JP2015518150A - グリコサミノグリカンを検出する方法 - Google Patents
グリコサミノグリカンを検出する方法 Download PDFInfo
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- JP2015518150A JP2015518150A JP2015505915A JP2015505915A JP2015518150A JP 2015518150 A JP2015518150 A JP 2015518150A JP 2015505915 A JP2015505915 A JP 2015505915A JP 2015505915 A JP2015505915 A JP 2015505915A JP 2015518150 A JP2015518150 A JP 2015518150A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
Description
本出願は、2012年4月11日出願の米国仮出願第61/622,720号の35U.S.C.§119(e)に基づく利益を主張し、その内容が、その全体において参照により本明細書に組み込まれる。
いくつかの実施形態では、本明細書に記載される方法および構成は、タンパク質サンプル、例えば濃縮されたタンパク質サンプル、例えば治療用のタンパク質のサンプルにおける他の成分からグリコサミノグリカンを区別するために有用である。
いくつかの実施形態では、グリコサミノグリカンは、6.8またはそれ以下のpHを有する移動相とともにサイズ排除クロマトグラフィを用いることにより、サンプルにおける他の成分から分離される。サイズ排除クロマトグラフィは、サンプルにおける成分のサイズおよび分子量に基づく分離技術である。サイズ排除カラムは、一般的に、サンプル成分が進む「迷路」をもたらす、特定の孔径のシリカ粒子を含む。より小さな粒子がカラムのポアに嵌って遅れ、一方、ポアに嵌らないより大きな粒子は最初にカラムから溶出する。
本明細書において提供される方法の一態様は、検出装置が、サンプルにおける関心成分の存在を測定するために使用される。いくつかの実施形態では、関心成分は、グリコサミノグリカン(例えば、デキストラン硫酸)である。いくつかの実施形態では、関心成分は、タンパク(例えば、遺伝子組換え型産生タンパク)である。いくつかの実施形態では、検出装置は、サイズ排除カラムを通って流れた後の移動相をモニターする。
いくつかの実施形態では、本明細書に開示される方法は、サイズ排除クロマトグラフィカラムに移動相を適用する行為を含む。一般的に、サイズ排除クロマトグラフィカラム上に適用されるサンプルは、カラムに移動相を適用することによりカラムを通って流れるであろう。カラムに適用される移動相は、サンプルがカラムを通って流れることを可能にする、例えば、塩、バッファ濃度、pHのような特性を有するであろう。
本明細書に記載される方法および構成は、サンプルを分析的に評価するため、およびサンプルにおける1つまたはそれ以上のグリコサミノグリカンの存在および/または量を測定するために使用されることとしてもよいことが理解されるべきである。いくつかの実施形態では、分析は、サンプルにおけるグリコサミノグリカン(例えば、デキストラン硫酸)の量が、所定の閾値を上回るまたは下回るかどうかを測定するために較正されることとしてもよい。例えば、閾値は、グリコサミノグリカンの基準量を用いて設定されることとしてもよい(例えば、10μg/ml、5μg/ml、2μg/ml、1μg/ml、またはそれ以下)。閾値は、タンパク調製物が拒絶されおよび/またはさらに処理されるべき上記の量を表すこととしてもよい。
実施例1:タンパク質サンプルからのデキストランの分離
因子IX‐Fcの製造のために部分的に精製されたタンパク製造サンプルは、デキストラン硫酸(6〜8kDa)が添加され、1μg/mlから50μg/mlのデキストラン硫酸の範囲のサンプルをもたらす。
上記明細書は、当業者が本発明を実施することを可能にするために十分であると考えられる。実施例は本発明の一態様の1つの例示として意図され、他の機能的に均等な実施形態が本発明の範囲内のものとなるため、本発明は提供された実施例により範囲を限定されるものではない。本明細書に示されおよび記載されるものに加えて、本発明の種々の改変は、上記明細書から、当業者にとって明らかとなり、添付の請求の範囲の範囲内に属するであろう。本発明の利点および目的は、本発明の各実施形態により包含される必要はない。
Claims (25)
- サンプルにおける1つまたはそれ以上の他の成分からグリコサミノグリカンを区別する方法であって、
サンプルを、6.8またはそれ以下のpHを有する移動相を用いて、サイズ排除クロマトグラフィに供することを含む方法。 - 前記サイズ排除クロマトグラフィに供される前記移動相におけるグリコサミノグリカンの量を測定するための検出装置を用いることにより、前記サンプルにおける前記グリコサミノグリカンの量を測定することをさらに含む、請求項1の方法。
- サンプルがグリコサミノグリカンを含むかどうかを測定する方法であって、
サイズ排除カラムにサンプルを適用し、
前記サイズ排除カラムを通って前記サンプルが流れるように移動相を適用し、
前記移動相は、6.8またはそれ以下のpHを有し、
前記カラムを流れた前記移動相に、グリコサミノグリカンが存在するかどうかを測定することにより、前記サンプルがグリコサミノグリカンを含むかどうかを測定するための検出装置を用いることを含む、方法。 - 前記サンプルにおける前記グリコサミノグリカンの量が閾値レベルを超えるかどうかを測定することをさらに含む、請求項2または請求項3の方法。
- 前記閾値レベルが2μg/mlである、請求項4の方法。
- 前記サンプルがタンパクを含む、請求項1から5のうちいずれか1つの方法。
- 前記サンプルが未精製である、請求項1から6のうちいずれか1つの方法。
- 前記サンプルが部分的に精製されている、請求項1から6のうちいずれか1つの方法。
- 前記サンプルが1つまたはそれ以上の血液カスケードタンパクを含む、請求項1から8のうちいずれか1つの方法。
- 前記サンプルが因子VIIIFcまたは因子IXFcを含む、請求項9の方法。
- 前記グリコサミノグリカンは負に帯電している、請求項1から10のうちいずれか1つの方法。
- 前記グリコサミノグリカンはデキストラン硫酸である、請求項1から11のうちいずれか1つの方法。
- 前記デキストラン硫酸は6〜8kDaのサイズを有する、請求項12の方法。
- 前記グリコサミノグリカンはヘパリン硫酸である、請求項1から11のうちいずれか1つの方法。
- 前記検出装置がUV検出器である、請求項2から14のうちいずれか1つの方法。
- 前記サイズ排除クロマトグラフィの充填材料と移動相が二次相互作用を可能にする、請求項1から15のうちいずれか1つの方法。
- 前記サイズ排除クロマトグラフィの充填材料はシリカ系であり、100から200オングストロームの間の孔径と、5ミクロンまたはそれ以下の粒子径を有する、請求項16の方法。
- 前記サイズ排除クロマトグラフィはシリカ系サイズ排除カラム上で行われる、請求項1から17のうちいずれか1つの方法。
- 前記移動相の前記pHは6.0またはそれ以下である、請求項1から18のうちいずれか1つの方法。
- 前記移動相の前記pHは5.0またはそれ以下である、請求項1から18のうちいずれか1つの方法。
- 前記移動相の前記pHは4.0またはそれ以下である、請求項1から18のうちいずれか1つの方法。
- 前記移動相の前記pHは3.0またはそれ以下である、請求項1から18のうちいずれか1つの方法。
- 前記移動相の前記pHは2.5である、請求項1から18のうちいずれか1つの方法。
- 前記移動相は塩を含む、請求項1から23のうちいずれか1つの方法。
- 前記塩は200mMのNaClである、請求項24の方法。
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