Method for producing recombinant hyaluronidase
Inventors
Park, Soon Jae • KIM, Kyuwan • YUN, Sang Hoon • CHO, Jeong Soo • PARK, Kibum • Byun, Minsoo • SONG, HYUNG-NAM • KIM, Ji-Sun • NAM, Ki Seok
Assignees
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Publication Number
US-12221638-B2
Publication Date
2025-02-11
Expiration Date
Abstract
Disclosed is a method for producing hyaluronidase or a variant thereof. Specifically, the method is capable of changing the N-glycan levels under culture conditions including a controlled concentration of glucose in the culture medium and a decreased culture temperature for a specific culture time period, thereby increasing the specific activity by 10% or more and improving the quality and production yield.
Core Innovation
The invention relates to a method for producing a recombinant protein that comprises at least one of hyaluronidase PH20 or a variant thereof. The method uses two-stage culturing of host animal cells expressing the recombinant protein, with first culturing within a first temperature range from 35°C to 38°C until an integral viable cell density reaches 20×10^6 to 120×10^6 cells·day/mL.
Afterward, the method proceeds to second culturing at least part of the host animal cells for 2 to 18 days, while the culture medium is controlled to maintain residual glucose within 0.001 g/L to 4.5 g/L and/or pH within 6.8 to 7.2. The produced recombinant protein is characterized by N-glycan sialylation of 1% to 38% and hyaluronidase enzymatic activity at 10,000 units/mL or higher.
The disclosure links N-glycosylation, especially limiting N-glycan sialylation, together with culture glucose and temperature control to obtain PH20 or variants with defined N-glycan sialylation, galactosylation, and mannosylation ranges and increased hyaluronidase specific activity. The document also describes purification and evaluation aspects, including purification based on ion exchange and/or hydrophobic interaction and enzyme kinetics or assay evaluation.
Claims Coverage
The independent claim covers a two-stage host-cell culturing method for producing recombinant hyaluronidase PH20 or a variant, where second culturing controls residual glucose and/or pH, and the resulting protein is characterized by N-glycan sialylation and high hyaluronidase enzymatic activity. The claim set includes additional dependent limitations that further narrow glycosylation profiles, refine culture-medium conditions, and specify non-affinity purification modes.
Two-stage host-cell culturing with defined temperature ranges
A method for producing a recombinant protein comprising at least one of hyaluronidase PH20 or a variant thereof, comprising first culturing host animal cells expressing the recombinant protein within a first temperature range from 35°C to 38°C until an integral viable cell density of 20×10^6 to 120×10^6 cells·day/mL is reached, and subsequently second culturing at least part of the host animal cells for 2 to 18 days.
Second culturing control of residual glucose and/or pH
The second culturing maintains a residual glucose concentration within 0.001 g/L to 4.5 g/L and/or pH within 6.8 to 7.2 in a culture medium.
Produced protein characterized by N-glycan sialylation range
The produced recombinant protein has N-glycan sialylation of 1% to 38%.
Produced protein with hyaluronidase enzymatic activity at or above threshold
The produced recombinant protein has a hyaluronidase enzymatic activity at 10,000 units/mL or higher.
Refined N-glycan galactosylation and mannosylation constraints
The method is characterized by N-glycan galactosylation of 1% to 68% and N-glycan mannosylation of 40% to 63%.
N-glycan sialylation narrowed upper range
The method is characterized by sialylation of N-glycan of 1% to 30%.
Culture-medium conditions including ammonia threshold and/or specified additions/omissions
At least one of the first and/or second culturing steps uses culture-medium conditions including maintaining ammonia at 5 mM or higher, adding one or more specified substances, and/or omitting galactose and manNAc from the culture medium.
Increased enzymatic activity relative to wild human PH20
The method includes using a recombinant protein whose hyaluronidase enzymatic activity is at least 10% higher than the enzymatic activity of a wild human PH20.
Purification by ionic bond and/or hydrophobic interaction rather than affinity binding
The produced recombinant protein is separated and purified using ionic bond and/or hydrophobic interaction properties rather than affinity binding.
Across the claim family, the core inventive coverage is the two-stage culturing approach with defined temperature ranges, combined with second-culturing control of residual glucose and/or pH, producing PH20 or variants characterized by specified N-glycan sialylation and hyaluronidase activity at 10,000 units/mL or higher. Dependent claims further narrow N-glycan galactosylation and mannosylation ranges, refine culture-medium conditions, optionally require increased activity relative to wild human PH20, and specify purification using ionic bond and/or hydrophobic interaction rather than affinity binding.
Stated Advantages
Produces the recombinant protein with hyaluronidase enzymatic activity at 10,000 units/mL or higher.
Maintains specific N-glycan sialylation of 1% to 38% and, in dependent forms, 1% to 30%.
Produces recombinant protein having defined N-glycan galactosylation and mannosylation ranges.
Enables increased enzymatic activity relative to a wild human PH20 by at least 10%.
Uses purification by ionic bond and/or hydrophobic interaction rather than affinity binding.
Documented Applications
Not explicitly described in patent.
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