Downstream processing of an alkaline phosphatase
Inventors
Jonk, Luigi Johannes Cornelius • Connor, Stephen Edward • Van Den Berg, Erik Jan • Van Elsas, Andrea • SHUKLA, Abhinav Alok • HORNE, Heather Bethea • Cook, Susan • Kelly, Timothy Martin • DOWLING, Victoria Anne • RAMAROSON, Mialy Fanjamalala
Assignees
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Publication Number
US-10570380-B2
Publication Date
2020-02-25
Expiration Date
Abstract
The invention relates to the field of downstream processing (DSP) of an alkaline phosphatase (AP). More specifically, it relates to a method for reducing host cell protein content in a composition comprising AP. The invention further relates to a composition comprising an AP and a reduced content of a host cell protein.
Core Innovation
The invention relates to isolating an alkaline phosphatase (AP) comprising an amino acid sequence 100% identical to SEQ ID NO: 1 from a composition that also contains host cell protein (HCP). A solid phase is provided that comprises a ligand of formula (I), where R is a spacer molecule linking the ligand to the solid phase, and the AP is contacted with the ligand so that AP is captured on the solid phase. After contacting, two or more wash steps are performed, followed by eluting the AP with an elution buffer.
At least one wash step uses a wash buffer comprising between 20–100 mM arginine (Arg), between 0.5–2 M urea, between 5–15% ethylene glycol, or any combination thereof. The eluate comprises less than 100 ppm HCP, and the described approach addresses impurity reduction while maintaining the isolation of the AP.
A related formulation and composition aspect is directed to producing a composition comprising an isolated AP with an amino acid sequence 100% identical to SEQ ID NO: 1, in which the composition has a pH between 6.5 and 7.5 and comprises between 200–300 mM sorbitol and/or between 10–40% glycerol and/or between 5–40 mM histidine and/or between 10–40 mM citrate, or any combination thereof. The composition is characterized by no visible particle formation during stability testing at 2–8°C for 2 months, and a composition is also claimed that comprises less than 100 ppm HCP and does not show visible particle formation during the same stability test, where the AP is obtained from a cell-based expression system.
Claims Coverage
The partial content includes three independent claims: clm-00001, clm-00009, and clm-00011. Across these, the coverage centers on ligand-based AP isolation with defined spacer-linked ligand (formula (I)) and wash buffer conditions to reach <100 ppm HCP in eluates, a formulation defined by pH, excipient ranges, and a stability/visible particle criterion, and a composition definition combining <100 ppm HCP with no visible particle formation after stability testing for AP obtained from a cell-based expression system.
Spacer-linked ligand of formula (I) for AP isolation
A method isolates an AP by providing a solid phase comprising a ligand of formula (I) in which R is a spacer molecule that links the ligand to the solid phase, contacting the ligand with an AP comprising an amino acid sequence 100% identical to SEQ ID NO: 1 and HCP, performing two or more wash steps, and eluting the AP.
Arg/urea/ethylene glycol intermediate washes to reach <100 ppm HCP
The contacting-and-washing method performs at least one wash step using a wash buffer comprising between 20–100 mM arginine (Arg), between 0.5–2 M urea, between 5–15% ethylene glycol, or any combination thereof, such that the resulting eluate comprises less than 100 ppm HCP.
Formulated isolated AP composition without visible particles after stability testing
A method produces a composition comprising an isolated AP with an amino acid sequence 100% identical to SEQ ID NO: 1 by dissolving or diluting the AP in a buffer to yield a composition having a pH between 6.5 and 7.5 and comprising between 200–300 mM sorbitol and/or between 10–40% glycerol and/or between 5–40 mM histidine and/or between 10–40 mM citrate, wherein there is no visible particle formation in the composition after stability testing at 2–8°C for 2 months.
Isolated AP composition with <100 ppm HCP and no visible particle formation
A composition comprises an isolated alkaline phosphatase obtained from a cell-based expression system, wherein the alkaline phosphatase comprises an amino acid sequence 100% identical to SEQ ID NO: 1, wherein the composition comprises less than 100 ppm HCP and wherein the composition does not show visible particle formation during stability testing at 2–8°C for 2 months.
Overall, the claim coverage ties spacer-linked ligand-based capture with specific arginine/urea/ethylene glycol wash steps to obtain eluates containing less than 100 ppm HCP, and defined pH and excipient composition ranges that result in no visible particle formation after stability testing, culminating in a composition that combines the <100 ppm HCP impurity limit with the visible-particle stability criterion.
Stated Advantages
The eluate comprises less than 100 ppm HCP.
The composition does not show visible particle formation during stability testing at 2–8°C for 2 months.
Documented Applications
No documented applications found
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