Stable formulations of polypeptides and uses thereof

a polypeptide and stable technology, applied in the field of single variable domain formulations, can solve the problems of short shelf life of liquid antibody preparations, physical instability, loss of biological activity of antibodies, etc., and achieve the effects of increasing the melting temperature and/or increasing the solubility and increasing the stability of single variable domains

Inactive Publication Date: 2012-09-27
ABLYNX NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The present invention provides improved formulations (also referred to as “formulation(s) of the invention”) of polypeptides comprising one or more single variable domains that show good solubility of the single variable domains and retain increased stability of the single variable domains under a variety of different transportation, storage and in-use conditions. The present invention is based on the finding that the presence in the formulation of certain buffers, certain excipients and / or certain surfactants may increase the solubility, the melting temperature and / or the stability of the single variable domains present in the formulation.
[0009]The present invention provides solubility data for formulations with polypeptides comprising one or more single variable domains (also referred to as “polypeptide(s) of the invention”) up to 150 mg / mL and higher. The invention further shows that such formulations can be transported, manipulated through various administration devices and retain activity, purity and potency under different stress conditions: mechanical stress conditions; storage of the formulation at various stress conditions such as different freeze / thaw cycles, at 2-8° C., at 25±5° C. and at elevated temperature.

Problems solved by technology

Certain prior liquid antibody preparations have shown short shelf lives and loss of biological activity of the antibodies resulting from chemical and / or physical instabilities during the transportation and storage.
Chemical instability may be caused by deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange, and physical instability may be caused by antibody denaturation, aggregation, precipitation or adsorption.

Method used

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  • Stable formulations of polypeptides and uses thereof
  • Stable formulations of polypeptides and uses thereof
  • Stable formulations of polypeptides and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Formulation and Stability Studies with RANKL008a

example 1.1

Materials and Methods Used in the Study

[0676]1.1.1 Single variable domains

[0677]RANKL008a (SEQ ID NO: 4; EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVS SITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS GGGGSGGGSEVOLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKG RFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLR LSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC AAYIRPDTYLSRDYRKYDYWGQGTLVTVSS) has been described as SEQ ID NO: 759 in WO 2008 / 142164. RANKL008a is a trivalent bispecific Nanobody consisting of three humanized variable domains of a heavy-chain llama antibody, of which two identical subunits are specific for binding to RANKL while the remaining subunit binds to HSA. The subunits are fused head-to-tail with a G / S linker in the following format: RANKL13H5-9GS-Alb8-9GS-RANKL13H5.

[0678]RANKL008a was expressed in Pichia pastoris and purified on SP Sepharose as a capturing step a...

example 1.2

Stability of the Nanobody in Different Buffers after Different Freeze / Thaw Cycles

[0697]A freeze / thaw stability study was performed to determine the effect of repetitive freeze and thawing on the recovery, physical stability and chemical stability of RANKL008a. Aliquots of batch RANKL008a formulated at ˜60-85 mg / mL in the buffers 1-12 given in Table 1 were subjected to 10 freeze / thaw (F / T) cycles at −20° C. One F / T cycle is defined by freezing the sample for 1 hour in a freezer at −20° C. followed by thawing at room temperature for 30 minutes. The stressed samples were compared with reference material (stored at 4° C.) using SE-HPLC (FIG. 1 (A); representative figure of the experiments performed in phosphate buffer), RP-HPLC (FIG. 2; representative figure of the experiments performed in phosphate buffer) and the ELISA potency assays (Table 2). All other data of the freeze thaw experiments demonstrate similar patterns as given in FIGS. 1 and 2 (except FIG. 1 (B) see below).

[0698]Subje...

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Abstract

Formulations are provided that contain single variable domains with a good solubility and good stability under different storage, transportation and stress conditions. The formulations are useful as pharmaceutical formulation. The formulation comprises an aqueous carrier with a pH of 5.5 to 8.0, a buffer selected from the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0; an excipient; and / or a surfactant selected from Tween 80, Tween 20 and poloxamers. The formulation is further characterized that it has an inorganic salt concentration of 150 mM or lower. The invention further relates to containers and pharmaceutical units comprising such formuSations and to methods for preparing and prophylactic and therapeutic uses of the formulations and pharmaceutical units of the invention.

Description

FIELD OF THE INVENTION[0001]The present invention relates to formulations of single variable domains. More specifically the present invention provides formulations that contain single variable domains with a good solubility and good stability under different storage and stress conditions. The formulations of the invention are suitable for administration to human subjects.[0002]The invention further relates to containers and pharmaceutical units comprising such formulations and to prophylactic and therapeutic uses of the formulations and pharmaceutical units of the invention.[0003]Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.BACKGROUND ART[0004]Nanobodies (as further described herein) are characterized by formation of the antigen binding site by a single variable domain, which does not require interaction with a further domain (e.g. in the form of VH / VL interaction) for antigen recognition. Nanobodies h...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395
CPCA61K39/39591A61K9/0019A61K47/26A61K47/14A61K47/02A61P19/08A61P19/10
Inventor BRIGE, ANNDE BRABANDERE, VERONIQUE
Owner ABLYNX NV
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