Large Molecule Drugs Research Articles

Nanotracing and cavity-ring down spectroscopy: A new ultrasensitive approach in large molecule drug disposition studies.

New therapeutic biological entities such as bispecific antibodies targeting tissue or specific cell populations form an increasingly important part of the drug development portfolio. However, these biopharmaceutical agents bear the risk of extensive target-mediated drug disposition or atypical pharmacokinetic properties as compared to canonical antibodies. Pharmacokinetics and bio-distribution studies become therefore more and more important during lead optimization. Biologics present, however, greater analytical challenges than small molecule drugs due to the mass and selectivity limitation of mass spectrometry and ligand-binding assay, respectively. Radiocarbon (14C) and its detection methods, such as the emerging 14C cavity ring down spectroscopy (CRDS), thus can play an important role in the large molecule quantitation where a 14C-tag is covalently bound through a stable linker. CRDS has the advantage of a simplified sample preparation and introduction system as compared to accelerator mass spectrometry (AMS) and can be accommodated within an ordinary research laboratory. In this study, we report on the labeling of an anti-IL17 IgG1 model antibody with 14C propionate tag and its detection by CRDS using it as nanotracer (2.1 nCi or 77.7 Bq blended with the therapeutic dose) in a pharmacokinetics study in a preclinical species. We compare these data to data generated by AMS in parallel processed samples. The derived concentration time profiles for anti-IL17 by CRDS were in concordance with the ones derived by AMS and γ-counting of an 125I-labeled anti-IL17 radiotracer and were well described by a 2-compartment population pharmacokinetic model. In addition, antibody tissue distribution coefficients for anti-IL17 were determined by CRDS, which proved to be a direct and sensitive measurement of the extravascular tissue concentration of the antibody when tissue perfusion was applied. Thus, this proof-of-concept study demonstrates that trace 14C-radiolabels and CRDS are an ultrasensitive approach in (pre)clinical pharmacokinetics and bio-distribution studies of new therapeutic entities.

Quantitative Estimation of the Effect of Nasal Mucociliary Function on in Vivo Absorption of Norfloxacin after Intranasal Administration to Rats.

Nasal drug delivery has attracted significant attention as an alternative route to deliver drugs having poor bioavailability. Large-molecule drugs, such as peptides and central nervous system drugs, would benefit from intranasal delivery. Drug absorption after intranasal application depends on the nasal retention of the drug, which is determined by the nasal mucociliary clearance. Mucociliary clearance (MC) is an important determinant of the rate and extent of nasal drug absorption. The aim of the present study was to clarify the effect of the changes in MC on in vivo drug absorption after nasal application, and to justify the pharmacokinetic model to which the MC parameter was introduced, to enable prediction of bioavailability after intranasal administration. The pharmacokinetics of norfloxacin (NFX) after intranasal administration were evaluated following the modification of nasal MC by pretreatment with the MC inhibitors propranolol and atropine and the MC enhancers terbutaline and acetylcholine chloride. From the relationship between nasal MC and bioavailability after nasal application, prediction of drug absorption was attempted on the basis of our pharmacokinetic model. Propranolol and atropine enhanced the bioavailability of NFX by 90 and 40%, respectively, while the bioavailability decreased by 30% following terbutaline and 40% following acetylcholine chloride. As a result of changes in the MC function, nasal drug absorption was changed depending on the nasal residence time of the drug. On the basis of our pharmacokinetic model, the nasal drug absorption can be precisely predicted, even when the MC is changed. This prediction system allows the quantitative evaluation of changes in drug absorption due to changes in nasal MC and is expected to contribute greatly to the development of nasal formulations.

Bimetallic Nanostructures: Shape-Controlled Synthesis for Catalysis, Plasmonics, and Sensing Applications

The formulation of novel drug delivery systems using biopolymers has been standard practice for over half a century. Polyethylene glycol (PEG) has been the “go-to” polymer in many successful instances resulting in the creation of many blockbuster pharmaceutical drugs. Recent reports have shown that PEG has many chemical and biological limitations and the need to create other biopolymers is of renewed interest in industry and academia. Poly(oxazoline) or POZ is a versatile type of polymer with many industrial and pharmaceutical applications. Pharmaceutical-grade POZ has stealth-like properties similar to PEG, but has many advantages in the areas of synthesis, solubility, stability, viscosity, drug loading, tissue targeting, immunogenicity, and toxicity. POZ has been successfully conjugated to small and large molecule drugs and the first POZ conjugated small molecule has been successfully tested in a Phase I clinical study. This chapter describes the chemistry and properties of POZ and its application in delivering a protein and a small molecule.

Concerted Application of LC–MS and Ligand Binding Assays to Better Understand Exposure of a Large Molecule Drug

A ligand-binding assay (LBA) was used to measure exposure of PRM-151, the recombinant form of human pentraxin-2 (PTX-2), a complex pentamer with multiple binding partners. However, the assay showed a lack of dose-dependent exposure in select preclinical species and it could not differentiate the infused PRM-151 from the endogenous PTX-2 in nonhuman primates. Instead of assessing interference from its multiple binding partners, which could be time consuming and laborious, a LC-MS assay avoid of these interference was implemented to measure 'total' drug without the use of immunoaffinity capture reagents. The resultant LC-MS data confirmed the original data and the lack of dose-dependent exposure is now understood to be due to the multiple and diverse targets and functions and resultant complex biodistribution rather than an assay artifact.

High Throughput Transfection of HEK293 Cells for Transient Protein Production.

Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply recombinant protein for research and large molecule drug development. Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30mL tubespins using polyethylenimine (PEI) as a transfection reagent. An automated liquid handler can be used to perform pipetting steps for transfecting batches of 96 tubespins, and septa in the tubespin caps allow for rapid processing without decapping. The addition of valproic acid (VPA) to transfection cultures enhances recombinant protein production. The thawing and passaging operations for HEK293 cultures to source the transient transfections are also described.

Delivery of Biologics Across the Blood–Brain Barrier with Molecular Trojan Horse Technology

Biologics are potential new therapeutics for many diseases of the central nervous system. Biologics include recombinant lysosomal enzymes, neurotrophins, decoy receptors, and therapeutic antibodies. These are large molecule drugs that do not cross the blood-brain barrier (BBB). All classes of biologics have been tested, without success, in clinical trials of brain disease over the last 25years. In none of these past clinical trials was the biologic re-engineered to enable transport across the BBB. If the biologic does not cross the BBB, the drug cannot reach the target site in brain, and success in a clinical trial is not expected. Biologics can be re-engineered for BBB transport with the use of molecular Trojan horse technology. A BBB molecular Trojan horse is a monoclonal antibody (MAb) against an endogenous BBB receptor transporter, such as the insulin receptor or transferrin receptor. The receptor-specific MAb penetrates the brain via transport on the endogenous BBB receptor. The MAb acts as a molecular Trojan horse to deliver across the BBB the biologic pharmaceutical that is genetically fused to the MAb. The lead Trojan horse is a MAb against the human insulin receptor (HIR), and HIRMAb-derived fusion proteins have entered clinical trials for the treatment of brain disease.

Addressing the challenges of biomarker calibration standards in ligand-binding assays: a European Bioanalysis Forum perspective.

The analysis of biomarkers by ligand-binding assays offers significant challenges compared with the bioanalysis of small and large molecule drugs. The presence of endogenous analyte is a commonly cited issue. Also the sourcing and application of appropriate calibration or reference standards can present many issues. One of the main challenges is ensuring the continuity and validity of biomarker data when the source or lot number of calibration standard changes within or between studies. Several strategies exist in attempting to deal with this and standardize the biomarker data through the assay life or looking for ways to compare and normalize biomarker data. In this manuscript, the European Bioanalysis Forum view on dealing with calibration standards in biomarker assays is described.

Endocytic Uptake, Transport and Macromolecular Interactions of Anionic PAMAM Dendrimers within Lung Tissue

PurposePolyamidoamine (PAMAM) dendrimers are a promising class of nanocarrier with applications in both small and large molecule drug delivery. Here we report a comprehensive evaluation of the uptake and transport pathways that contribute to the lung disposition of dendrimers.MethodsAnionic PAMAM dendrimers and control dextran probes were applied to an isolated perfused rat lung (IPRL) model and lung epithelial monolayers. Endocytosis pathways were examined in primary alveolar epithelial cultures by confocal microscopy. Molecular interactions of dendrimers with protein and lipid lung fluid components were studied using small angle neutron scattering (SANS).ResultsDendrimers were absorbed across the intact lung via a passive, size-dependent transport pathway at rates slower than dextrans of similar molecular sizes. SANS investigations of concentration-dependent PAMAM transport in the IPRL confirmed no aggregation of PAMAMs with either albumin or dipalmitoylphosphatidylcholine lung lining fluid components. Distinct endocytic compartments were identified within primary alveolar epithelial cells and their functionality in the rapid uptake of fluorescent dendrimers and model macromolecular probes was confirmed by co-localisation studies.ConclusionsPAMAM dendrimers display favourable lung biocompatibility but modest lung to blood absorption kinetics. These data support the investigation of dendrimer-based carriers for controlled-release drug delivery to the deep lung.

Use of mathematics to guide target selection in systems pharmacology; application to receptor tyrosine kinase (RTK) pathways

A key element of the drug discovery process is target selection. Although the topic is subject to much discussion and experimental effort, there are no defined quantitative rules around optimal selection. Often ‘rules of thumb’, that have not been subject to rigorous exploration, are used. In this paper we explore the ‘rule of thumb’ notion that the molecule that initiates a pathway signal is the optimal target. Given the multi-factorial and complex nature of this question, we have simplified an example pathway to its logical minimum of two steps and used a mathematical model of this to explore the different options in the context of typical small and large molecule drugs. In this paper, we report the conclusions of our analysis and describe the analysis tool and methods used. These provide a platform to enable a more extensive enquiry into this important topic.

Minimum Transendothelial Electrical Resistance Thresholds for the Study of Small and Large Molecule Drug Transport in a Human in Vitro Blood-Brain Barrier Model.

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here, human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ω·cm2. By assessing the permeabilities of several known drugs, a benchmarking system to evaluate brain permeability of drugs was established. Furthermore, relationships between TEER and permeability to both small and large molecules were established, demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype, and the benchmarks established here are useful for assessing functionality of other in vitro BBB models.

Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography

Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

Comparison of atmospheric microplasma and plasma jet irradiation for increasing of skin permeability

Atmospheric plasma is attracting interest for medical applications such as sterilization, treatment of cancer cells and blood coagulation. Application of atmospheric plasma in dermatology has potential as a novel tool for wound healing, skin rejuvenation and treatment of wrinkles. In this study, we investigated the enhancement of percutaneous absorption of dye as alternative agents of transdermal drugs. Hypodermic needles are often the only way to deliver large-molecule drugs into the dermis, although a safe transdermal drug delivery method that does not require needles would be desirable. We therefore explored the feasibility of using atmospheric microplasma irradiation to enhance percutaneous absorption of drugs, as an alternative delivery method to conventional hypodermic needles. Pig skin was used as a biological sample, exposed to atmospheric microplasma, and analyzed by attenuated total reflection-Fourier transform infrared spectroscopy. A tape stripping test, a representative method for evaluating skin barrier performance, was also conducted for comparison. Transepidermal water loss (TEWL) was measured and compared with and without atmospheric microplasma irradiation, to quantify water evaporation from the inner body through the skin barrier. The results show that the stratum corneum, the outermost skin layer, could be chemically and physically modified by atmospheric microplasma irradiation. Physical damage to the skin by microplasma irradiation and an atmospheric plasma jet was also assessed by observing the skin surface. The results suggest that atmospheric microplasma has the potential to enhance percutaneous absorption.

Abstract 2092: Characterizing NIR dye-IL13RA2 antibody conjugates for biodistribution studies in xenograft tumor models by fluorescence molecular tomography (FMT)

Abstract Background: The antibody based therapies are promising anti-cancer therapeutic modalities with minimal toxicity and maximum efficacy. The efficacy of these agents is regulated by their biodistribution and targeting. The contemporary methods of testing the biodistriution of large molecule drugs are expensive and tedious. The development of simple and rapid methodology, such as optical imaging, can enable effective screening of a larger number of compounds. Here we have used Fluorescence Molecular Tomography (FMT), an optical imaging technique to study biodistribution and tumor targeting of IL3RA2 antibody (Ab). Methods: Different near infrared (NIR) fluorophores (λmax:650-800nm) were conjugated to the Ab. The fluorophore conjugation protocol was optimized to achieve a degree of labeling (DOL) of 1-3 for all the conjugates. The properties of Ab-fluorophore conjugate (Ab-F) were compared to unlabeled Ab using SEC-HPLC and cell binding assays in three cell lines with varying expression of IL13RA2: A375(+++), U87MG(+) and H460(-). For in vivo evaluation, Ab-F conjugates were administered intravenously at a dose of 2 nmol fluorophore to nu/nu mice bearing A375. Similar studies were also conducted in U87MG and H460 xenografts with selected Ab-F. The mice were imaged longitudinally (6 time-points) for up to 96 hrs using FMT4000 and the data were analyzed using TrueQuant software. Results: The SEC-HPLC and flow cytometry studies demonstrated that conjugation of Ab with most of the fluorophores did not change its stability or functionality. The in vivo fluorescence data from all the NIR dyes showed a peak tumor accumulation of the Ab-F at 6h and was maintained until 96 hrs. Quantitation of various Ab-F conjugated revealed that 2-6% of the injected dose was accumulated in the A375 tumors. In contrast to tumor profile, there was a steep decline in heart signal (a surrogate for blood/ plasma concentration), suggesting fast clearance from blood. The in vivo and ex vivo data suggested that there was 15-80pmol of Ab-F conjugate accumulated in the tumors at 96h. In addition, Alexa Flour® (AF)680 and AF750 showed minimal non-specific accumulation in other organs, whereas VivoTag® (VT)680 and BODIPY®630 showed a significantly higher non-specific accumulation in liver. Conclusions: These results show that the biological properties of Ab were not changed by conjugation with various NIR fluorophores at DOL <3. Optical imaging using fluorescent tags can effectively track and quantitate the tumor targeting/ biodistribution of large molecule drugs. Citation Format: Parul Gupta, Dangshe Ma, Rachel Roach, Mary Spilker, Mauricio Leal, Cedo Bagi, Anand Giddabasappa. Characterizing NIR dye-IL13RA2 antibody conjugates for biodistribution studies in xenograft tumor models by fluorescence molecular tomography (FMT). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2092.

Nuclear-targeting TAT-PEG-Asp8-doxorubicin polymeric nanoassembly to overcome drug-resistant colon cancer.

Aim:Drug efflux-associated multidrug resistance (MDR) is a main obstacle to effective cancer chemotherapy. Large molecule drugs are not the substrates of P-glycoprotein, and can circumvent drug efflux and be retained inside cells. In this article we report a polymer-drug conjugate nanoparticulate system that can overcome MDR based on size-related exclusion effect.Methods:Doxorubicin was coupled with the triblock polymeric material cell-penetrating TAT-PEG-poly(aspartic acid). The amphiphilic macromolecules (termed TAT-PEG-Asp8-Dox) could self-assemble into nanoparticles (NPs) in water. The antitumor activity was evaluated in drug-resistant human colon cancer HCT8/ADR cells in vitro and in nude mice bearing HCT8/ADR tumor.Results:The self-assembling TAT-PEG-Asp8-Dox NPs were approximately 150 nm with a narrow particle size distribution, which not only increased the cellular uptake efficiency, but also bypassed P-glycoprotein-mediated drug efflux and improved the intracellular drug retention, thus yielding an enhanced efficacy for killing drug-resistant HCT8/ADR colon cancer cells in vitro. Importantly, the TAT-PEG-Asp8-Dox NPs enhanced the intranuclear disposition of drugs for grater inhibition of DNA/RNA biosynthesis. In nude mice bearing xenografted HCT8/ADR colon cancers, intravenous or peritumoral injection of TAT-PEG-Asp8-Dox NPs for 22 d effectively inhibited tumor growth.Conclusion:TAT-PEG-Asp8-Dox NPs can increase cellular drug uptake and intranuclear drug delivery and retain effective drug accumulation inside the cells, thus exhibiting enhanced anticancer activity toward the drug-resistant human colon cancer HCT8/ADR cells.

Application of High-Resolution MS for Development of Peptide and Large-Molecule Drug Candidates

For quantitative bioanalysis utilizing MS, the instrument of choice is typically a triple quadruple mass spectrometer. However, advances in high-resolution MS have allowed sensitivity and dynamic ranges to approach that of triple quadrupole instruments. A matrix-free protein digest, a digested therapeutic protein and the intact peptide therapeutic liraglutide were each analyzed on high-resolution and triple quadrupole mass spectrometers with data compared. Samples from a mouse PK study with liraglutide were analyzed using the two different instruments, and equivalent PK exposure data were demonstrated. High-resolution and triple quadrupole mass spectrometers can generate data resulting in identical PK parameters from an in-life sample set, thus giving confidence in either technique in support of biotherapeutic PK exposure studies.

The Impact of the Entry of Biosimilars: Evidence from Europe

Biologic drugs (therapeutic proteins or “large-molecule drugs”) represent large and growing share of all drug spending in the United States, accounting for less than 1% of prescriptions filled but nearly 28% of drug spending. Whereas traditional (chemically-synthesized, “small-molecule”) drugs have historically faced price competition from generic drugs after patent expiration, biosimilars – biologic drugs that have been shown to be therapeutically equivalent to an already approved original biologic drug – have only been approved in the United States since 2015. Europe has had biosimilar entry since 2006. This paper considers how competition from biosimilars may impact the U.S. biosimilar market by examining data from the first eight years of biosimilar competition in 23 European countries. A major contribution of this project is the completion of a detailed survey, allowing us to precisely characterize European biologic drug procurement institutions over time. Using data from three classes of biosimilar drugs, we analyze how market features and public policies predict entry, market prices, and penetration of biosimilars. We find significant heterogeneity across countries and drug classes in all of these outcomes. While we observe that effective buyer institutions (in particular, committed tenders) are associated with increased biosimilar entry and penetration, price patterns are more difficult to glean from the available data. Our estimates can inform ongoing policy discussions on both sides of the Atlantic about the economic implications of biosimilar policies.

Stability assessment in ligand-binding assays: a critical parameter for data integrity.

Stability assessment in ligand-binding assays: a critical parameter for data integrity.

A novel engineered VEGF blocker with an excellent pharmacokinetic profile and robust anti-tumor activity.

BackgroundRelatively poor penetration and retention in tumor tissue has been documented for large molecule drugs including therapeutic antibodies and recombinant immunoglobulin constant region (Fc)-fusion proteins due to their large size, positive charge, and strong target binding affinity. Therefore, when designing a large molecular drug candidate, smaller size, neutral charge, and optimal affinity should be considered.MethodsWe engineered a recombinant protein by molecular engineering the second domain of VEGFR1 and a few flanking residues fused with the Fc fragment of human IgG1, which we named HB-002.1. This recombinant protein was extensively characterized both in vitro and in vivo for its target-binding and target-blocking activities, pharmacokinetic profile, angiogenesis inhibition activity, and anti-tumor therapeutic efficacy.ResultsHB-002.1 has a molecular weight of ~80 kDa, isoelectric point of ~6.7, and an optimal target binding affinity of <1 nM. The pharmacokinetic profile was excellent with a half-life of 5 days, maximal concentration of 20.27 μg/ml, and area under the curve of 81.46 μg · days/ml. When tested in a transgenic zebrafish embryonic angiogenesis model, dramatic inhibition in angiogenesis was exhibited by a markedly reduced number of subintestinal vessels. When tested for anti-tumor efficacy, HB-002.1 was confirmed in two xenograft tumor models (A549 and Colo-205) to have a robust tumor killing activity, showing a percentage of inhibition over 90% at the dose of 20 mg/kg. Most promisingly, HB-002.1 showed a superior therapeutic efficacy compared to bevacizumab in the A549 xenograft model (tumor inhibition: 84.7% for HB-002.1 versus 67.6% for bevacizumab, P < 0.0001).ConclusionsHB-002.1 is a strong angiogenesis inhibitor that has the potential to be a novel promising drug for angiogenesis-related diseases such as tumor neoplasms and age-related macular degeneration.

Sandoz v. Amgen: Why the Biologics Price Competition and Innovation Act of 2009 Allows Biosimilar Makers to Pursue Pre-Application Declaratory Judgment Actions and Will Enhance Competition

The generic drug industry is shifting towards biosimilars. In the coming years, first-generation blockbuster biologic drugs will come off patent protection. However, unlike the small-molecule chemical drug market where exact generic copies can be produced, the complexity of large-molecule biologic drugs makes exact replicates impossible. As a result, biologic copies aim to be as similar as possible to their blockbuster counterparts – thus, the name: biosimilars. The Biologics Price Competition and Innovation Act of 2009 (BPCIA) regulates biosimilar approval.Submarine patents threaten the development of biosimilars. These types of patents are a product of manipulative continuation practices by the applicant, and can result in patent issuance more than a decade after the filing date. Submarine patents are meant to surprise an industry and preclude competition. While some believe submarine patents are a thing of the past, a pending case before the Federal Circuit shows they are not. In Sandoz v. Amgen, the Federal Circuit’s interpretation of the BPCIA will determine what litigation strategies are available to biosimilar makers to combat submarine patents. This is the first case to arise in the biosimilar industry. The issue is one of pure statutory interpretation: does the BPCIA bar a biosimilar maker from filing a declaratory judgment (DJ) action before submitting an application for approval.This article argues that the BPCIA does not preclude pre-application DJ actions. In addition, the article posits that pre-application DJ actions are an important tool in the fight against submarine patents and will enhance competition. The biosimilar industry is fast approaching and the legal community must take notice. This article seeks to further that conversation.

Insulin receptor antibody‐iduronate 2‐sulfatase fusion protein: Pharmacokinetics, anti‐drug antibody, and safety pharmacology in Rhesus monkeys

Mucopolysaccharidosis (MPS) Type II is caused by mutations in the gene encoding the lysosomal enzyme, iduronate 2-sulfatase (IDS). The majority of MPSII cases affect the brain. However, enzyme replacement therapy with recombinant IDS does not treat the brain, because IDS is a large molecule drug that does not cross the blood-brain barrier (BBB). To enable BBB penetration, IDS has been re-engineered as an IgG-IDS fusion protein, where the IgG domain is a monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor-mediated transport on the endogenous BBB insulin receptor, and the HIRMAb domain of the fusion protein acts as a molecular Trojan horse to ferry the fused IDS into brain from blood. The present study reports on the first safety pharmacology and pharmacokinetics study of the HIRMAb-IDS fusion protein. Juvenile male Rhesus monkeys were infused intravenously (IV) weekly for 26 weeks with 0, 3, 10, or 30 mg/kg of the HIRMAb-IDS fusion protein. The plasma clearance of the fusion protein followed a linear pharmacokinetics profile, which was equivalent either with measurements of the plasma concentration of immunoreactive HIRMAb-IDS fusion protein, or with assays of plasma IDS enzyme activity. Anti-drug antibody (ADA) titers were monitored monthly, and the ADA response was primarily directed against the variable region of the HIRMAb domain of the fusion protein. No infusion related reactions or clinical signs of immune response were observed during the course of the study. A battery of safety pharmacology, clinical chemistry, and tissue histopathology showed no signs of adverse events, and demonstrate the safety profile of chronic treatment of primates with 3-30 mg/kg weekly IV infusion doses of the HIRMAb-IDS fusion protein.