalent carbamate linker with amine groups on the protein. PEGINTRON is a mono-PEGylated protein conjugate and consists of 14 positional isomers. The distribution of positional isomers is dependent on the PEGylation reaction conditions and activity of resultant positional isomer conjugates depends on the position of

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PEG coupling on the protein. The final product PEG-Intron is modified at various lysines, histidines, cysteine, serine, and tyrosine [75] and therefore represents a quite heterogeneous PEGylated protein, although almost 50% of PEG is coupled to histidine 34. The acute and chronic toxicity of PEG-Intron was assessed in mice, rats, and monkeys. A 4-week monkey toxicity study with subcutaneous dosing every other day and several studies with subcutaneous and intramuscular administration was conducted. In addition, different to most toxicology programs conducted for the marketed drugs, mPEG alone was tested in acute intravenous (IV) and subcutaneous studies in mice and rats, in 13-week toxicology studies in rats (subcutaneous administration twice weekly at doses up to 2276 mg/m2/week) and monkeys ( subcutaneous administration twice weekly at doses up to 2276 mg/m2/week) and in embryoefetal development studies in rats and rabbits. No cellular vacuolation or other effects related to PEG were seen (PEGINTRON EMA EPAR, FDA SBA). KRYSTEXXA (pegloticase) is another PEGylated protein modified with an acylating PEG reagent. Krystexxa is mammalian engineered uricase (urate oxidase) used to treat gout. Humans do not naturally express the enzyme and nonmammalian sources of the enzyme are highly immunogenic. PEGylation of the mammalian-derived enzyme was therefore intended to mitigate some of the immunogenicity but does not eliminate it completely [76e78]. The exact number of PEGs bound to the protein varies according to the literature. The protein is a tetramer with a molecular weight of 136 kDa consisting of four 34 kDa subunits. According to the Biological License Applications (BLA) submission [79], each subunit has approximately nine molecules of 10 kDa PEG after lysine conjugation, resulting in approximately 360 kDa of total PEG per drug molecule with a total conjugate size of approximately 500 kDa. PEGylation of uricase in KRYSTEXXA uses modification with an acylating PEG reagents resulting in a carbamate linkage between PEG and the enzyme [3,77]. Pivotal nonclinical IV toxicology studies were conducted in dogs, while shorter-term studies were conducted after subcutaneous and/or intramuscular administration in rats and dogs [80]. Dose- and time-dependent cellular vacuolation of some cells attributed to PEG was observed. In dogs, observations of vacuolated cells in the splenic red pulp (RES) were found at all doses (0.5, 1.5, 5.0 mg/kg/week) in a 12-week study without reversal after 6 weeks of treatment-free period. Partially reversible vacuolation was also seen in the spleen of the rat. Vacuolation was considered nonadverse because of the lack of cell damage or inflammatory infiltration. However, another review concluded that the effect should be evaluated further. In a 39-week study, followed by 12 weeks of recovery in dogs (0.4, 1.5, 10 mg/kg/week intravenously), cellular vacuolation was observed in hematoxylin and eosin stained tissues, mainly in the high dose in the adrenal cortex, duodenum, jejunum, liver Kupffer cells, and heart without recovery of vacuolation after 12 weeks. Detailed immunohistochemistry (IHC) evaluation determined that PEG and uricase were present in vacuoles and tissue of macrophages in the gastrointestinal tract both in duodenum and jejunum, liver, and spleen. Vacuoles observed in the adrenal cortex and heart may not be

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associated with macrophages. There was also an indication of uricase and/or PEG staining in these organs not inside vacuoles. Importantly, IHC staining determined that the presence of uricase and PEG in vacuoles and tissues at the end of the recovery period was absent or reduced. It was assumed that vacuoles in macrophages were not of toxicological relevance while the impact of the vacuoles in adrenal cortex and heart was unclear and additional information may be needed. In the same experiment, intravascular and interstitial proteinaceous fluid was collected after the animals were sacrificed, from organs where vacuolation was seen and analyzed for uricase and PEG staining (KRYSTEXXA FDA, SBA). At the end of the dosing period, a dose-dependent increase in staining was seen for both the enzyme and PEG. Staining was mostly absent after 12 weeks of recovery. Vacuolation was also observed in a rat study in one group treated with a dose of 10 mg/kg and in all groups at 40 mg/kg. No other effects or any health impact because of the vacuolization was seen in any of the animals. To further assess vacuolation, cells from rats dosed for 4 weeks with Krystexxa doses of 4.3, 10.2, and 34 mg/kg were tested in vitro. Significant decreases in the tumor necrosis factor (TNF) response to a lipopolysaccharide (LPS) challenge were seen in total splenocytes and macrophages. The effects were most prominent in the high-dose group but were also observed for the lower dose groups. As there were no vacuoles reported in the low- and middose groups, it was difficult to establish a functional relationship between TNF release upon LPS challenge and the presence of vacuoles. Vacuolation of nonphagocytic cells of the aortic endothelium and the adrenal cortex along with the reduced TNF response to LPS was discussed in the available regulatory information and resulted in the US prescribing information. CIMZIA (certolizumab pegol, CDP879): CIMZIA is a PEGylated anti-TNF recombinant antibody Fab fragment approved for treatment of chronic, moderate-to-severe rheumatoid arthritis, Crohn’s disease, and axial spondyloarthritis. CIMZIA has an engineered thiol for PEGylation. The antibody fragment produced in a microbial E. coli system is monovalent and then bound to a 40 kDa PEG. A PEG2MAL 40 kDa PEG reagent is bound covalently through an inserted cysteine moiety, located three amino acids from the C-terminus of the heavy chain antibody fragment [81e85]. CIMZIA is one of two thiol engineered PEG conjugate currently marketed. Acute and repeated-dose toxicity studies (acute, 4, 13, 26, and 52 weeks with either IV or subcutaneous dosing once weekly) were conducted in cynomolgus monkeys. PEGrelated histological changes were observed mainly in the RES. Macrophage vacuolation with presence of foamy macrophages in several organs like lymph nodes, injections sites, red pulp of spleen, adrenal cortex, uterus, cervix, and choroid plexus of the brain was seen after 26 weeks at 100 mg/kg and after 52 weeks at 50 and 100 mg/kg CIMZIA. These changes did not lead to functional deficits and were reversible within 13 weeks, except at the high dose of 100 mg/kg in the 52-week study (CIMZIA EMA EPAR, FDA SBA). Similar macrophage changes were seen in the rat, where CIMZIA is not pharmacologically active. Therefore, it can be concluded that the macrophage changes were

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caused by PEG and not by exaggerated pharmacological action of the antibody. CIMZIA was cleared from the circulation via proteolysis of the protein component Fab0 and renal excretion of PEG polymers [86]. In vitro macrophage function assays with CIMZIA indicated that the PEG moiety alone reduced phagocytosis of bacteria and fungi at high concentrations above the intended pharmacological use (no-observed-effect level [NOEL]: 1.0 mg/mL); some inhibition of T cell proliferation to a toxoid challenge in a human cell system was also seen (NOEL: 1.0 mg/mL) [85]. Although vacuole formation at high doses was seen in vivo, no adverse effects were attributable to the vacuoles [3].

6. Chemistry and toxicity of three PEGylated recombinant human coagulation factor VIII products modified with three different polyethylene glycol reagents Replacement therapy with human coagulation factor VIII (FVIII) has been standard of care for patients with hemophilia A since the 1960s. FVIII is a low abundant labile protein in human plasma present in a concentration of approximately 100 ng/mL. It is therefore difficult to purify and represents a precious fraction derived from human plasma. Consequently, at the beginning of replacement therapy, it had been only used in so-called on-demand treatment protocols to treat acute bleeds. Following cloning of the FVIII gene, it became one of the first proteins ever expressed in mammalian cell lines to produce a recombinant human protein analog. Recombinant FVIII (rFVIII) products were introduced into hemophilia therapy in the early 1990s, thus increasing availability of the drug. With the increased supply of FVIII products and the evolvement of therapeutic protocols, it became more and more used in prophylactic therapy to very effectively prevent bleeding in hemophilia patients and to improve long-term treatment outcomes [87]. FVIII is a relatively short-lived protein with a plasma half-life of approximately 11e14 h [88]. Therefore, prophylaxis with FVIII required redosing at least every second day which makes treatment cumbersome for patients. As consequence, next-generation rFVIII with extended circulatory half-life were developed and introduced into therapy starting around 2015. Meanwhile, several such products had been approved by regulatory authorities for use in hemophilia patients. PEGylation was the preferred approach to improve and prolong the plasma half-life of rFVIII. At this moment, three different rFVIII products are available which are used for very similar indications. Besides differences in the rFVIII protein moiety, these differ primarily in the PEG reagents and coupling chemistries used to generate rFVIII products with extended half-lives (EHL) [89e91]. Similarities and differences between the three products are summarized in Table 4.2. All three products have been tested in comparable clinical trial protocols. Summaries of the product characteristics and clinical trial results are described in Chapter 8 by Siekmann and Turecek in this same book. Outcomes of these clinical studies and the clinical trial enabling nonclinical in vitro and animal studies and the resulting licensed therapeutic labels allow comparison of these products with emphasis on the influence of the different PEG reagents on safety and efficacy.

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Comparison of PEGylated human coagulation factor VIII products.

Product name/INN

ADYNOVATE, ADYNOVI/ rurioctocog alfa pegol

JIVI/damoctocog alfa pegol

ESPEROCT/turoctocog alfa pegol

Year of first approval 2015 Manufacturer Takeda/Baxalta Human FVIII source Full length rFVIII

2018 Bayer B domainedeleted rFVIII

Modification strategy PEG conjugated to primary amine residues PEG size and 20 kDa branched structure No PEG size above theoretical threshold for vacuolation Indication Hemophilia A patients (adults and children), treatment and prophylaxis Intended clinical dose 40e50 IU (IU rFVIII/kg bodyweight) Route-duration IV-chronic

PEG conjugated via cysteine substitution in A3-domain 60 kDa branched

2019 NovoNordisk rFVIII with truncated Bdomain GlycoPEGylation to 21 aa Bdomain 40 kDa branched

Yes

Yes

Hemophilia A patients (12 years of age and older), treatment and prophylaxis 60 IU

Hemophilia A patients (adults and children), treatment and prophylaxis 25e75 IU

IV-chronic

IV-chronic

The first EHL rFVIII product introduced into hemophilia therapy employing PEGylation technology was ADYNOVATE. The product was first licensed for human use in the USA in 2015 and in 2017 also by EMA under the brand name ADYNOVI. Preceding initiation of human clinical studies, the product underwent a comprehensive nonclinical safety assessment with several pharmacological and toxicological studies. The product was tested against ADVATE as a comparator because ADVATE has the same full-length rFVIII protein component while ADYNOVATE is the PEGylated version of this protein modified with a 20 kDa branched PEG reagent resulting in a conjugate of an average molar ratio between PEG and protein of approximately 2 (mol/mol) [92]. Coupling of the PEG reagent is performed by an amine targeting chemistry by which lysine residues of rFVIII having Ɛ-amino groups form stable amide attachments with the 20 kDa branched PEG consisting of two 10 kDa arms per attachment site. With an array of biochemical assays, it was proven that functionality of rFVIII remains unchanged upon PEGylation. Pharmacokinetics of ADYNOVATE was determined in FVIII knockout mice, rats, macaques, and inbred dogs with hemophilia A. An approximately 1.5-fold increased half-life of ADYNOVATE was seen when compared with ADVATE in different animal models. To a similar extent, efficacy to control bleeding was maintained longer in FVIII knockout mice. Safety pharmacology was assessed by measuring thrombogenicity in the Wessler test in rabbits with a maximum dose of 900 IU FVIII/kg. The compound was found being not thrombogenic and comparable to ADVATE [93]. The product was well-tolerated at a maximum dose of 600 IU FVIII/kg in macaques to assess

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cardiovascular and respiratory safety with no adverse test itemerelated findings [93]. In a 4-week repeated dose toxicity study after IV application of 700 IU FVIII/kg in rats which were dosed 15 times every other day, no signs of toxicity or test itemerelated adverse effects could be observed. The same result was seen also in macaques [94]. A comparative immunogenicity study of ADYNOVATE and ADVATE was performed in mice and macaques which received 8 and 40 mg protein per kg once weekly in eight consecutive doses. These studies showed a similar immunogenicity profile as the non-PEGylated ADVATE [95]. A distribution, metabolism, and excretion study with tritium (3H) radiolabeled PEGylated rFVIII investigated the distribution and excretion of the conjugate after a single high dose given intravenously to male and female rats. Radiolabeling of the PEGeprotein conjugate with a tritium label directly on the PEG was achieved by a pioneering chemical synthesis. This approach was in contrast to previous studies with radiolabeled PEGylated biotherapeutics where the radiolabel had been on the protein moiety or the linker between PEG and the biologically active constituent. Therefore, this study with ADYNOVATE for the first time allowed to monitor how a PEGylated protein PEG was distributed and eliminated traced by the PEG moiety [96]. Single doses of 1 and 2 mg PEGylated FVIII protein per kg body weight (BW) corresponded to FVIII activities of 2088 and 4176 IU per kg BW, respectively. These activity doses were accompanied by PEG doses of approximately 0.12 and 0.24 mg PEG per kg BW, respectively. Urine, feces, and selected tissues were collected up to 1008 h (6 weeks) postdose. PEG-rFVIII was well-tolerated in all rats. The distribution of drug-derived radioactivity was extensive, with the highest concentrations of radioactivity observed in plasma, blood, mesenteric lymph nodes, spleen, liver, adrenal glands, and kidneys. However, after high doses of radiolabeled PEG-rFVIII, which were 25e50-fold of the maximum clinical dose of 80 IU FVIII per kg, only marginal radioactivity levels were measured in the brain and spinal cord over 6 weeks. These data demonstrated that even at very high doses of ADYNOVATE, PEG did not reach the brain or related tissues at clinically relevant levels. Furthermore, radioactivity was completely excreted within 6 weeks, indicating that PEG metabolized to the respective PEG acid derivative of the branched PEG reagent used for modification of rFVIII that had been distributed into tissues was subsequently redistributed to the circulation and excreted. Based on these results, the authors established a model for degradation and elimination of PEG-rFVIII (Fig. 4.2). Besides the low toxicity of PEG, it was known from previous studies with other PEGylated drugs that there was a remaining concern about PEG stemming from animal model toxicological studies where repetitive dosing at very high supratherapeutic levels led to vacuolation in particular cells and tissues. PEG-related toxicity and formation of vacuoles were therefore also specifically addressed in the toxicology program for ADYNOVATE/ADYNOVI. In single and repeated dose toxicity studies in rats and monkeys, the product was well-tolerated at doses up to 700 IU FVIII/kg where assessment of toxicology was based on BW determination, clinical signs, ophthalmic examinations, hematology, coagulation, clinical chemistry, urine analysis, macroscopic findings at necropsy, and histopathological findings. Toxicokinetics and antibody assessment were

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FIGURE 4.2 Hypothetical degradation and elimination pathways of polyethylene glycol (PEG)-rFVIII conjugate of a branched 20 kDa PEG covalently attached to full-length rFVIII using an amine modification chemistry. Following the degradation of the proteinaceous FVIII moiety to peptides and amino acids, PEG acid is the final degradation product, which is primarily eliminated via the kidney and liver. From Stidl R, Fuchs S, Bossard M, Siekmann J, Turecek PL, Putz M. Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates. Haemophilia 2016;22(1):54e64.

also included. ADYNOVATE did not reveal any adverse clinical symptoms or findings that could be clearly attributed to the test item. No vacuoles were found in the choroid plexus of PEG-rFVIII exposed animals. However, two animals showed vacuolation in the kidney, but only in the middose group (350 IU FVIII), which did not recover after 2 weeks. It was notable that no vacuolation was detected in the low- and high-dose groups. Observation of unspecific vacuoles is a typical background finding mainly in monkey studies. In other repeated dose toxicity studies with the same PEG-rFVIII in rats and cynomolgus monkeys, no macroscopic, microscopic, or clinical findings suggestive

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of CNS toxicity were observed at any time point during the studies [97]. In addition, a study had been performed with the aforementioned PEG acid, the expected final degradation product of PEG-rFVIII. This compound was investigated in a 28-day repeated dose toxicity study in rats. Animals received IV doses of 0.65, 6.5, and 65 mg per kg of PEG acid twice weekly for a total of eight doses. Dose levels selected were based on an estimation of the cumulative lifetime exposure of w6.5 mg PEG per kg BW considering an ADYNOVATE dose of 80 IU FVIII per kg BW twice weekly over 70 years. All doses were well-tolerated with no negative clinical observations, effects on BW or food consumption, ophthalmoscopy, or clinical or anatomical pathology. There were no macroscopic or microscopic findings including no signs of cell vacuolation with 10,000fold exposure compared with PEG administered during prophylactic therapy with ADYNOVATE moiety [96]. The nonclinical studies for ADYNOVATE described here enabled human clinical studies in pediatric and adult patients with hemophilia A as further described in Chapter 8 in this book. These studies so far include five completed studies, Phase 1, pivotal Phase 2/3, pediatric Phase 3, Phase 3 surgery, and a Phase 3 PK-guided dosing study, and two ongoing studies, the Phase 3b continuation and the Phase 3 study in previously untreated patients (PUPs) [98e100]. These studies revealed a safety profile of ADYNOVATE as expected for a recombinant FVIII protein used for replacement therapy in humans with hemophilia A and seemed comparable to the parent non-PEGylated product ADVATE, although in most of the studies not directly compared. No adverse reactions associated with PEG accumulation were seen in the submitted clinical trials. With regard to the safety of PEG and PEGylated pharmaceuticals, the EMA Committee for Medicinal Products for Human Use (CHMP) Safety Working Party discussed the risk for ependymal cell vacuolation caused by PEG (mainly >40 kDa) at an exposure of 0.4 mmol/kg/month. For ADYNOVI, the clinical PEG exposure of 6.4 mg PEG/kg BW (70 IU) per day ranges 125 times below the threshold of 0.4 mmol/kg/month [97]. All FVIII products generate antibodies against FVIII as these are xenogeneic proteins for patients who are deficient in FVIII and do not produce cross-reacting protein making them tolerant against FVIII [101]. Immunogenicity towards FVIII in ADYNOVATE cannot be finally assessed, as the PUP study has not been completed. However, antibodies against PEG occurred only rarely in clinical trials with ADYNOVATE. According to an interim updated safety data set (because clinical studies were ongoing at the time when this chapter was written), most subjects (238/243) in an integrated analysis did not develop a persistent binding antibody response against FVIII, PEG-FVIII, PEG, or host proteins during the studies. Only 5 of the 243 subjects developed binding antibodies to PEG, PEG-FVIII, or FVIII at study completion, at several visits including study completion, or at data cutoff, but are still under evaluation [97]. Based on the current data, no conclusion could be drawn whether these antibodies were of transient or persistent nature, but none seemed to be directly triggered by PEG alone.

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Ultimately clinical trials performed with ADYNOVATE resulted in market authorization with country-specific labels. According to US FDA ADYNOVATE, antihemophilic factor (recombinant), PEGylated, is a human antihemophilic factor indicated in children and adults with hemophilia A with congenital factor VIII deficiency for on-demand treatment and control of bleeding episodes, perioperative management, and routine prophylaxis to reduce the frequency of bleeding episodes, without specific warnings related to the products nature as a PEGylated protein [102]. Concerns about safety related to PEG upon lifelong chronic use came up when results were published indicating that another coagulation factor product with EHL, glycoPEGylated recombinant factor IX (nonacog beta pegol, N9-GP, REFIXIA), resulted in a vacuole formation when tested in supratherapeutic doses in the rats [103]. Nonacog beta pegol is a 40-kDa polyethylene glycosylated human recombinant coagulation factor IX, intended for the treatment of hemophilia B. Long-term toxicity of this compound was investigated in an immune-deficient, athymic rat (Rowett nude; Crl:NIH-Foxn1rnu). Rats (n ¼ 216) were given IV nonacog beta pegol 0, 40, 150, 600, or 1200 IU FIX/kg every fifth day for 26 weeks. Standard toxicity end points were unaffected by treatment. However, IHC staining revealed PEG in choroid plexus epithelial cells in a dose-dependent manner. Transmission electron microscopy showed that PEG was distributed in cytoplasmic vesicles of these cells, with no apparent effect on cellular organelle structures. Despite PEG containing vacuoles were seen in brain tissue of the rats, nonacog beta pegol was generally well-tolerated, with no adverse effect of PEG on choroid plexus epithelial cells. This prompted an in-depth investigation of the corresponding rFVIII product, ESPEROCT (turoctocog alfa pegol, N8-GP), which is a recombinant FVIII with a truncated B-domain modified by the same enzymatic glycoPEGylation technology with a 40 kDa PEG reagent as REFIXIA [104]. ESPEROCT was tested in hemophilia A dogs. A dose level of 125 IU FVIII/kg normalized prolonged whole blood clotting time (WBCT) and thromboelastogram (TEG) reaction time from greater than 40 min to within the normal range (5e12 min) for canines. The return to baseline WBCT and TEG values were delayed for HA dogs administered ESPEROCT compared to NOVOEIGHT, the nonPEGylated B domainetruncated rFVIII counterpart, suggesting a prolonged duration of activity for ESPEROCT. These findings were supported by additional pharmacology studies conducted in standard tail vein transection, tail clipping, joint bleed, saphenous vein and -induced bleeding models in HA mice. Repeat administration of ESPEROCT once every fourth day in immunocompromised rats over 52 weeks followed by a 12-week recovery period did not result in notable toxicities at dose levels up to 1200 IU/kg. IHC staining did not indicate the presence of the PEG moiety in brain tissues, including the choroid plexus or brain blood vessels, of animals after 52 weeks of repeat administration of 1200 IU/kg of ESPEROCT. However, the lowest detection limit for IHC was not determined and the presence of PEG below this detection limit could not be excluded. No dose-dependent test article-related histopathological changes were noted in any tissues compared to control animals. The no-observed-adverse-effect level (NOAEL) was

80 Polymer-Protein Conjugates

established at 1200 IU/kg for ESPEROCT administered intravenously every fourth day. This dose level is approximately 20-fold higher than the proposed prophylactic clinical dose levels (50e60 IU/kg twice weekly). Genotoxicity, carcinogenicity, and reproductive and developmental toxicity studies were not conducted with ESPEROCT. This was found acceptable based on the product class and the absence of adverse histopathological findings in the reproductive organs in the toxicology studies [105]. Like glyco-PEGylated recombinant factor IX, REFIXIA, pharmacokinetics, tissue distribution, and excretion of ESPEROCT were studied in detail in animals. In addition, the 40 kDa PEG reagent had been included as another test article in these studies [106]. The biologic fate of the 3H-PEG-moiety incorporated into N8-GP was evaluated based on single IV bolus doses to rats. Furthermore, the 40 kDa 3H PEG-moiety was given separately to rats by single IV bolus doses to investigate if the pharmacokinetics were dose-dependent. For both compounds, plasma pharmacokinetics, distribution, and excretion pathways were investigated based on total radioactivity measurements in doses of 3HeN8-GP: 0.17e4.1 mg/kg, equivalent to about 1300e30,000 U FVIII/kg with a PEG load of approximately 0.03e0.7 mg/kg. The 3H-PEG reagent was applied to rats in doses of 0.6, 1, 12, 100, and 200 mg/kg. After single IV administration to rats, both 3 HeN8-GP and 3H-PEG were shown to be widely distributed, mainly in highly vascularized tissues, with the lowest levels of radioactivity found in the CNS. Although a slow elimination of radioactivity was observed over the 12-week study period, approximately half of the radioactive dose of either compound was removed from the body 1-week postdose. The radioactivity was eliminated mainly not only via the kidney into urine but also via the liver into feces, with a larger fraction found in the feces for 3H-N8-GP. Elimination of the 40 kDa PEG-moiety was shown to be dose-dependent with faster elimination at lower dose levels. The clinical dose of N8-GP of 50e75 IU FVIII/kg provides a substantially lower PEG exposure, equivalent to a PEG load of <0.002 mg/kg, when compared to the PEG doses investigated in this study (0.03e200 mg/kg). This may imply an even faster clearance of the PEG-moiety after N8-GP administration of clinically relevant doses. Overall, the study showed that the finding of PEG containing vesicles in neuronal tissues in animal toxicology studies with glyco-PEGylated FIX was specific for the modified FIX protein. It would not be unlikely that the extravascular appearance of FIX triggered by an affinity to collagen type IV, which seems to be unique for coagulation FIX [107], caused deposition of PEG in the choroid plexus of the brain in rats at very high doses while a similar phenomenon was absent for PEG-FVIII. In contrast to FIX, FVIII circulates in blood tightly bound to its chaperon protein von Willebrand factor (VWF) (many references, e.g., Ref. [108]), preventing the FVIII molecule and PEGylated FVIII proteins from uptake in neuronal tissues. Further experimental verification would be needed to foster this hypothesis. Pharmacokinetics and pharmacodynamics of N8-GP in comparison to nonPEGylated B domainetruncated rFVIII NOVOEIGHT were investigated in hemophilia A dogs [109] and in hemophilic mice model, as well as in rabbits and cynomolgus monkeys [104]. In both FVIII- and VWF-deficient mice, the half-life was found to

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increase with the size of PEG. In vivo potency and efficacy of N8-GP conjugated with a 40-kDa PEG and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys. However, the prolongation was less pronounced in rats. On average, half-life improvement compared to the same nonPEGylated truncated rFVIII was about 1.5-fold, common with the other two PEGylated rFVIII products. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. Following favorable nonclinical safety assessments, human clinical trials with ESPEROCT were performed as described in Chapter 8 in this book. Safety and efficacy of ESPEROCT have been evaluated in five multinational, open-label trials in male subjects with severe hemophilia A (<1% endogenous factor VIII activity). One trial was subsequently partially randomized to evaluate two different prophylaxis regimens. All subjects were previously treated, which was defined as having received other factor VIII products for 150 exposure days for adolescents and adults and 50 exposure days for pediatric subjects. The key exclusion criteria across trials included known or suspected hypersensitivity to trial or related products and known history of factor VIII inhibitors or current inhibitor 0.6 Bethesda units (BU) [110]. The clinical trial program was largely similar as described above for ADYNOVATE. It consisted of a two phase 1 PK studies, two studies in adult 12 years (n ¼ 186) and pediatric (n ¼ 68) previously treated patients (PTPs), and a surgery study, enabling approval for human use [111e115]. Phase 3 extension studies in adult and pediatric PTPs and PUPs are ongoing. The product was also tested for subcutaneous administration in 50 subjects. This study was completed but publication of results is pending. Therefore, a conclusive safety assessment including evaluation of immunogenicity in PUPs cannot be made at this stage. However, the clinical profile indicated safe use in humans comparable to other unmodified and modified rFVIII products. Five severe adverse events (SAEs) in four patients (5.9%)dtwo patients had treatment related SAEsdwithdrew from the study (allergic reaction and moderate hemorrhagic symptoms). All patients who had SAEs recovered. No FVIII inhibitor development was observed, two patients had positive nonneutralizing, N8-GPbinding antibodies, 1 of these patients had low-titer antibodies before N8-GP exposure. About 31.4% of patients tested positive for anti-PEG antibodies before the first N8-GP exposuredmatching the prevalence of anti-PEG-antibodies described in paragraph “Immunogenicity of PEG” of this chapterdone of these patients became positive after N8-GP exposure, with low-titer anti-PEG antibodies. Anti-PEG antibodies were not shown to affect FVIII activity or treatment response. Other than that, no specific safety concerns were raised about PEG. ESPEROCT was approved for use in humans with an unrestricted label like ADYNOVATE with no specific warnings and precautions related to PEG [110]. The third PEGylated rFVIII product, JIVI (damoctocog alfa pegol, BAY 94-9027) uses the largest PEG of 60 kDa. It is based on a recombinant B domainedeleted rFVIII protein

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produced in baby hamster kidney cells, which is covalently coupled to a single branched PEG-maleimide with a molecular weight of 60 kDa. The PEG reagent is site-specifically attached to an engineered cysteine residue inserted by mutation [116]. Multiple nonclinical studies in healthy rodents and nonrodents and in hemophilia A mice were conducted to evaluate the activity and safety profiles of JIVI, as well as the safety profile for the PEG-60-maleimide-cysteine linker moiety of JIVI. Single IV administration of JIVI (4e60 IU/kg) in hemophilia A (HemA) mice resulted in dose-dependent reduced bleeding times and blood loss like those generated in mice injected with the comparator product Kogenate FS, a nonmodified full-length rFVIII, following tail vein resection. Single IV administration of JIVI (50 IU/kg) in hemophilia A dogs resulted in equivalent or better clotting activity compared to dogs injected with Kogenate FS. This activity correlated with the longer half-life displayed in JIVI-injected dogs. Repeat-dose IV toxicity studies in healthy adult male rats and rabbits administered JIVI every other day did not result in any significant adverse effects on the coagulation system or any organ systems. The NOAEL of JIVI was 2250 IU FVIII/kg/injection (37.5-fold higher than the maximum recommended prophylactic clinical dose level of JIVI). A 52-week repeat-dose safety study in immune-deficient male rats to characterize the PEG tissue distribution/accumulation profile and any adverse findings following longterm repeat dosing with JIVI is ongoing. Rats were IV injected with up to 1200 IU/kg/ injection of JIVI twice weekly for 26 weeks. Subgroups were sacrificed at 13 and 26 weeks. Remaining animals will be sacrificed at 52 weeks, following a 26-week recovery period. Based on the in-life data for all animals out to 26 weeks, and the histopathology data for the animals sacrificed at 13 and 26 weeks, no adverse clinical signs, clinical pathology, or organ histopathology findings were observed. In addition, there was no evidence of tissue vacuolation in any tissue examined and no presence of PEG in the brain (including the choroid plexus), kidney, spleen, or cerebrospinal fluid (CSF) [117]. Genotoxicity, carcinogenicity, and developmental and reproductive toxicity studies were not conducted with JIVI. The 60 kDa PEG moiety used to modify rFVIII had been toxicologically assessed in animals. In order to prepare a non-reactive molecule the PEG-maleimide reagent, which binds to sulfhydryl groups, was reacted with cysteine to obtain PEG-60-Mal-Cys closely resembling the PEG moiety of JIVI (BAY 1025662). Single-dose and repeat-dose IV toxicity studies (up to 4 weeks of dosing) with BAY 1025662 in healthy adult male rats and rabbits and repeat-dose toxicity assessment in healthy neonatal male rats (twice weekly up to 4 weeks) were conducted at dose levels that were significantly higher than the amount of PEG (0.004 mg/kg) in a single administration of 60 IU/kg of JIVI. No significant adverse findings were seen. No immunohistochemical assessment of PEG was performed for the brain or any other organs, tissues, or body fluids. Cellular vacuolation was sporadically observed in some organs and tissues (testes, kidney, pancreas, parotid glands, choroid plexus, stomach, and adrenal cortex) of control- and BAY 1025662injected animals but was considered incidental by the pathologist. Plasma concentrations in male rats after a single 11-mg/kg IV dose of BAY 1025662, approximating the cumulative PEG-60 exposure in patients during 30 years of BAY 94-9027 treatment,

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decreased with an initial half-life of 119 h (5 days) in the interval of 114e336 h postadministration. Single-dose mass balance studies using 14C radiolabeled BAY 1025662 showed that 30.4% of radioactivity was excreted within 1 week and 79.1% by day 168, primarily in urine. The terminal half-life of radioactivity elimination was approximately 24 days in blood and plasma and was 31e68 days in most other organs up to day 168. Elimination was nearly complete at the end of the experiment on day 168. Only w4% of residual radioactivity was present in the animal body. There was no irreversible binding of radioactivity to any tissues and no penetration of the bloodebrain barrier. Based on these results, very low steady-state concentrations of 60-kDa PEG were predicted in patients treated with BAY 94-9027, and the validity of these predictions was supported by clinical studies in which almost all 179 patients receiving BAY 94-9027 for prophylaxis had undetectable PEG in plasma for up to >5 years; those with detectable PEG levels demonstrated concentrations within the predicted range. These combined preclinical and clinical observations suggested that excretion processes are in place for high molecular weight PEGs such as the PEG-60 moiety used in BAY 94-9027, for which based on molecular size the risk for accumulation could have been assumed [118]. A series of in vitro and in vivo genotoxicity studies conducted with BAY 1025662 showed no evidence of mutagenicity [117]. The clinical development program included studies to evaluate the safety and efficacy of JIVI to support a traditional approval. Clinical studies were initiated with a Phase 1 PK and dose escalation study [119], followed by Phase 3 studies in adult and pediatric PTPs. The two license enabling studies were (1) a study to evaluate the efficacy and safety of JIVI for on-demand, routine prophylaxis, and perioperative management in PTPs, adults, and adolescents (12 years of age), with severe hemophilia A [120], and (2) a study to evaluate the efficacy and safety of JIVI for treatment of bleeding and routine prophylaxis in pediatric PTPs (<12 years of age) with severe hemophilia A [118]. The clinical pharmacological profile of JIVI related to efficacy and PK was largely comparable to the profiles found for the other two PEGylated FVIII products. In contrast to ADYNOVATE and ESPEROCT, PEG-related safety signals were observed in the JIVI trials, mainly in small children. In the European Public Assessment Report (EPAR), a detailed discussion of clinical safety was published [121]. The clinical part of the dossier was based on three fully completed studies, the Phase 1 first-in-man study and two Phase 2/3 studies in adult and pediatric hemophilia A patients, and from the ongoing extension studies in adolescents/adults and in children. Safety assessment was done according to the CHMP Guideline on clinical investigation of recombinant factor VIII products. A total of 184 AEs were reported in 148 (83.1%) and 73 (83.6%) of the patients 12 years and <12 years of age, respectively. Most of the AEs were of mild severity and considered unrelated to JIVI. The nature and frequency of AEs did not give rise to concern and did not reveal unexpected safety signals. About 15 (10.1%) patients 12 years reported AEs which were considered possibly or probably related to JIVI. Of these, the cases of overdose and pelvic hemorrhage were classified as SAEs. The pelvic hemorrhage occurred in course of the Phase 1 study, 6 days after the last JIVI dose.

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Among PTPs <12 years of age, 13 patients had TEAEs classified as drug-related, 11 patients <6 years (25.0%) and 2 patients 6 to <12 years of age (6.9%). The most frequently reported adverse reactions in clinical trials in PTPs were headache, cough, and pyrexia. The most common AEs were the following: hypersensitivity, insomnia, headache, dizziness, cough, abdominal pain, nausea, vomiting, erythema, rash, infusion site reactions, and pyrexia. There were no deaths reported during the clinical development program. No thromboembolic event has been reported, but this risk needs to be considered with dosing regimens as high as 6000 IU FVIII/day. In patients with existing cardiovascular risk factors, substitution therapy with factor VIII may increase the cardiovascular risk. No major clinically relevant change in laboratory investigations has been detected. There were no events of suspected transmission of an infectious agent. No patient developed a new or confirmed inhibitor. No AEs were reported in relation to overdose. Several hypersensitivity-related study discontinuation cases were reported. They appear to be related to innate immune responses mediated by B1a cells, B1b cells, and marginal zone cells and mainly to occur in young children (<6 years). Only one adult experienced a hypersensitivity reaction, which was not serious and which he recovered fully from. It is known that the side effects of all FVIII products are FVIII inhibitor development (with a low incidence in PTPs) and allergic type of hypersensitivity reactions which may progress to anaphylaxis including shock. In the current clinical studies, no new and confirmed FVIII inhibitor was observed in PTPs treated with JIVI, no anaphylaxis was reported, and no serious cardiovascular event was observed. Allergic type hypersensitivity reactions are possible with JIVI. CHMP said, “The medicinal product may contain traces of mouse and hamster proteins. Hypersensitivity reactions could also be related to antibodies against PEG. If symptoms of hypersensitivity occur, patients should be advised to discontinue the use of the medicinal product immediately and contact their physician. Patients should be informed of the early signs of hypersensitivity reactions including hives, generalized urticaria, tightness of the chest, wheezing, hypotension, and anaphylaxis. Symptomatic treatment for hypersensitivity should be instituted as appropriate. In case of anaphylaxis or shock, the current medical standards for treatment should be implemented.” Besides the general risk of formation of neutralizing antibodies (inhibitors) to factor VIII common to all FVIII products, immunogenicity of PEG seemed to be a special issue with JIVI. The risk of an immune response to PEG has been identified ,which resulted in loss of efficacy observed as bruising or bleeding not responsive to treatment with JIVI and lower than expected recovery following JIVI infusion in the absence of FVIII inhibitors and occurred mainly in children <6 years. The immune response was characterized by the development of IgM antibodies directed against the PEG, which were neutralizing for JIVI activity and resulting in loss of efficacy of the study medication and decreased JIVI activity postinjection. This immune response was accompanied by hypersensitivity reactions in some patients. In several cases, the affected patients were

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found to have anti-PEG IgM antibodies before first exposure to the study drug. The immune reaction is considered as immune response of the innate immune system. The overall AE profile for patients 12 years was similar to what would be expected for long-term studies in the study population and is consistent with previous studies in this target patient population. For pediatric patients <12 years, an increase in hypersensitivity reactions and AEs related to lack of efficacy was observed particularly in children <6 years of age. An unexpected high drop-out rate due to these two events occurred in this age group and demonstrated an age-dependent difference in the AE profile of JIVI. Hypersensitivity reactions can be expected in 0%e2% of the patients >12 years of age. EMA requested special warnings and precautions added to the smPC and the prescribing information. JIVI was therefore only recommended by CHMP in patients 12 years of age. In the United States, however, the applicant did not wish to seek any indication in pediatric patients, although the Pediatric Research Equity Act (PREA) requirements were met [117]. From nonclinical studies with other PEGylated products, there is a possible risk of accumulation of PEG causing vacuolation in tissues such as in the brain structures and kidney as seen in animal studies with REFIXIA, glycoPEGylated rFIX, as explained above. The implication of this finding in humans is unclear. As shown with JIVI’s current preclinical study data, there is probably no indication of this affecting adult tissue as there were no such JIVI-related changes in the brain or kidney, including any potential cell vacuolation in ependymal cells of the choroid plexus or other cells. Furthermore, it is claimed that JIVI does not appear to cross the blood barrier, as is also the case with the other PEGylated rFVIII products. Safety data from clinical trials including data from extension studies with a treatment time up to 5 years did not show any signs of cerebral or nervous toxicity (e.g., tremors). Nevertheless, CHMP expressed the following opinion: “The risk associated to PEG accumulation, which might be observed only after years of exposure, cannot be completely excluded. A potential accumulation of PEG moiety in the nervous system tissue or other human tissue is therefore a possibility as well as the potential clinical impact on brain development in children e.g., on cognitive, functional or metabolic properties.” Despite that, there is currently no indication of vacuolation or accumulation of JIVI CHMP requested from the applicant to add the following statement indicating an important potential risk to the risk management plan: “Long-term potential effects of PEG accumulation in the choroid plexus of the brain and other tissues/organs”. In addition, the applicant was requested to commit to conducting a PostApproval Safety Study (PASS) investigating the long-term safety of the PEG moiety in JIVI as a follow-measure. Study completion is expected for 2027. Another aspect of product safety is measurability with FVIII assays determining function as results determine dosing and endpoints and therefore treatment outcomes. Here it seems that quality and structure of PEG reagents have an influence as the nonPEGylated FVIII counterparts of the three PEGylated FVIII products display normal functionality in standard FVIII assays. In contrast to ADYNOVATE which has a comparable FVIII functional activity in different assays, JIVI and to a lower extent ESPEROCT

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show aberrant test results in FVIII activity assays. The reason for these discrepancies has not been completely elucidated for FVIII, but from studies on glyco-PEGylated FIX, REFIXIA, it can be assumed that PEG interferes with reagents used in the activity assay by adsorption to the APTT reagent critical for the reliability of the assay, which might be dependent on the PEG size [122]. With ADYNOVATE using the smallest PEG of 20 kDa among the three PEGylated FVIII products, no differences had been seen, which would be relevant for monitoring FVIII replacement [123,124], while larger PEGs cause discrepant assay results with JIVI using the largest PEG of 60 kDa size showing the highest impact [125,126] and to a lower but relevant extent also with ESPEROCT [127e129]. ADYNOVATE, the PEGylated rFVIII product using a 20 kDa PEG, is now in routine clinical use for several years. No safety concerns including none for PEG were popping up and favorable real-world use data had been reported [130]. PEGylation of rFVIII with differently sized PEG reagents having molecular sizes of 20, 40, and 60 Da resulted in products used for on-demand and prophylactic therapy in hemophilia A with comparable pharmacological profiles related to pharmacokinetics and efficacy. The PEG size seems to influence the safety profile of the products. In addition to typical side effects of the rFVIII moiety in the conjugates, which were common for all three products, aberrant safety signals appeared with PEGs larger than 20 kDa. Branched PEG reagents of 40 and 60 kDa influenced the measurability of FVIII function by interfering with the reagents used in the activity assay. The 60 kDa PEG had a more pronounced impact than the 40 kDa PEG. Only the product using the largest PEG of 60 kDa size resulted in increased number of hypersensitivity reactions and AEs related to lack of efficacy and pronounced anti-PEG antibody formation. The mechanism of these reactions so far remains unclear but could be influenced by recognition of large PEG polymers by phagocytic cells and cells of the immune system not occurring with PEGs <40 kDa. As these reactions primarily occurred in small children, consequently, the products in some geographies were approved only for use in children above 12 years of age.

7. Relevance of cellular vacuolation observed in nonclinical studies and potential risk for human therapies Phagocytosis is a cellular function relevant for host defense against infection, tissue turnover, and other aspects of human physiology. Phagocytosis is also representative of functions wherein external stimuli activate motile events in the cell. Recognition of suitable objects by the plasma membrane of the phagocyte initiates phagocytosis. The work of phagocytosis that causes pseudopodia to enclose objects in vacuoles is ascribable to metabolic energy-dependent interactions between actin filaments and other contractile proteins in the peripheral cytoplasm. These interactions may also regulate the fusion of lysosomes with phagocytic vacuoles, an event important for the processing

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of ingested objects after phagocytosis [131]. This process is referred to as vacuolation, sometimes also called vacuolization. Vacuoles can be seen under the light microscope, which is a morphological observation giving no information about their genesis. Phagocytic cells containing vacuoles after administration of PEG or PEGylated therapeutics include Kupffer cells in the liver, reticuloendothelial cells in the spleen, and macrophages in a variety of tissues such as lung, skin after subcutaneous injection, lymph nodes, bone marrow, thymus, adipose tissue, ovary, testis, lamina propria of the urinary bladder, vagina, cervix, endometrial stroma of the uterus, brain, adrenal cortex, pituitary, and the choroid plexus. As removal of foreign bodies or materials from circulation is a normal function of macrophages and histiocytes, the resulting vacuolation is considered a normal physiological response in phagocytic cells. PEG-associated vacuoles have been observed by light microscopy in hematoxylin and eosin stained sections in a limited spectrum of nonphagocytic cell types including proximal tubular epithelial cells, glomerular podocytes, and glomerular endothelial cells within the kidney, adrenal cortical cells, choroid plexus epithelial cells, and endothelial cells in cerebral cortical capillaries and the aorta [132,133]. Vacuolation has also been observed in parenchymal (epithelial) cells of other tissues including joints (synovium), liver, pancreatic islets, prostate, epididymides, urinary bladder, mammary gland, thyroid gland, and ciliary body, as well as neurons in the dorsal root ganglia and brain [7]. The article of Ivens et al. reviews the current knowledge of PEG-related vacuolation [7]. The authors concluded from a comprehensive review of all available published information and an industry survey performed between 2013 and 2014 that the only effect attributed to PEG seen in nonclinical toxicology studies was cellular vacuolization observed with approximately 50% of approved PEGylated biopharmaceuticals. This vacuolation is seen mainly in phagocytic but sometimes also in nonphagocytic cells. Phagocytic cells likely contribute to the clearance of larger PEGylated biologicals, while small PEG molecules and PEG-conjugates are eliminated via the kidney and liver because of their hydrophilicity and size [3]. As cellular vacuolation is a nonspecific finding that can occur in response to a variety of influences and stimuli, the occurrence of vacuolation in toxicology studies of PEGylated proteins may not always be due to the presence of PEG in tissues. Presence of PEG in vacuoles can be confirmed by IHC with anti-PEG detection antibodies. The vacuoles associated with PEG are morphologically consistent with lysosomes by light microscopy, but these observations have not been confirmed by ultrastructural studies. For those PEGylated therapeutics, where tissue vacuolation is observed, phagocytic cells such as macrophages are usually involved. Occasionally, vacuoles have been observed in nonphagocytic cell types including renal tubular epithelium, endothelium, synovial epithelium, adrenal cortical cells, choroid plexus epithelium, and neurons. Historically, endocytic uptake into phagocytic cells has been classified as pinocytosis or phagocytosis (large particles >250 nm) based on particle size, energy requirements, and morphologic attributes observed by electron microscopy [134,135]. In the cells these phagosomes and endosomes fuse with lysosomes where the ingested materials are recycled or degraded. Indigestible materials are

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exocytosed or remain within the lysosome forming residual bodies. Several factors influence the presence of vacuoles in animal tissues. Different patterns of tissue distribution of PEG-associated cytoplasmic vacuolation likely reflect the total amount of PEG administered over the treatment period minus the amount excreted, as well as differences in pharmacology, pharmacokinetics, and physiologic filtration barriers. These variations are dependent on the molecular weights of both the PEG and the PEGylated therapeutic [136]. Importantly, for any given PEGylated therapeutic associated with tissue vacuolation, the occurrence, incidence, and severity of vacuolation increase with dose, dosing frequency, and dosing duration in toxicology studies. Whether the dominant mechanism of PEG uptake by phagocytic cells is via engulfment of free drug, drug complexed to antibodies, phagocytosis of damaged or senescent cells, or other potential mechanisms unique to phagocytic cells remained unclear. PEG vacuoles within the choroid plexus epithelium, cerebral microvasculature, or rarely in brain tissue are sometimes observed and their generation is not yet well understood. For PEG present in brain tissue, nonsaturable paracellular diffusion or transcytosis across the blood brain barrier or bloodeCSF barrier is a possibility. However, current literature suggests that these routes are highly inefficient resulting in very low penetration [137,138]. While histochemical evidence of PEG contained within macrophages in the choroid plexus exists, the mechanisms underlying this observation are not clear. It is possible that PEG could enter macrophages that are resident within the choroid plexus or that macrophages already containing PEG may enter the choroid plexus. In addition, there could be receptor-mediated transport when the PEGylated ligand’s receptor is expressed on the cerebral microvasculature [6]. PEG-related vacuolation in animal studies has been observed more frequently at PEG molecular weights 30 kDa [134]. Smaller PEG molecules are excreted readily [44,48] by glomerular filtration in relation to their half-life and therefore systemic exposure is lower. However, vacuolation could be found in toxicology studies with PEGylated proteins also with lower molecular weight PEG, for example, Krystexxa, where 8e10 PEG 10 kDa molecules are attached to each subunit of the tetramer protein uricase resulting in a large PEG dose (see also Section 5). It was demonstrated that upon administration of PEG conjugated tumor necrosis factor binding protein (TNF-bp) conjugates with 20 or 50 kDa PEG, the severity of renal tubular vacuolation was inversely related to molecular weight and PEG complexity [132]. In this study, the 50 kDa PEGeprotein dimer was associated with the least severe vacuolation and the 20 kDa PEGeprotein monomer was associated with the greatest severity. Renal tubular vacuolation was not observed when PEG alone was administered. The observed differences may have been related to reuptake triggered by the protein moiety. Although most PEG-related vacuolation is noted when PEGs of at least 30 kDa are used, the exceptions described above illustrate the difficulties in generalizing about PEG-related vacuolation and the contribution of PEG size because dose, dosing frequency, and pharmacological properties influence exposure and pattern of vacuolation.

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As anti-PEG antibodies suitable for IHC determination have become available, there are a few studies that describe the cellular localization of PEG or the relationship between observations of vacuoles and observation of PEG immunoreactivity. The assumption that these vacuoles represent the presence of PEG is supported by the similarity between the pattern of tissues with vacuoles and the pattern of tissues that are positive in IHC for PEG [133,139,140]. However, IHC studies using anti-PEG antibodies have also demonstrated that PEG-related immunoreactivity occurs in the cytoplasm of renal cortical tubule epithelial cells that were not vacuolated, and although the presence of vacuoles in toxicology studies of PEG or PEGylated proteins generally correlated with dose, the vacuoles were not always immunoreactive for PEG [132]. More studies using methods with higher sensitivity and specificity would be required to further elucidate the question of vacuole content by IHC. Furthermore, an understanding of the mechanism of formation of vacuoles, where they are located within the cytoplasm, the content of these vacuoles, and their relationship to PEG remains a gap in the understanding of the biologic effects of PEG and PEGylated proteins [7]. After IV administration of high doses of unconjugated 10, 20, and 40 kDa PEG over 3 months, PEG immunoreactivity and vacuolation were observed only with 40 kDa PEG in choroid plexus macrophages and choroid plexus epithelial cells but not in brain parenchyma, suggesting that PEG alone did not cross the bloodebrain barrier [133]. In rats, subcutaneous recombinant human IGF-1 conjugated to a 40 kDa PEG was reported to reach higher steady-state concentrations in brain tissue and CSF than non-PEGylated IGF-1, although it entered the CNS more slowly. In mice, administration of the same PEGylated IGF-1 subcutaneously resulted in dose-dependent increases in human IGF-1 in hippocampal CA1 neurons as assessed by IGF-I immunoreactivity in frozen brain sections [141]. Immunoreactivity for localization of PEG and microscopic analysis for assessment of vacuolation were not performed in these studies, so the presence of PEG or potentially PEG-associated vacuolation was not evaluated. Baumann et al. [136] describe neuronal vacuolation after administration of a different PEGylated polypeptide, with confirmation of PEG content in the vacuoles using IHC that was only partially reversible. It appears while unconjugated PEG does not enter the CNS, there may be transport of PEG across the bloodebrain barrier when it is administered conjugated to some therapeutic proteins, probably depending to the target, as shown by the published example with PEGylated rFIX [103]. PEG-associated vacuolation observed in toxicological studies may be partially or completely reversible after a treatment-free period, although often the recovery periods used in nonclinical toxicology studies were too short to demonstrate reversibility [132]. In the study by Bendele et al., cytoplasmic vacuolation in renal tubular epithelial cells remained after a 2-month recovery period, despite resolution of immunoreactivity for the protein to which the PEG was conjugated, suggesting that lysosomal enzymes may be able to process the protein but not the PEG. Larger vacuoles or multiple vacuoles may take longer to resolve during a treatment-free period. Additionally, retention of vacuoles

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may reflect the slow turnover of the cell population in which the vacuoles reside. In a retrospective review of BLAs for nine marketed PEG conjugated biologics, increasing dose and dosing duration was associated with reduced reversibility [7]. Current published data therefore suggest that vacuoles are reversible, given enough time to recover. This is also supported by recent studies showing that even larger PEGs as large as 60 kDa can be eliminated completely [118]; see also Section 6. Published studies indicate that despite significant cytoplasmic vacuolation of cells such as macrophages or renal tubular epithelial cells, adverse effects on organ function were not seen [4,141]. Rudmann et al. [133] proposed that the cytoplasmic vacuolation seen after PEG administration is an adaptive rather than a toxic response and related to the increased demand for clearance of PEG [133]. In vitro effects on macrophage function were reported for two approved PEGylated drugs, certolizumab pegol (CIMZIA) and pegloticase (KRYSTEXXA) (see also Section 5). In vitro macrophage function assays indicated that the PEG moiety alone reduced phagocytosis of bacteria and fungi at high concentrations above the intended pharmacological use. Administration of pegloticase to rats or dogs results in vacuolation of macrophages. Immunohistochemical methods confirmed that PEG was present within the vacuoles of macrophages from dogs. Although the conclusion was that vacuolation did not result in adverse effects to the overall health of the animals, vacuolated macrophages isolated from spleen of rats, which had received pegloticase, had a reduced response to an LPS challenge. Besides this in vivo test, no in vivo host resistance studies have been conducted with certolizumab pegol or pegloticase to determine if the in vitro results translate in vivo [7]. A specific question is the potential impact of PEG-related vacuolation on the function of epithelium of the choroid plexus, particularly in pediatric populations and on the ability of ependymal cells to generate CSF or transport hormones, also because of the slow regeneration of neuronal cells. It is unclear how to reliably evaluate effects on ependymal function as there are no readily available biomarkers. It can be postulated that impairment of the choroid plexus ependymal function would lead to clinical symptoms in the animals (and humans). However, nonclinical toxicology studies of PEGylated drugs up to chronic duration did not report abnormalities in animals presenting choroid plexus vacuolation. PEG present in neurons after administration of a 40 kDa PEGylated therapeutic protein did not reverse over the recovery period. However, the presence of PEG in neurons did not affect function or viability as assessed by nerve conduction velocity and labeling of degenerating neurons in ex vivo tissue samples [136]. In summary, the only effect attributed to PEG seen in nonclinical toxicology studies was cellular vacuolization observed with 5 of 11 approved PEGylated biopharmaceuticals [3]. This vacuolation is seen mainly in phagocytic but sometimes also in nonphagocytic cells. Phagocytic cells likely contribute to the clearance of larger PEGylated proteins, while small PEG molecules and PEG conjugates are eliminated via the kidney and liver because of their hydrophilicity and size. The occurrence of vacuoles in animals in toxicological studies with supratherapeutic doses of PEGeprotein conjugates is unlikely to impair cell, tissue, and organ function of the compartments affected by vacuoles.

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FIGURE 4.3 Hypothesized mechanism of cellular polyethylene glycol (PEG) uptake and processing of a large branched PEG reagent with intermediate vacuole formation. Modified according to Baumann A, Tuerck D, Prabhu S, Dickmann L, Sims J. Pharmacokinetics, metabolism and distribution of PEGs and PEGylated proteins: quo vadis? Drug Discov Today 2014;19(10):1623e31.

Fig. 4.3 shows a schematic hypothetical mechanism of cellular PEG uptake and processing where formation of vacuoles is an intermediate stage of the process.

8. Toxicological guidance and regulatory recommendations to support clinical development of polyethylene glycoleprotein conjugates PEGeprotein conjugates are biotechnology-derived pharmaceuticals. In Chapter 7 of this book, regulatory requirements for characterization of PEGylated proteins are described although not entirely related to toxicological evaluation of PEG and PEGeprotein conjugates. There, M. Bossard reviews historical and current regulatory guidelines specific to PEGylated proteins illustrated by examples from currently marketed products. Here attention is exclusively turned on toxicological guidance. Major guidance on the toxicity assessment of biotechnology-derived pharmaceuticals is given by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) in so-called ICH guidelines. ICH guideline S6 (R1) describes preclinical safety evaluation of biotechnology-derived pharmaceuticals [142]. Scope of this guidance is to recommend a basic framework for the preclinical safety evaluation of biotechnology-derived pharmaceuticals. The primary goals of preclinical safety evaluation are to identify an initial safe dose and subsequent dose escalation schemes in humans, to identify potential target organs for toxicity and for the study of whether such toxicity is reversible, and to identify safety parameters for clinical monitoring. Although not directly mentioned, PEGylated proteins are covered by ICH S6, but no specific guidance is given on how to evaluate toxicity related to PEG. Another ICH

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guideline that is applicable is ICH M3 (R2) [143]. This guideline describes nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, usually synthetic and/or small molecules. Scope of the guideline is the nonclinical safety assessment for marketing approval of a pharmaceutical, which usually includes pharmacology studies, general toxicity studies, toxicokinetic and nonclinical pharmacokinetic studies, reproduction toxicity studies, genotoxicity studies, and, for drugs that have special cause for concern or are intended for a long duration of use, an assessment of carcinogenic potential. The guideline says that other nonclinical studies to assess phototoxicity, immunotoxicity, juvenile animal toxicity, and abuse liability should be conducted on a case-by-case basis. The need for nonclinical safety studies and their relation to the conduct of human clinical trials is delineated in this guidance. For biotechnology-derived products as dealt with in ICH S6 ICH M3, it only provides guidance regarding timing of nonclinical studies relative to clinical development. Like ICH S6, this guideline does not give any specific information on how PEG or PEGylated drug molecules should be toxicologically assessed and how effects of systemic PEG exposure should be investigated, such as cellular vacuolation. Two other toxicological guidelines, which specifically address PEGylated drug products, have been issued. FDA published Guidance for Industry on Immunogenicity Assessment of Therapeutic Protein Products [144]. This guideline acknowledges that PEGylation of therapeutic protein products has been found to diminish their immunogenicity via similar mechanisms as glycosylation by indirectly altering protein immunogenicity by minimizing protein aggregation, as well as by shielding immunogenic protein epitopes from the immune system. It also recognizes that immune responses to PEG itself have been seen and have caused loss of product efficacy and adverse safety consequences, and that anti-PEG antibodies have also been found to be cross-reactive between PEGylated products. The guideline, thus, recommends that the antidrugantibody assay used in nonclinical and clinical studies should be able to detect both the antiprotein antibodies and antibodies against the PEG moiety. More details on the role of anti-PEG-antibodies are described in Section 4 of this chapter. The only regulatory guidance document which specifically addresses effects of longterm exposure to PEG and the phenomenon of vacuolation was published in 2012 [145]. CHMP Safety Working Party responded to the Pediatric Committee of EMA regarding the use of PEGylated drug products in the pediatric population intended for long-term treatment. The CHMP response acknowledged that, while vacuolation in cells of the RES may represent the organism’s normal response to remove a foreign body, there was a concern for other cells and tissues such as the renal tubular epithelium or the choroid plexus epithelium (ependymal cells). The CHMP summarized that vacuolation in these cells was observed in animal studies following certain conditions including PEG molecular weight >40 kDa, study duration of at least 4 weeks, and a cumulative PEG dose of >0.4 mmol/kg/month. It was recommended that before conducting clinical trials

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of more than 4 weeks duration with a PEGylated drug product, it should be addressed whether ependymal cell vacuolation has been observed in the nonclinical studies, and whether the PEGylated drug product may undergo active transport across the bloodeCSF barrier, e.g., via a literature review elucidating to what extent there is a physiological role for active transport of the PEGylated protein into the CSF. Further, the biodistribution of the PEGylated drug product should be addressed via applying a method enabling detection of the PEG moiety unless the monthly PEG exposure is significantly lower than the cases where ependymal cell vacuolation has been observed (>0.4 mmol/kg/month). In case ependymal vacuolation is observed in the nonclinical studies for a drug product intended for long-term treatment, clinical development for pediatric patients should only be initiated in case a sufficiently large safety margin can be established in a long-term toxicity study applying animals representative for the pediatric population in question and the finding can be demonstrated to be reversible. CHMP also suggested that “if feasible, ex vivo/in vitro data evaluating the effect of PEG vacuolation on the viability and function of choroid plexus ependymal cells could contribute to the risk assessment process”. Since this guidance document was issued, all PEGylated therapeutic proteins contain such assessments in their nonclinical and clinical study programs (see also the other sections of this chapter). Despite recognition by drug regulatory agencies and their request to investigate this in animal toxicology studies, accurate detection of PEG-related vacuoles remains a challenge. In a recent review article, Irizarry Rovira et al. [146] provided a detailed pictorial review of PEG-associated microscopic findings and points to consider when evaluating and reporting the extent, severity, and significance defined as adversity or lack of adversity of PEG-associated cytoplasmic vacuolation in safety assessment studies. The authors identified gaps in the pathological assessments of vacuoles, both in the methods used and the standardized interpretation of data. They suggested that the information they provided would help practicing toxicologic pathologists who grapple with several key analytical and reporting issues, as they evaluate the qualitative nature, extent, and significance of cytoplasmic vacuolation in various organs consequent to the repeated administration of PEGylated biopharmaceuticals during nonclinical safety studies. They acknowledged that establishing an industry-wide, standardized severity grading system for any morphologic finding associated with a xenobiotic is a challenging task because of the inherent subjective nature of microscopic evaluation and other factors. Nevertheless, the authors summarized their knowledge by saying that “the collective experience gathered from multiple nonclinical toxicology studies of PEGylated biopharmaceuticals indicates that in general, PEG-related vacuolation is not associated with demonstrable cell and tissue damage or dysfunction and is reversible with sufficient duration of drug-free periods” [146].

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References [1] Stidl R, Denne M, Goldstine J, Kadish B, Korakas KI, Turecek PL. Polyethylene glycol exposure with antihemophilic factor (recombinant), PEGylated (rurioctocog alfa pegol) and other therapies indicated for the pediatric population: history and safety. Pharmaceuticals (Basel) 2018;11(3). [2] Milla P, Dosio F, Cattel L. PEGylation of proteins and liposomes: a powerful and flexible strategy to improve the drug delivery. Curr Drug Metab 2012;13(1):105e19. [3] Turecek PL, Bossard MJ, Schoetens F, Ivens IA. PEGylation of biopharmaceuticals: a review of chemistry and nonclinical safety information on approved drugs. J Pharm Sci 2016;105(2):460e75. [4] Ivens IA, Baumann A, McDonald TA, Humphries TJ, Michaels LA, Mathew P. PEGylated therapeutic proteins for haemophilia treatment: a review for haemophilia caregivers. Haemophilia 2013; 19(1):11e20. [5] Swierczewska M, Lee KC, Lee S. What is the future of PEGylated therapies? Expert Opin Emerg Drugs 2015;20(4):531e6. [6] Webster R, Didier E, Harris P, Siegel N, Stadler J, Tilbury L, et al. PEGylated proteins: evaluation of their safety in the absence of definitive metabolism studies. Drug Metab Dispos 2007;35(1):9e16. [7] Ivens IA, Achanzar W, Baumann A, Bra¨ndli-Baiocco A, Cavagnaro J, Dempster M, et al. PEGylated Biopharmaceuticals: current experience and considerations for nonclinical development. Toxicol Pathol 2015;43(7):959e83. [8] European pharmacopoeia monograph 01/2008:1444, Macrogols. [9] Brent J. Current management of ethylene glycol poisoning. Drugs 2001;61(7):979e88. [10] Eder AF, McGrath CM, Dowdy YG, Tomaszewsky JE, Rosenberg FM, Wilson RB, et al. Ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis. Clin Chem 1998;44(1):168e77. [11] McChesney EW, Golberg L, Parekh CK, Russell JC. Reappraisal of the toxicology of ethylene glycol. II. Metabolism studies in laboratory animals. Food Cosmet Toxicol 1971;9(1):21e38. [12] Milles G. Ethylene glycol poisoning; with suggestions for its treatment as oxalate poisoning. Arch Pathol 1946;41:631e8. [13] Berman LB, Schreiner GE, Feys J. The nephrotoxic lesion of ethylene glycol. Ann Intern Med 1957; 46(3):611e9. [14] Bove KE. Ethylene glycol toxicity. Am J Clin Pathol 1966;45(1):46e50. [15] Budaveri S, editor. Merck index. 11th ed. Rahway (NJ): Merck & Co; 1989. [16] Schep LJ, Slaughter RJ, Temple WA, Beasley DM. Diethylene glycol poisoning. Clin Toxicol (Phila) 2009;47(8):525e35. Erratum in: Clin Toxicol (Phila) 2009, 840. [17] Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 2002;54(4):459e76. [18] Kemptner J, Marchetti-Deschmann M, Siekmann J, Turecek PL, Schwarz HP, Allmaier G. GEMMA and MALDI-TOF MS of reactive PEGs for pharmaceutical applications. J Pharm Biomed Anal 2010; 52(4):432e7. [19] Kawai F. Microbial degradation of polyethers. Appl Microbiol Biotechnol 2002;58(1):30e8. [20] Wong DW. Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol 2009;157(2):174e209. [21] Jenkins CM, Yang K, Liu G, Moon SH, Dilthey BG, Gross RW. Cytochrome c is an oxidative stressactivated plasmalogenase that cleaves plasmenylcholine and plasmenylethanolamine at the sn-1 vinyl-ether linkage. J Biol Chem 2018;293(22):8693e709.

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