Research Articles
Immunogenicity of aggregates of recombinant human growth hormone in mouse models

https://doi.org/10.1002/jps.21834Get rights and content

Abstract

Aggregation of recombinant therapeutic protein products is a concern due to their potential to induce immune responses. We examined the immunogenicity of protein aggregates in commercial formulations of recombinant human growth hormone produced by freeze-thawing or agitation, two stresses commonly encountered during manufacturing, shipping and handling of therapeutic protein products. In addition, we subjected each preparation to high-pressure treatment to reduce the size and concentration of aggregates present in the samples. Aggregates existing in a commercial formulation, as well as aggregates induced by freeze-thawing and agitation stresses enhanced immunogenicity in one or more mouse models. The use of high-pressure treatment to reduce size and concentrations of aggregates within recombinant human growth hormone formulations reduced their overall immunogenicity in agreement with the “immunon” hypothesis. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3247–3264, 2009

Section snippets

INTRODUCTION

Therapeutic proteins are susceptible to aggregation in response to a wide variety of stresses encountered during their manufacture, storage and delivery to patients.1 In turn, aggregates of therapeutic proteins may compromise their safety and efficacy.2, 3, 4, 5 The primary safety concern is that aggregates in therapeutic protein products may induce immune responses,6,7 which can have consequences ranging from reduction of product efficacy to patient fatality.8 In extreme cases, parenterally

Materials

The two commercial formulations of rhGH Nordiflex® (Novo Nordisk®, Bagsvaerd, Denmark) and Saizen® (Serono, Rockland, MA), were purchased from the University of Colorado apothecary, and are hereafter referred to as Product A and Product B, respectively. Sterile water for injection (SWFI) (Hospira, Inc., Lake Forest, IL) and 0.9% sodium chloride for injection (Hospira, Inc.) were also purchased form the University of Colorado apothecary. Histidine and mannitol were purchased from JT Baker

Stressing of rhGH Samples

The responses of the Product A and Product B formulations to the various stresses (agitation, freeze-thawing) were different. The agitated Product A samples were cloudy by the end of the 72 h of agitation whereas the agitated Product B samples were still clear. Similarly, the FT Product A samples began to become cloudy around the 12–15th freeze–thaw cycle; however, the FT Product B never showed signs of cloudiness even after the 20th freeze–thaw cycle. The contrast in aggregates produced in the

DISCUSSION

Parenteral administration of aggregates of a therapeutic protein can induce immune responses to the monomeric protein. However, little is known about the characteristics of aggregates that are capable of inducing immunogenicity and the mechanism by which they provoke the response.5 B cells can be stimulated to produce antibodies in T-cell independent mechanism that requires an antigen with a repetitive structure.39 Dintzis et al.40 determined that in order for polymeric antigens to activate

CONCLUSIONS

This study has shown that the immunogenicity of protein aggregates may depend on their size and the manner and solution conditions by which they are produced. Aggregates found in existing commercial formulations were immunogenic in the naïve adult and neonatally primed mice, as were aggregates generated by freeze-thawing- or agitation-induced stresses applied to the formulations, two types of stresses that are routinely encountered during production, handling, and storage of protein

Acknowledgements

Financial support for this study was provided by BaroFold, Inc., a company in which TWR and JFC hold financial interest.

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