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. Author manuscript; available in PMC: 2023 Apr 15.
Published in final edited form as: Toxicol Lett. 2022 Jan 29;359:22–30. doi: 10.1016/j.toxlet.2022.01.011

Toxicity of high-molecular-weight polyethylene glycols in Sprague Dawley rats

Jia-Long Fang 1,*, Michelle M Vanlandingham 1, Frederick A Beland 1, Robert P Felton 2, Mackean P Maisha 2, Greg R Olson 3, Ralph E Patton 3, Amy S Rosenberg 4, Gonçalo Gamboa da Costa 1
PMCID: PMC8932377  NIHMSID: NIHMS1779302  PMID: 35092809

Abstract

Polyethylene glycol (PEG) is present in a variety of products. Little is known regarding the accumulation of high-molecular-weight PEGs or the long-term effects resulting from PEG accumulation in certain tissues, especially the choroid plexus. We evaluated the toxicity of high-molecular-weight PEGs administered to Sprague Dawley rats. Groups of 12 rats per sex were administered subcutaneous injections of 20, 40, or 60 kDa PEG or intravenous injections of 60 kDa PEG at 100 mg PEG/kg body weight/injection once a week for 24 weeks. A significant decrease in triglycerides occurred in the 60 kDa PEG groups. PEG treatment led to a molecular-weight-related increase in PEG in plasma and a low level of PEG in cerebrospinal fluid. PEG was excreted in urine and feces, with a molecular-weight-related decrease in the urinary excretion. A higher prevalence of anti-PEG IgM was observed in PEG groups; anti-PEG IgG was not detected. PEG treatment produced a molecular-weight-related increase in vacuolation in the spleen, lymph nodes, lungs, and ovaries/testes, without an inflammatory response. Mast cell infiltration at the application site was noted in all PEG-treated groups. These data indicate that subcutaneous and intravenous exposure to high-molecular-weight PEGs produces tissue vacuolation without inflammation and anti-PEG IgM antibody responses.

Keywords: Polyethylene glycol, anti-PEG antibody, vacuolation, choroid plexus

Introduction

Polyethylene glycol (PEG) is a biocompatible synthetic polymer of ethane-1,2-diol found in a variety of products, such as skin creams, bath and shower products, and medicines, that are used or consumed by the US public (Fruijtier-Pölloth 2005; Turecek et al. 2016). These uses extend from topically applied to ingested materials with different polymeric lengths of PEG, for example PEG 400 (~ 0.4 kDa) in cosmetics and Macrogol 6000 (~ 6 kDa), in medicines (Chang et al. 2019; Fruijtier-Pölloth 2005). The Joint Food and Agriculture Organization of the United Nations/WHO Expert Committee on Food Additives has considered 10 mg/kg body weight (bw) to be an acceptable daily oral intake of PEG from food for humans (EFSA 2007).

PEGylation is the process of either covalently or non-covalently linking a high-molecular-weight PEG to a drug or therapeutic protein or peptide, such as Plegridy (interferon beta-1a coupled to 20 kDa PEG) and Jivi (recombinant antihemophilic factor coupled to 60 kDa branched PEG) (Chang et al. 2019). PEGylation is used to increase the serum half-life of drugs or therapeutic proteins or peptides, decrease frequency of drug administration, improve efficacy, and reduce the immunogenicity of the proteins or peptides (Morpurgo and Veronese 2004). At least 20 PEGylated biologicals have been marketed over the last decade to treat various diseases (Chang et al. 2019).

PEGs can be absorbed by the gastrointestinal tract in a molecular weight-dependent manner. PEG-8 (~ 0.4 kDa) is well absorbed after oral administration, while PEG-150 (~ 6.7 kDa) is not absorbed at all (Fruijtier-Pölloth 2005). When applied to intact skin, PEGs with lower molecular weights are only minimally absorbed, and PEGs with molecular weights of 4 kDa or greater are not absorbed (Fruijtier-Pölloth 2005). Renal toxicity has been associated with oral and intravenous administration of high concentrations of PEGs to humans (Webster et al. 2009).

PEGs with molecular weights of up to 7.5 kDa demonstrate a low level of toxicity in acute, short-term, and long-term studies using oral, intraperitoneal, and intravenous routes in a wide range of animal species (Fruijtier-Pölloth 2005; Ivens et al. 2015; Webster et al. 2007). Little is known about the tissue distribution and toxicity of the higher molecular weight PEGs. High-molecular-weight PEGs show slow renal clearance and consequently have a greater potential to accumulate within cells (Baumann et al. 2014; Caliceti and Veronese 2003).

The accumulation of PEGs in cells has been linked to cellular vacuolation in five of nine approved PEGylated biologicals (Ivens et al. 2015). Cellular vacuolation occurred in certain organs and tissues, including lymph nodes, spleen, liver, kidney, uterus, and choroid plexus (Ivens et al. 2015; Stidl et al. 2016; Turecek et al. 2016). Rudman et al. (2013) demonstrated that the cellular vacuolation is markedly influenced by the molecular weight of the PEG. After repeated intravenous injection of 40 kDa PEG at a dose of 100 mg/kg bw to male rats for 90 days, PEG was detected, along with the formation of cytoplasmic vacuoles in the choroid plexus ependymal cells, renal interstitial macrophages, and cerebral cortex capillaries. Vacuolization did not occur in the brains of the rats receiving 10 kDa or 20 kDa PEG. In addition, renal tubule basophilia and degeneration was associated with 40 kDa PEG (Rudmann et al. 2013). Similarly, PEG accumulation and vacuole formation occurred in ependymal cells of the choroid plexus and cortical adrenal tissue of monkeys administered 40 kDa PEG intravenously at 45 mg/kg bw/week for two or six weeks, but not at 7 mg/kg bw/week for 13 weeks (NovoNordisk 2017). In rats, intravenous doses of 45 or 117 mg 40 kDa PEG/kg bw/week for 6 weeks led to PEG accumulation and vacuole formation in the brain, spleen, mesenteric and mandibular lymph nodes, and liver, but no effects were observed after a two-week exposure to 45 mg 40 kDa PEG/kg bw/week (NovoNordisk 2017). No evidence of degeneration, inflammation, or necrosis was associated with the PEG-related vacuolation (NovoNordisk 2017).

Little is known regarding the effects of route administration on the tissue distribution of high-molecular-weight PEGs. After intraperitoneal, subcutaneous, or intramuscular injection of a single dose of 50 kDa [125I]PEG to female mice, the elimination rate of the PEG from the injection site increased in the order intraperitoneal > subcutaneous > intramuscular, with the peak radioactivity in the blood being observed at approximately 30, 60, and 120 min, respectively (Yamaoka et al. 1995). Recently, Knadler et al. reported that 20 kDa [14C]PEG was eliminated from the plasma of male rats, with a mean t1/2 of 202 and 165 h after subcutaneous and intravenous administration, respectively, at a level of 10 mg PEG/kg bw; the subcutaneous bioavailability for the PEG was 78% (Knadler et al. 2015).

Although PEGylation of proteins is designed, in part, to shield the proteins from immune access and was also considered to be non-immunogenic, such has not proven to be the case. Many reports have demonstrated immunogenicity of PEG species and of the proteins to which it is bound (Chang et al. 2019; Garay et al. 2012; Schellekens et al. 2013; Shiraishi and Yokoyama 2019). An early study observed antibody formation against PEG in rabbits immunized with PEG-linked proteins (Richter and Åkerblom 1983). A single intravenous administration of PEGylated bovine serum albumin, ovalbumin, or adenovirus produced an anti-PEG IgM response in Wistar rats (Shimizu et al. 2012). Repeat injections of PEGylated solid lipid nanoparticles in mice and beagles induced an anti-PEG IgM immune response that accelerated blood clearance (Abu Lila et al. 2013; Zhao et al. 2012).

There is significant concern that the accumulation of high-molecular-weight PEGs and cellular vacuolization may lead to adverse outcomes for PEGylated biologicals used at high dose, chronically and/or in pediatric populations (Ivens et al. 2015; Stidl et al. 2016; Turecek et al. 2016). However, there is a lack of data on the tissue and cellular accumulation of high-molecular-weight PEGs over time or the long-term effects resulting from high-molecular-weight PEG accumulation in certain tissues, especially the choroid plexus. Thus, assessment of the toxicity of high-molecular-weight PEG is important. We have conducted a toxicity study in Sprague-Dawley rats involving repeated subcutaneous injection of 20, 40, or 60 kDa PEG or intravenous injections of 60 kDa PEG weekly for 24 weeks. The toxicological characterization included assessments of gross and histopathological organ toxicity, hematology, and clinical chemistry. In addition, the induction of anti-PEG antibodies was determined and the levels of PEG in plasma, cerebrospinal fluid (CSF), urine, and feces were assessed.

Methods and materials

Test articles

PEGs with molecular weights 20, 40, and 60 kDa were purchased from JenKem Technology USA, Inc. (Plano, TX). All the PEGs were methoxy capped at one terminal end, had a hydroxyl group on the other terminal end, and had a narrow distribution in molecular weight. The polydispersity was 1.047, 1.031, and 1.047 for the 20, 40, and 60 kDa PEG, respectively, as assessed gel permeation chromatography. Sterile saline (0.9% sodium chloride) was used as the vehicle and was purchased from VWR International (Radnor, PA) at the highest quality available that meets U.S. Pharmacopeia guidelines.

Dose selection and formulation

Based on the approximate high dose in several published toxicological studies (NovoNordisk 2017; Rudmann et al. 2013) and recommendations by the US FDA’s Center for Biologics Evaluation and Research and Center for Drug Evaluation and Research, a dose was 100 mg PEG/kg bw/injection once a week. The PEGs were administered by subcutaneous and intravenous injections because these dosing routes are used to administer pegylated biologics to humans (Baumann et al. 2014). A 24-week dosing duration was selected to ensure that steady-state conditions would be obtained based on a reported half-life of approximately eight weeks for a 100 mg/kg bw of 40 kDa PEG (branched) (Bjørnsdottir et al. 2016). The PEG was dissolved in sterile saline to give 100 mg/kg bw/injection in a dose volume of 2 ml/kg bw.

The concentrations of dose solutions were determined according to the procedure of Sims and Snape (Sims and Snape 1980) with minor modifications. Briefly, dose solutions were diluted with saline to 7.5 μg PEG/ml; saline was used as the reagent blank. To a 1-ml sample, 15 μl 0.1 N iodine solution (EMD Millipore Corp., Billerica, MA) was added, the resulting solution was gently vortexed and incubated for 20 min at room temperature, and then the absorbance was read at 540 nm against the saline blank. PEG reference solutions (2 – 10 μg PEG/ml) were prepared and the standard curves were constructed by plotting the absorbance versus the concentration of PEG. The concentration of PEG dose solutions was calculated from the standard curve. All dose solutions were within 10% of the target concentrations and were stable for up to 81 days at room temperature.

Study design

Male and female Sprague Dawley rats [Crl:CD®(SD)IGS BR] were obtained from Charles River Laboratories (Wilmington, NC) and tail-tattooed for identification. Upon receipt from the supplier at 4–5 weeks of age and until weight ranking at 7–8 weeks of age, the rats were housed two per cage in standard NCTR polycarbonate rat cages with hardwood chip bedding. At 7–8 weeks of age the rats were singly housed for approximately 1 week for weight-ranking and then randomly assigned to each experimental group. Upon completion of this process, the rats were housed two per cage for the duration of the study until the collection of excreta, when rats were singly housed in metabolic cages for the last 24-hour period prior to sacrifice. NIH-41 irradiated pellets and Millipore-filtered drinking water were provided ad libitum. Urine and feces were stored at −80 °C before processing. The animal room was maintained on a 12-hour light/dark cycle, with 10–15 air changes per hour. Environmental controls were set to maintain the temperature at 22 ± 4 °C, with a relative humidity of 40–70%. Animal use was in accordance with the Institute for Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals. All experimental protocols were reviewed and approved by the Animal Care and Use Committee at the NCTR.

Groups of 12 rats per sex were administered subcutaneous injections of 20, 40, or 60 kDa PEG at 100 mg PEG/kg bw/injection once a week for 24 weeks; a vehicle control (12 rats per sex) received saline only. Based upon recommendations from scientists at the Center for Biologics Evaluation and Research, an additional group of 12 rats per sex received intravenous injections of 60 kDa PEG at 100mg/kg bw/injection once a week for 24 weeks and a vehicle control (12 rats per sex) received saline only. The subcutaneous dose site was the loose skin over the neck and the intravenous dose site was via the lateral tail vein. At the end of every 4-week exposure, approximately 400 μl of tail vein blood was collected into tubes containing EDTA and processed for plasma, which was stored at −80 °C until the analysis of anti-PEG antibodies. All animals were observed for mortality or morbidity twice daily; clinical observations were conducted weekly. Body weights were measured on the first treatment day, prior to the initial treatment, and weekly thereafter until study termination. The doses were adjusted weekly based on these body weight measurements. Food consumption was measured weekly.

Extraction of PEG from feces, urine, CSF, and plasma

Frozen fecal samples were freeze-dried using a Labconco FreeZone 4.5 freeze-dry system (Labconco Corporation, Kansas City, MO) until dry. Aliquots (1 g) of the lyophilized feces were resuspended in 8 ml of Millipore water with an end-over-end mixing for 2 h at room temperature, centrifuged at 4,000 g for 20 min at 4 °C, and the fecal supernatant was collected for the extraction of PEG.

The fecal supernatants (50 μl) and aliquots of urine (1.5 – 15 μl), CSF (25 μl), and plasma (0.3 – 15 μl) were diluted with Millipore water to obtain a final volume of 50 μl and then 100 μl of acetonitrile were added. The mixtures were vortex mixed for 2 min and centrifuged at 4000 g for 20 min at 4 °C. The extracts were then transferred to a new tube and evaporated using a Speed-Vac concentrator attached to a water aspirator. The residues were re-dissolved in 8.4 μl Millipore water for use in PEG analysis.

The recoveries of PEG after extraction with acetonitrile were determined by a series of experiments, in which blank samples and Millipore water (control sample) were spiked with 20, 40, or 60 kDa PEG to give a final concentration of 5 μg/ml and subjected to extraction with acetonitrile. For each experiment, triplicate samples were prepared. The amount of PEG was then measured by iodine staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Kurfürst 1992). The detection limit was 25 ng for 20 kDa PEG and 10 ng for 40 and 60 kDa PEG. The average extraction recovery of spiked PEG in plasma and feces was > 95% for all PEGs from three independent experiments. The average extraction recovery from urine was 86, 83, and 95% for 20, 40, and 60 kDa PEG, respectively.

Measurement of PEG in plasma, CSF, urine, and feces

The levels of PEG in plasma, CSF, urine, and feces were measured by SDS-PAGE and iodine staining (Kurfürst 1992) after extraction with acetonitrile. An aliquot of 20, 40, and 60 kDa was used as reference standard. The reference standard (500 ng/well) and samples were mixed with NuPAGE™ LDS sample buffer (ThermoFisher Scientific, Waltham, MA), loaded onto 6% Bis-Tris gels, and separated by gel electrophoresis. After electrophoresis, the gels were fixed in 5% glutaraldehyde for 30 min at room temperature. Afterward, the gels were soaked in 20 ml of 0.1 M perchloric acid for 30 min, and then stained with a solution containing 5 ml of 5% barium chloride and 2 ml of a 0.1 N iodine. After 30 min, the staining solution was replaced, and the gels were washed with water for another 30 min. The bands were detected using a FluorChem R system (ProteinSimple, San Jose, CA) and Digital Darkroom software for acquisition & analysis (ProteinSimple). The intensity of bands was quantified using ProteinSimple AlphaView SA and the levels of PEG were calculated using the PEG reference standard.

To determine whether there was blood contamination in the CSF, the amount of hemoglobin in the CSF was measured using a Nanodrop 8000 Spectrometer (ThermoFisher Scientific). The extent of blood contamination in the CSF was calculated by comparison to the levels of hemoglobin in a corresponding blood sample. For blood-contaminated CSF, the concentration of PEG in the CSF was determined after subtracting the amount of PEG contributed by the contaminating blood.

Clinical Pathology

At terminal sacrifice, animals were placed into a stereotactic frame under anesthesia with ~ 3 – 5% isoflurane and CSF was collected. The anesthetized animals were then exsanguinated via cardiac blood withdrawal followed by sequential whole-body perfusion through the heart with heparinized saline and 10% neutral buffered formalin (10% NBF). Aliquots of blood were placed into EDTA tubes for hematological analyses (Fang et al. 2015) and serum-separating tubes for serum preparation. Blood samples in serum-separating tubes were allowed to clot and then centrifuged. The serum was collected and stored at −80 °C until clinical chemistry measurements, which were conducted as previously described (Fang et al. 2015).

Necropsy and histopathology:

After whole-body perfusion through the heart with heparinized saline followed by 10% NBF, a complete necropsy was conducted on all animals from all groups (moribund or at the terminal necropsy). The brain, epididymis, heart, kidneys, liver, lung, ovaries, testes, and thymus were weighed. Complete histopathological evaluation of all tissues was performed on all animals. The tissues were fixed and preserved in 10% NBF, except for the eyes and testes, which were fixed in modified Davidson’s solution. After fixation, the tissues were trimmed, processed and embedded in infiltrating media (Formula R®), sectioned at approximately 5 microns, stained with hematoxylin and eosin, and examined histopathologically. The skin at the application site was examined in all rats treated by subcutaneous injection. When applicable, non-neoplastic lesions were graded for severity as 1 (minimal), 2 (mild), 3 (moderate), or 4 (marked).

Statistics

A Cox proportional hazard regression model (Cox 1972) was used to test the effect of PEG treatment on the animal survival probability. Body weights and food consumption were analyzed by a repeated measure, mixed model analysis of variance. Within-group correlations were modeled using a heterogeneous first-order autoregressive correlation structure, which allowed for correlated differences in variability across time points. Organ weights were analyzed using a one-way analysis of covariance model with PEG treatment fixed effects and receiving body weight as a covariate (Sellers et al. 2007).

Hematology and clinical chemistry data were analyzed by an analysis of variance using a nonparametric method with mid-ranks for ties and an unstructured covariance (Brunner et al. 2002).

Linear correlation analysis for the molecular-weight-related trend in the levels of PEG in plasma, urine, feces, and CSF was performed using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA).

For non-neoplasm incidence, Fisher’s exact test was used to compare dosed groups to the vehicle control group.

Results

Survival, body weights, and food consumption

Groups of 12 male and 12 female rats were administered subcutaneous injections of saline, 20, 40, or 60 kDa PEG or intravenous injections of saline or 60 kDa PEG at 100 mg PEG/kg bw/injection once a week for 24 weeks. Two female rats (one dosed subcutaneously and one intravenously) were removed before the scheduled terminal sacrifice due to trauma at the injection site that was unrelated to PEG treatment. All remaining animals exposed to PEG survived until the end of study. There were no treatment-related clinical findings. PEG treatment, by either subcutaneous or intravenous injection, did not produce any significant changes in the body weights of the rats (Supplement 1). Only sporadic changes in food consumption were observed in rats treated with PEG (Supplement 1).

Organ weights

Treatment with PEGs by subcutaneous injection had a significant molecular-weight-related trend on mean liver weights in both female and male rats and on mean kidney weights in female rats (Supplement 2). In males, the liver weights in the 40 and 60 kDa PEG groups were 8.5% and 9.6% lower than those in the control group. In contrast, the liver weights of female rats treated with 60 kDa PEG were 11.0% higher than those in the female control group. A similar increase in the kidney weights of female rats treated with 60 kDa PEG was also observed. Intravenous injection of 60 kDa PEG did not affect liver and kidney weights in either sex (Supplement 2).

In male rats, the left and right epididymis weights of rats administered 60 kDa PEG intravenously were 10.4% and 11.5% higher than the controls. In females, brain weights of rats administered 20 kDa PEG subcutaneously were 4.0% lower than the controls (Supplement 2).

Clinical pathology

Hematology and clinical chemistry data are presented in Supplement 2. Although, there were significant molecular-weight-related trends and pairwise comparisons for several hematological and clinical chemistry parameters, the changes tended to be modest in magnitude. The only consistent pattern across sexes and routes of administration was a decrease in triglycerides in the rats treated with the 60 kDa PEG, with the decrease varying between 26 and 50%, depending upon the treatment group.

Levels of PEG in the plasma, urine, feces, and CSF

Twenty-four hours before the scheduled terminal sacrifice, the rats were placed in metabolism cages to collect feces and urine. Blood was collected at the terminal sacrifice for the preparation of plasma and the levels of PEGs in plasma, urine, and feces were examined using SDS-PAGE with iodine staining. As shown in Figure 1a, PEG was detected in the plasma, urine, and feces from PEG treatment groups, but not in the vehicle controls. In both female and male rats treated subcutaneously with PEG, there was a molecular-weight-related increase in the levels of PEG in plasma (Figure 1b). Male rats had higher plasma levels of PEG than female rats after both subcutaneous and intravenous administration (Figure 1b).

Figure 1.

Figure 1.

Figure 1.

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(a) Detection of PEG in plasma, urine, and feces using SDS-PAGE with iodine staining assay. (b) The levels of PEG in plasma, urine, and feces. Each group consisted of 12 animals per sex, except for the female 60 kDa PEG subcutaneous injection group and the female saline intravenous injection group, each of which had 11 animals. Columns and bars are the mean and standard deviation. #Significant (p < 0.05) molecular-weight-related trend. ^Significant (p < 0.05) difference between subcutaneous and intravenous injection.

In contrast to plasma, a molecular-weight-related decrease in the levels of PEG was observed in urine of both males and females, suggesting that higher molecular weight PEG is retained in plasma duo to lack of renal mechanisms of excretion, while low-molecular-weight PEG is readily excreted by renal mechanisms thus reducing plasma levels of PEG. The levels of PEG (μg/ml) in urine were 0.7- to 7.5-fold greater than those (μg/g) in feces (Figure 1b). Male rats treated by intravenous injection with 60 kDa PEG had higher fecal levels of PEGs than that male rats treated by subcutaneous injection. There was no difference in the levels of PEG in plasma and urine between the subcutaneously and intravenously 60 kDa PEG treated rats of either sex (Figure 1b).

Of critical importance was the penetration of the central nervous system by PEG species; thus, the levels of PEG in CSF were evaluated. A very faint band of PEG was detected in some of the CSF samples from the PEG treatment groups (Figure 2a) while PEG was not detected in CSF from the vehicle controls. As shown in Figure 2b, PEG was present in the CSF from nearly all males treated with 40 and 60 kDa PEGs while very few females had detectable PEG in the CSF. The reasons for such a dramatic disparity between sexes are not clear.

Figure 2.

Figure 2.

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(a) Detection of PEG in CSF using SDS-PAGE with iodine staining assay. (b) The prevalence and level of PEG in CSF. The prevalence is reported as the number of animals with detectable PEG per number of animals examined. The concentrations of PEG (μg/ml) are presented as the mean and standard deviation if the number of PEG-positive samples was ≥ 3. n.d.: not detected.

Anti-PEG antibodies

Anti-PEG antibodies in plasma samples were analyzed using a previously described flow cytometry assay (Fang et al. 2021). As shown in Supplement 3, anti-PEG IgG was not detected in plasma at any time point while anti-PEG IgM was present in plasma of both controls and PEG treated groups (Supplement 3 and Table 1).

Table 1:

Prevalence of anti-PEG IgM in plasma from rats administered PEGsa

Weeks on study Subcutaneous Intravenous
Saline 20 kDa PEG 40 kDa PEG 60 kDa PEG Saline 60 kDa PEG
Male rats
4 0/12 7/12** 4/12* 5/12* 2/12 6/12
8 0/12 8/12*** 5/12* 4/12* 4/12 9/12*
12 1/12 8/12** 5/12 5/12 4/12 9/12*
16 1/12 8/12** 5/12 4/12 4/12 9/12*
20 1/12 7/12* 5/12 5/12 4/12 9/12*
24 1/12 8/12** 5/12 5/12 4/12 9/12*
Female rats
4 2/12 5/12 6/12 5/12 1/12 4/12
8 3/12 4/12 7/11 8/12* 2/12 7/12*
12 4/12 5/12 8/12 8/12 2/12 7/12*
16 4/12 5/12 8/12 7/12 2/12 7/12*
20 4/12 5/12 8/12 7/12 2/12 7/12*
24 4/12 5/12 8/12 6/11 1/11 7/12*
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The prevalence is reported as the number of animals with an anti-PEG IgM levels ≥ 15 ng/ml per number of animals assessed.

An asterisk in PEG columns indicates a significant pairwise comparison to the saline control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Histopathology

PEG treatment caused cellular vacuolation (Table 2). The vacuoles were predominantly in macrophages, but also present in non-phagocytic cell types, including various epithelial and other parenchymal cells. The vacuoles occurred in the cytoplasm and were single or multiple, clear, colorless, round, and variable in size (Figures 3 and 4).

Table 2.

Incidence and severity of tissue vacuolation in rats administered PEGs for 24 weeks.a

Subcutaneous
 Intravenous
Saline 20 kDa PEG 40 kDa PEG 60 kDa PEG Saline 60 kDa PEG
Male rats
Liver: hepatocyte 0/12 3/12 (1.6) 9/12 (2.1)** 7/12 (1.8)** 1/12 (2.0) 3/12 (2.0)
Adrenal cortex, zona reticularis: parenchymal cell 0/12 0/12 9/12 (2.5)*** 9/12 (3.0)*** 0/12 12/12 (3.0)***
Adrenal medulla: parenchymal cell 0/11 0/12 0/11 2/12 (3.0) 0/12 4/12 (2.5)*
Pituitary gland: parenchymal cell 0/12 1/12 (2.0) 11/12 (2.5)*** 8/12 (3.1)*** 0/12 12/12 (2.8)***
Testes: Leydig cell 0/12 2/12 (1.0) 12/12 (2.0)*** 12/12 (2.1)*** 0/12 12/12 (2.7)***
Lymph node, cervical, sinus: macrophage 0/12 12/12 (3.0)*** 12/12 (3.6)*** 12/12 (3.2)*** 0/12 11/11 (3.5)***
Lymph node, mandibular, sinus: macrophage 0/12 12/12 (2.3)*** 12/12 (3.0)*** 12/12 (3.0)*** 0/12 12/12 (3.5)***
Lymph node, mesenteric, sinus: macrophage 0/12 12/12 (1.8)*** 10/12 (2.7)*** 12/12 (2.5)*** 0/12 12/12 (3.3)***
Spleen, red pulp: macrophage 0/12 11/12 (1.1)*** 12/12 (3.1)*** 12/12 (3.5)*** 0/12 12/12 (4.0)***
Thymus: macrophage 0/12 3/12 (1.0) 9/12 (1.4)*** 12/12 (1.8)*** 0/12 11/12 (2.0)***
Mammary gland: epithelial cell 0/12 2/12 (2.0) 6/12 (2.5)** 11/12 (2.9)*** 0/12 12/12 (3.5)***
Skin, site of application: fibrocyte 0/12 12/12 (3.9)*** 12/12 (4.0)*** 12/12 (4.0)***
Skin, site of application: macrophage 0/12 11/12 (3.9)*** 12/12 (4.0)*** 11/12 (4.0)***
Bone, femur: synovial tissue 0/12 0/12 2/12 (1.0) 5/12 (1.2)* 0/12 4/12 (1.0)*
Brain, choroid plexus: epithelial cell 8/12 (1.4) 10/12 (1.3) 12/12 (2.1)* 12/12 (2.0)* 9/12 (1.3) 12/12 (2.7)
Lung: macrophage 0/12 3/12 (1.0) 10/12 (1.1)*** 12/12 (1.0)*** 0/12 12/12 (1.2)***
Eye, ciliary body: epithelial cell 0/12 0/12 8/12 (1.0)*** 10/12 (1.5)*** 0/12 11/12 (1.7)***
Female rats
Liver: hepatocyte 0/12 1/12 (2.0) 2/12 (2.5) 2/12 (3.0) 0/12 4/12 (2.0)
Adrenal cortex, zona reticularis: parenchymal cell 0/12 2/12 (2.0) 12/12 (2.2)*** 12/12 (2.8)*** 0/12 12/12 (2.7)***
Adrenal medulla: parenchymal cell 2/12 (2.0) 0/12 5/12 (1.8) 2/12 (2.5) 0/12 7/12 (2.3)**
Pituitary gland: parenchymal cell 0/12 4/12 (3.0)* 9/12 (2.4)*** 10/12 (2.9)*** 0/12 12/12 (3.0)***
Ovary: parenchymal cell 0/12 5/12 (1.6)* 12/12 (1.7)*** 12/12 (2.6)*** 1/12 (1.0) 12/12 (3.0)***
Uterus: parenchymal cell 0/12 3/12 (1.6) 9/12 (2.0)*** 12/12 (2.8)*** 0/12 12/12 (2.8)***
Lymph node, cervical, sinus: macrophage 0/12 12/12 (3.7)*** 12/12 (3.5)*** 12/12 (3.2)*** 0/12 12/12 (4.0)***
Lymph node, mandibular, sinus: macrophage 0/12 12/12 (2.4)*** 12/12 (3.3)*** 12/12 (3.5)*** 0/12 12/12 (3.9)***
Lymph node, mesenteric, sinus: macrophage 1/12 (4.0) 12/12 (2.5)*** 10/12 (3.0)*** 12/12 (3.4)*** 0/12 12/12 (3.9)***
Spleen, red pulp: macrophage 0/12 12/12 (1.0)*** 12/12 (3.1)*** 12/12 (3.9)*** 0/12 12/12 (4.0)***
Thymus: macrophage 1/12 (3.0) 8/12 (1.2)** 8/12 (1.7)** 11/12 (2.1)*** 1/12 (1.0) 12/12 (2.5)***
Mammary gland: epithelial cell 0/12 6/12 (2.5)** 4/12 (3.2)* 3/12 (2.6) 0/12 8/12 (2.6)***
Skin, site of application: fibrocyte 0/12 12/12 (3.5)*** 12/12 (4.0)*** 12/12 (4.0)***
Skin, site of application: macrophage 0/12 12/12 (3.5)*** 12/12 (4.0)*** 12/12 (4.0)***
Bone, femur: synovial tissue 0/12 0/12 5/12 (1.2)* 5/12 (2.0)* 0/12 7/12 (1.7)**
Brain, choroid plexus: epithelial cell 8/12 (1.1) 10/12 (1.1) 11/12 (2.0) 11/12 (2.2) 7/12 (1.5) 12/12 (2.8)*
Lung: macrophage 0/12 0/12 9/12 (1.2)*** 12/12 (1.0)*** 0/12 11/12 (1.4)***
Eye, ciliary body: epithelial cell 0/12 1/12 (1.0) 4/12 (1.5)* 9/12 (1.5)*** 1/12 (1.0) 10/12 (2.3)***
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a

The incidence is reported as the number of animals with lesions observed microscopically. The average severity is given in parentheses. The severity of tissue vacuolation was scored and evaluated as follows: 1 = minimal – very few vacuoles noted at high magnification and were generally small; 2 = mild – increased numbers of vacuoles were clearly present at high magnification with an admixture of some larger vacuoles; 3 = moderate – vacuoles were discernible at lower magnification with more larger vacuoles being visible along with smaller ones; and 4 = marked – vacuoles evident at lowest magnification with increased numbers and/or size of vacuoles which sometimes caused tissue distortion. An asterisk in the PEG column indicates a significant pairwise comparison of the PEG group to the saline control group for the incidence.

*

p < 0.05.

**

p < 0.01.

***

p < 0.001.

Figure 3.

Figure 3.

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The choroid plexuses from a saline control rat and rats administered 20, 40, and 60 kDa PEGs subcutaneously at 100 mg PEG/kg once a week for 24 weeks. Each group consisted of 12 animals per sex and all animals were evaluated histopathologically. Arrows indicate vacuoles. All images are 40X objective and H&E stain.

Figure 4.

Figure 4.

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The skin at the application site from a saline control rat and rats administered 20, 40, and 60 kDa PEGs subcutaneously at 100 mg PEG/kg once a week for 24 weeks. Each group consisted of 12 animals per sex and all animals were evaluated histopathologically. Arrows indicate vacuoles and arrow heads indicate mast cell infiltration. All images are 40X objective and H&E stain.

Vacuolization was not detected in male rats treated subcutaneously with the vehicle except for choroid plexus. Male rats treated subcutaneously with 20, 40, or 60 kDa PEG had an 83 – 100% incidence of vacuolization in the cervical, mandibular, and mesenteric lymph nodes, spleen, and skin at the site of application (Table 2). In the liver, adrenal cortex, pituitary gland, testes, thymus, mammary gland, bone, lung, and eye, the prevalence of vacuolization tended to increase as the molecular weight of the PEG increased. Vacuolization in the choroid plexus occurred in 67% of the control male rats treated subcutaneously and this was significantly increased to 100% in 40 and 60 kDa PEG-treated male rats. The high percentage of vacuolization in the choroid plexus in control animals may reflect the normal physiologic vacuolation that occurs in this tissue (Kaufmann et al. 2012).

Male rats treated intravenously with 60 kDa PEG had a 92 – 100% incidence of vacuolization in adrenal cortex, pituitary gland, testes, cervical, mandibular, and mesenteric lymph nodes, spleen, thymus, mammary gland, lung, and eye. In the choroid plexus, the incidence of vacuolization increased from 75% in the control group to 100% in the male rats treated intravenously with 60 kDa PEG, an increase that was not statistically significant.

Female rats treated subcutaneously with 20, 40, or 60 kDa PEG had an 83 – 100% incidence of vacuolization in the cervical, mandibular, and mesenteric lymph nodes, spleen, and skin at the site of application (Table 2). In the pituitary gland, ovary, uterus testes, thymus, bone, lung, and eye, the prevalence of vacuolization tended to increase as the molecular weight of the PEG increased. Except for the choroid plexus, only a low incidence of vacuolization (0 – 17%) was detected in female rats treated subcutaneously with the vehicle. Vacuolization in the choroid plexus occurred in 67% of the control female male rats treated subcutaneously and this increased to 83 – 92%% in PEG-treated female rats, an increase that was not statistically significant.

Female rats treated intravenously with 60 kDa PEG had a 100% incidence of vacuolization in adrenal cortex, pituitary gland, ovary, uterus, cervical, mandibular, and mesenteric lymph nodes. A high incidence of vacuolization also occurred in the adrenal medulla, mammary gland, bone lung, and eye. In the choroid plexus, the incidence of vacuolization increased from 67% in the control group to 100% in the female rats treated intravenously with 60 kDa PEG, an increase that was statistically significant.

The route of administration did not result in any marked differences in incidence or severity in cellular vacuolation. Apart from the mammary gland in female rats administered 60 kDa PEG, there was no difference between the subcutaneous and intravenous saline controls or between the subcutaneous and intravenous 60 kDa PEG rats (Table 2).

Cellular vacuolation in the affected tissues did not result in any histopathological changes such as degeneration, inflammation, or necrosis. Whether a more prolonged study would reveal evidence of such changes deserves consideration of additional long term studies. However, a few additional non-neoplastic findings were noted and are summarized in Table 3. In skin at the site of application, there was a 100% incidence of mast cell infiltration (Figure 4) in both male and female rats treated subcutaneously with the PEGs. Subcutaneous injection of males with 60 kDa PEG caused an increased incidence of lymphocytic infiltration of the prostate. Male rats treated subcutaneously with 40 or 60 kDa PEG had an increased incidence of hemorrhage of the lung, and female treated intravenously with 60 kDa had an increased in brain stem compression.

Table 3.

Incidence and severity of non-neoplastic lesions in rats administered PEGs for 24 weeks.a

Subcutaneous
 Intravenous
Saline 20 kDa PEG 40 kDa PEG 60 kDa PEG Saline 60 kDa PEG
Male rats
Skin, site of application, mast cell infiltration 0/12 12/12 (2.1)*** 12/12 (2.1)*** 12/12 (2.1)***
Liver, mixed cell infiltration 6/12 (1.0) 5/12 (1.0) 9/12 (1.0) 5/12 (1.0) 3/12 (1.0) 8/12 (1.0)*
Prostate, lymphocyte infiltration 2/12 (2.0) 2/12 (1.5) 2/12 (1.0) 7/12 (1.4)* 7/12 (1.0) 3/12 (1.3)
Lung, hemorrhage 3/12 (4.0) 6/12 (2.0) 9/12 (1.9)* 8/12 (2.1)* 2/12 (3.5) 5/12 (2.2)
Female rats
Skin, site of application, mast cell infiltration 0/12 12/12 (2.1)*** 12/12 (2.1)*** 12/12 (2.0)***
Brain stem, compression 1/12 (1.0) 2/12 (1.0) 1/12 (1.0) 3/12 (1.0) 1/12 (1.0) 5/12 (1.2)*
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a

The incidence is reported as the number of animals with lesions observed microscopically. The average severity is given in parentheses. The severity was scored as 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked. An asterisk in the PEG column indicates a significant pairwise comparison of the PEG group to the saline control group for the incidence.

*

p < 0.05.

***

p < 0.001.

Discussion

In this study, nearly all rats survived until their scheduled sacrifice time and had no signs of toxicity, such as treatment-related changes in body weight, food consumption, or organ weights. Following the 24-week exposure, PEG was detected in plasma and increased in a molecular-weight-related manner. PEG was excreted in both urine and feces; urinary levels of PEGs decreased with an increase in molecular weight. Low levels of PEGs were detected in CSF. Anti-PEG IgM was detected in control and treated groups, with the prevalence being greater in the PEG groups. Cellular vacuolization occurred in multiple tissues/organs including the choroid plexus, but not in the kidney, urinary bladder, or heart. The presence of PEG-related vacuoles did not result in inflammation, degeneration, or necrosis.

Although PEG has been regarded as non-immunogenic and PEGylation of therapeutic proteins has been used to diminish the immunogenicity of the conjugated immunogenic proteins, several studies have observed that IgG and IgM antibodies, which specifically recognize the PEG moiety of various PEGylated biologicals, are induced both in animals (Ichihara et al. 2011; Ishida et al. 2006; Ishida et al. 2007; Mima et al. 2015; Środa et al. 2005) and in humans (Armstrong et al. 2007; Ganson et al. 2006; Hershfield et al. 2014). However, there are few studies on the antibody response to high-molecular-weight unconjugated PEG. Richter and Åkerblom showed that the subcutaneous administration of 10 and 100 kDa PEG in Freund’s complete adjuvant was nonimmunogenic in rabbits and that a 5,900 kDa PEG elicited a weak and transitory immune response to PEG in mice (Richter and Åkerblom 1983). In a recent study, a single intravenous injection of 2 and 20 kDa PEG to mice induced low levels of anti-PEG IgM, but not anti-PEG IgG, with the level of anti-PEG IgM being at least 2-fold lower than that induced by PEGylated ovalbumin or PEGylated liposome (Mima et al. 2015). Our study is consistent with previous findings that the generation of anti-PEG IgM is not succeeded by isotype switching to IgG either by subcutaneous or intravenous administration of high-molecular-weight PEG to rats. The mechanism underlying the selective IgM antibody response and failure of isotype switching in this context is not clear as IgG antibodies to PEG have been observed in numerous clinical studies of PEGylated biologics (Armstrong 2009; Sundy et al. 2011) pointing to the possible role of the immune response to the protein moiety as the driver of isotype switching.

Animal studies have shown that anti-PEG IgM antibodies accelerate the clearance of PEGylated proteins in mice (Cheng et al. 2000; Cheng et al. 1999; Mima et al. 2015). Several pre-clinical studies have indicated that anti-PEG antibodies can alter the pharmacokinetics, including half-life, clearance, maximum concentration, and area-under-the-curve, and affect the efficacy of the PEGylated biologicals (Armstrong 2009; Armstrong et al. 2007; Ganson et al. 2006; Hsieh et al. 2018; Sundy et al. 2011). Anti-PEG antibodies have been associated with acute severe allergic reactions to the PEGylated RNA aptamer pegnivacogin (Ganson et al. 2016; Povsic et al. 2016) as well as with other adverse effects, such as gout flares and mild-to-moderate pain, cellulitis, and urticaria at the injection site following subcutaneous injection of PEG-uricase (Ganson et al. 2006). Thus, anti-PEG antibodies are clearly a concern for the efficacy and safety of PEGylated therapeutics.

The analysis of high-molecular-weight PEG is challenging as it does not contain active chromophores for UV characterization. Several methods, including SDS-PAGE with iodine staining (Kurfürst 1992), immunohistochemical (IHC) analyses (Rudmann et al. 2013), and size exclusion chromatography with different detectors (Corona charged aerosol, refractive index, evaporative light scattering, or mass spectrometry) (Kou et al. 2009; Liu et al. 2004), have been utilized. In this study, the levels of PEG in plasma and CSF and excretion of high-molecular-weight PEGs were successfully determined by a slightly modified SDS-PAGE with iodine staining (Kurfürst 1992). The assay was also used to examine the cellular PEG uptake by the human monocytic cell line THP-1 (data not shown). To measure the tissue accumulation of high-molecular-weight PEG in the formalin-fixed paraffin-embedded tissue specimens, an IHC assay is preferred although this method is not quantitative. However, the tissue accumulation of the PEG was not reported in our study due to high background levels when attempting to develop an IHC assay.

The choroid plexus actively secretes CSF and is responsible for maintaining the homeostasis of nutrients and osmotic load in the CSF. The choroid plexus consists of epithelial cells that surround capillaries and connective tissue, and these epithelial cells form the blood-brain barrier. Animals administered 40 kDa PEG had PEG accumulation and the formation of vacuolation in choroid plexus epithelial cells (NovoNordisk 2017; Rudmann et al. 2013). Examination of the blood-brain barrier indicated the PEG staining was on the circulatory and not brain epithelial side of the barrier suggesting no transfer across the blood-brain barrier (Rudmann et al. 2013). In our study, PEG was detected at low levels in CSF, suggesting the possibility that PEG could leak into the CSF; however, the mechanism underlying such leaking of PEG remains unknown.

Nonclinical studies on PEGylated biologicals and PEG have shown cellular vacuolation in some animals in various tissues/organs and with various time points and dosing regimens (Ivens et al. 2015; NovoNordisk 2017; Rudmann et al. 2013; Stidl et al. 2016; Turecek et al. 2016). There appear to be a correlation of cellular vacuolization with molecular weight of PEG (Rudmann et al. 2013). Consistent with these reports, we observed PEG-associated vacuolization in macrophages and parenchymal cells of multiple tissues/organs associated with its molecular weight. In contrast, we did not detect PEG-associated vacuolization in kidney, urinary bladder, and heart. The reasons for this discrepancy are not clear. Moreover, there are many factors that are likely to impact the occurrence and degree of vacuolization following treatment with PEG and PEGylated biologicals and include distribution of drug target, drug dose, dose frequency and duration of treatment, distribution of the drug, amount of nonspecific uptake versus specific drug, receptor mediated uptake, potential immunogenicity, the molecular weight of PEG, and types of clearance/removal mechanisms for drug or PEG (Ivens et al. 2015).

The formation of cellular vacuoles is probably the result of transport of the PEG into the cell and sequestration in the vacuoles, as PEG is not biodegradable. Upon reviewing a number of pre-clinical studies, a working group concluded that PEG-related vacuolation was not associated with cell or tissue damage or dysfunction and was reversible with sufficient recovery periods (Irizarry Rovira et al. 2018). Under our experimental conditions, there was no systemic toxicity following PEG exposure. Furthermore, there were no detectable degenerating neurons using Fluro-Jade staining of the brain, spinal cord, or trigeminal nerve with ganglia of rats exposed to PEGs (data not shown). Vacuolation in tissues/organs is generally considered a consequence of the natural PEG removal process (Ivens et al. 2015; Stidl et al. 2016); however, the genesis of vacuolization and the potential impact of the cellular vacuoles with PEG requires further understanding. As there is a particular accumulation of PEG within cells of the immune system with antigen presenting activity, evaluation of immune responses in PEG treated animals to antigenic challenge is a critical follow up study.

Supplementary Material

1
2
3

Highlights.

  • A higher prevalence of anti-PEG IgM was observed in PEG groups.

  • PEG treatment led to a molecular-weight-related increase in the levels of PEG in plasma and a low level of PEG in cerebrospinal fluid.

  • PEGs were excreted in both urine and feces, with a molecular-weight-related decrease in urine excretion.

  • PEG treatment produced a molecular-weight-trend in vacuolation in the spleen, lymph nodes, lungs, and ovaries/testes.

  • Vacuolation was not accompanied by an inflammatory response.

Acknowledgements

We thank Mercedes A. Serabian, Center for Biologics Evaluation and Research, for valuable comments and suggestions during the conceptualization of the protocol and the study; Alan Warbritton, Toxicologic Pathology Associates, Inc., NCTR, for the preparation the figures showing tissue vacuolation; Levan Muskhelishvili, Toxicologic Pathology Associates, Inc., NCTR, for the conducting Fluro-Jade C/4′,6-diamidino-2-phenylindole (DAPI) staining of the brain, spinal cord, or trigeminal nerve with ganglia; Andy Matson and Mark Moore for providing animal diets and animal care. This work was funded by the National Institute of Environmental Health Sciences (NIEHS) under an Interagency Agreement between the U.S. Food and Drug Administration (FDA) and NIEHS (FDA IAG #224-17-0502 and NIH IAG #AES12013).

Footnotes

Conflict of interest statement

The authors declare that there are no conflicts of interest.

This article reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug Administration.

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References

  1. Abu Lila AS, Kiwada H, Ishida T (2013) The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage. J Control Release 172(1):38–47 doi: 10.1016/j.jconrel.2013.07.026 [DOI] [PubMed] [Google Scholar]
  2. Armstrong JK (2009) The occurrence, induction, specificity and potential effect of antibodies against poly(ethylene glycol). In: Veronese FM (ed) PEGylated Protein Drugs: Basic Science and Clinical Applications. Birkhäuser, Basel, p 147–168 [Google Scholar]
  3. Armstrong JK, Hempel G, Koling S, et al. (2007) Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients. Cancer 110(1):103–111 doi: 10.1002/cncr.22739 [DOI] [PubMed] [Google Scholar]
  4. Baumann A, Tuerck D, Prabhu S, Dickmann L, Sims J (2014) Pharmacokinetics, metabolism and distribution of PEGs and PEGylated proteins: quo vadis? Drug Discov Today 19(10):1623–1631 doi: 10.1016/j.drudis.2014.06.002 [DOI] [PubMed] [Google Scholar]
  5. Bjørnsdottir I, Sternebring O, Kappers WA, et al. (2016) Pharmacokinetics, tissue distribution and excretion of 40kDa PEG and PEGylated rFVIII (N8-GP) in rats. Eur J Pharm Sci 87:58–68 doi: 10.1016/j.ejps.2015.10.020 [DOI] [PubMed] [Google Scholar]
  6. Brunner E, Domhof S, Langer F (2002) Nonparametric Analysis of Longitudinal Data in Factorial Experiments. John Wiley & Sons, Inc., New York [Google Scholar]
  7. Caliceti P, Veronese FM (2003) Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 55(10):1261–1277 [DOI] [PubMed] [Google Scholar]
  8. Chang T-C, Chen B-M, Lin W-W, et al. (2019) Both IgM and IgG antibodies against polyethylene glycol can alter the biological activity of methoxy polyethylene glycol-epoetin beta in mice. Pharmaceutics 12(1):15 doi: 10.3390/pharmaceutics12010015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng T-L, Chen B-M, Chern J-W, Wu M-F, Roffler SR (2000) Efficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy. Bioconjug Chem 11(2):258–266 doi: 10.1021/bc990147j [DOI] [PubMed] [Google Scholar]
  10. Cheng T-L, Wu P-Y, Wu M-F, Chern J-W, Roffler SR (1999) Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM. Bioconjug Chem 10(3):520–528 doi: 10.1021/bc980143z [DOI] [PubMed] [Google Scholar]
  11. Cox DR (1972) Regression models and life-tables. J R Stat Soc B34:187–220 [Google Scholar]
  12. EFSA (2007) Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to an application on the use of polyethylene glycol (PEG) as a film coating agent for use in food supplement products. EFSA Journal 5(1):414 doi: 10.2903/j.efsa.2007.414 [DOI] [Google Scholar]
  13. Fang J-L, Beland FA, Tang Y, Roffler SR (2021) Flow cytometry analysis of anti-polyethylene glycol antibodies in human plasma. Toxicol Rep 8:148–154 doi: 10.1016/j.toxrep.2020.12.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fang J-L, Vanlandingham MM, Juliar BE, Olson GR, Patton RE, Beland FA (2015) Dose–response assessment of the dermal toxicity of triclosan in B6C3F1 mice. Toxicol Res 4(4):867–877 doi: 10.1039/C4TX00152D [DOI] [Google Scholar]
  15. Fruijtier-Pölloth C (2005) Safety assessment on polyethylene glycols (PEGs) and their derivatives as used in cosmetic products. Toxicology 214(1–2):1–38 doi: 10.1016/j.tox.2005.06.001 [DOI] [PubMed] [Google Scholar]
  16. Ganson NJ, Kelly SJ, Scarlett E, Sundy JS, Hershfield MS (2006) Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase. Arthritis Res Ther 8(1):R12 doi: 10.1186/ar1861 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ganson NJ, Povsic TJ, Sullenger BA, et al. (2016) Pre-existing anti-polyethylene glycol antibody linked to first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer. J Allergy Clin Immunol 137(5):1610–1613 doi: 10.1016/j.jaci.2015.10.034 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Garay RP, El-Gewely R, Armstrong JK, Garratty G, Richette P (2012) Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEG-conjugated agents. Expert Opin Drug Deliv 9(11):1319–1323 doi: 10.1517/17425247.2012.720969 [DOI] [PubMed] [Google Scholar]
  19. Hershfield MS, Ganson NJ, Kelly SJ, Scarlett EL, Jaggers DA, Sundy JS (2014) Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients. Arthritis Res Ther 16(2):R63 doi: 10.1186/ar4500 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hsieh Y-C, Wang H-E, Lin W-W, et al. (2018) Pre-existing anti-polyethylene glycol antibody reduces the therapeutic efficacy and pharmacokinetics of PEGylated liposomes. Theranostics 8(11):3164–3175 doi: 10.7150/thno.22164 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ichihara M, Shimizu T, Imoto A, et al. (2011) Anti-PEG IgM response against PEGylated liposomes in mice and rats. Pharmaceutics 3(1):1–11 doi: 10.3390/pharmaceutics3010001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Irizarry Rovira AR, Bennet BM, Bolon B, et al. (2018) Scientific and regulatory policy committee points to consider: histopathologic evaluation in safety assessment studies for PEGylated pharmaceutical products. Toxicol Pathol 46(6):616–635 doi: 10.1177/0192623318791801 [DOI] [PubMed] [Google Scholar]
  23. Ishida T, Ichihara M, Wang XY, et al. (2006) Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes. J Control Release 112(1):15–25 doi: 10.1016/j.jconrel.2006.01.005 [DOI] [PubMed] [Google Scholar]
  24. Ishida T, Wang XY, Shimizu T, Nawata K, Kiwada H (2007) PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner. J Control Release 122(3):349–355 doi: 10.1016/j.jconrel.2007.05.015 [DOI] [PubMed] [Google Scholar]
  25. Ivens IA, Achanzar W, Baumann A, et al. (2015) PEGylated biopharmaceuticals: current experience and considerations for nonclinical development. Toxicol Pathol 43(7):959–983 doi: 10.1177/0192623315591171 [DOI] [PubMed] [Google Scholar]
  26. Kaufmann W, Bolon B, Bradley A, et al. (2012) Proliferative and nonproliferative lesions of the rat and mouse central and peripheral nervous systems. Toxicol Pathol 40(4 Suppl):87S–157S doi: 10.1177/0192623312439125 [DOI] [PubMed] [Google Scholar]
  27. Knadler MP, Ellis BB, Brown-Augsburger PL, Murphy AT, Martin JA, Wroblewski VJ (2015) Disposition of basal insulin peglispro compared with 20-kDa polyethylene glycol in rats following a single intravenous or subcutaneous dose. Drug Metab Dispos 43(10):1477–1483 doi: 10.1124/dmd.115.064337 [DOI] [PubMed] [Google Scholar]
  28. Kou D, Manius G, Zhan S, Chokshi HP (2009) Size exclusion chromatography with Corona charged aerosol detector for the analysis of polyethylene glycol polymer. J Chromatogr A 1216(28):5424–5428 doi: 10.1016/j.chroma.2009.05.043 [DOI] [PubMed] [Google Scholar]
  29. Kurfürst MM (1992) Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 200(2):244–248 [DOI] [PubMed] [Google Scholar]
  30. Liu M, Xie C, Xu W, Lu W (2004) Separation of polyethylene glycols and their amino-substituted derivatives by high-performance gel filtration chromatography at low ionic strength with refractive index detection. J Chromatogr A 1046(1–2):121–126 doi: 10.1016/j.chroma.2004.06.005 [DOI] [PubMed] [Google Scholar]
  31. Mima Y, Hashimoto Y, Shimizu T, Kiwada H, Ishida T (2015) Anti-PEG IgM is a major contributor to the accelerated blood clearance of polyethylene glycol-conjugated protein. Mol Pharm 12(7):2429–2435 doi: 10.1021/acs.molpharmaceut.5b00144 [DOI] [PubMed] [Google Scholar]
  32. Morpurgo M, Veronese FM (2004) Conjugates of peptides and proteins to polyethylene glycols. Methods Mol Biol 283:45–70 doi: 10.1385/1-59259-813-7:045 [DOI] [PubMed] [Google Scholar]
  33. NovoNordisk (2017) Novo Nordisk, Inc. Coagulation Factor IX (Recombinant), GlycoPEGylated N9-GP, BLA125611 FDA Briefing Document (April 4, 2017) https://wwwfdagov/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/BloodProductsAdvisoryCommittee/ucm543914htm. [Google Scholar]
  34. Povsic TJ, Lawrence MG, Lincoff AM, et al. (2016) Pre-existing anti-PEG antibodies are associated with severe immediate allergic reactions to pegnivacogin, a PEGylated aptamer. J Allergy Clin Immunol 138(6):1712–1715 doi: 10.1016/j.jaci.2016.04.058 [DOI] [PubMed] [Google Scholar]
  35. Richter AW, Åkerblom E (1983) Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins. Int Arch Allergy Appl Immunol 70(2):124–131 [DOI] [PubMed] [Google Scholar]
  36. Rudmann DG, Alston JT, Hanson JC, Heidel S (2013) High molecular weight polyethylene glycol cellular distribution and PEG-associated cytoplasmic vacuolation is molecular weight dependent and does not require conjugation to proteins. Toxicol Pathol 41(7):970–983 doi: 10.1177/0192623312474726 [DOI] [PubMed] [Google Scholar]
  37. Schellekens H, Hennink WE, Brinks V (2013) The immunogenicity of polyethylene glycol: facts and fiction. Pharm Res 30(7):1729–1734 doi: 10.1007/s11095-013-1067-7 [DOI] [PubMed] [Google Scholar]
  38. Sellers RS, Morton D, Michael B, et al. (2007) Society of Toxicologic Pathology position paper: organ weight recommendations for toxicology studies. Toxicol Pathol 35(5):751–755 doi: 10.1080/01926230701595300 [DOI] [PubMed] [Google Scholar]
  39. Shimizu T, Ichihara M, Yoshioka Y, Ishida T, Nakagawa S, Kiwada H (2012) Intravenous administration of polyethylene glycol-coated (PEGylated) proteins and PEGylated adenovirus elicits an anti-PEG immunoglobulin M response. Biol Pharm Bull 35(8):1336–1342 doi: 10.1248/bpb.b12-00276 [DOI] [PubMed] [Google Scholar]
  40. Shiraishi K, Yokoyama M (2019) Toxicity and immunogenicity concerns related to PEGylated-micelle carrier systems: a review. Sci Technol Adv Mater 20(1):324–336 doi: 10.1080/14686996.2019.1590126 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sims GEC, Snape TJ (1980) A method for the estimation of polyethylene glycol in plasma protein fractions. Anal Biochem 107(1):60–63 doi: 10.1016/0003-2697(80)90492-3 [DOI] [PubMed] [Google Scholar]
  42. Środa K, Rydlewski J, Langner M, Kozubek A, Grzybek M, Sikorski AF (2005) Repeated injections of PEG-PE liposomes generate anti-PEG antibodies. Cell Mol Biol Lett 10(1):37–47 [PubMed] [Google Scholar]
  43. Stidl R, Fuchs S, Bossard M, Siekmann J, Turecek PL, Putz M (2016) Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates. Haemophilia 22(1):54–64 doi: 10.1111/hae.12762 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sundy JS, Baraf HSB, Yood RA, et al. (2011) Efficacy and tolerability of pegloticase for the treatment of chronic gout in patients refractory to conventional treatment: two randomized controlled trials. JAMA 306(7):711–720 doi: 10.1001/jama.2011.1169 [DOI] [PubMed] [Google Scholar]
  45. Turecek PL, Bossard MJ, Schoetens F, Ivens IA (2016) PEGylation of biopharmaceuticals: a review of chemistry and nonclinical safety information of approved drugs. J Pharm Sci 105(2):460–475 doi: 10.1016/j.xphs.2015.11.015 [DOI] [PubMed] [Google Scholar]
  46. Webster R, Didier E, Harris P, et al. (2007) PEGylated proteins: evaluation of their safety in the absence of definitive metabolism studies. Drug Metab Dispos 35(1):9–16 doi: 10.1124/dmd.106.012419 [DOI] [PubMed] [Google Scholar]
  47. Webster R, Elliott V, Park BK, Walker D, Hankin M, Taupin P (2009) PEG and PEG conjugates toxicity: towards an understanding of the toxicity of PEG and its relevance to PEGylated biologicals. In: Veronese FM (ed) PEGylated Protein Drugs: Basic Science and Clinical Applications. Birkhäuser Verlag, Basel, Switzerland, p 127–146 [Google Scholar]
  48. Yamaoka T, Tabata Y, Ikada Y (1995) Fate of water-soluble polymers administered via different routes. J Pharm Sci 84(3):349–354 [DOI] [PubMed] [Google Scholar]
  49. Zhao Y, Wang L, Yan M, et al. (2012) Repeated injection of PEGylated solid lipid nanoparticles induces accelerated blood clearance in mice and beagles. Int J Nanomedicine 7:2891–2900 doi: 10.2147/IJN.S30943 [DOI] [PMC free article] [PubMed] [Google Scholar]

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