Granted Patent
B2
US 12,398,434 · App. 18/061,712 · Granted Aug 26, 2025
Compositions and methods for detecting a biological contaminant
Inventors:
Serge Monpoeho (East Greenbush, NY); Sheldon Mink (Rensselaer, NY); Paul Vescio (Malta, NY)
Assignee:
Regeneron Pharmaceuticals, Inc.
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Abstract
Provided are compositions and methods useful to the determination of whether a microbial contaminant is present in a biological therapeutic production process. Specifically, an artificial positive amplification control plasmid and unique quantitative PCR detection probe are provided, which enables the rapid and real-time detection of a false positive result.
Claims (18)
1. A positive amplification control (PAC) plasmid comprising:
a unique artificial plasmid-specific sequence (UAPS) comprising SEQ ID NO: 10, and
a target amplification polynucleotide (TAP) sequence, wherein said TAP comprises all or part of a parvovirus NS-1 sequence.
2. The PAC plasmid of claim 1 , wherein said UAPS does not recognize or anneal to any natural parvovirus.
3. The PAC plasmid of claim 1 , wherein the parvovirus NS-1 sequence is at least 88% identical to any one of SEQ ID NOs: 12-37 or 97% identical to SEQ ID NO: 9.
4. The PAC plasmid of claim 1 , wherein the parvovirus NS-1 sequence comprises SEQ ID NO: 9 or any one of SEQ ID NOs: 12-37.
5. The PAC plasmid of claim 1 , wherein the parvovirus NS-1 sequence comprises SEQ ID NO: 37.
6. The PAC plasmid of claim 1 , comprising a nucleic acid extraction control (NEC) nucleotide sequence, wherein said NEC comprises an M13 bacteriophage nucleotide sequence.
7. The PAC plasmid of claim 6 , wherein said M13 bacteriophage nucleotide sequence is a M13K07 nucleotide sequence.
8. The PAC plasmid of claim 7 , wherein said M13K07 nucleotide sequence comprises SEQ ID NO. 8.
9. The PAC plasmid of claim 1 , further comprising binding sequences for one or more oligonucleotide primers.
10. The PAC plasmid of claim 9 , wherein said one or more oligonucleotide primers are selected from the group consisting of a forward oligonucleotide primer, a reverse oligonucleotide primer, and a combination thereof.
11. The PAC plasmid of claim 9 , wherein said one or more oligonucleotide primers comprise rodent parvovirus oligonucleotide primers.
12. The PAC plasmid of claim 11 , wherein said rodent parvovirus oligonucleotide primers are selected from the group consisting of a forward oligonucleotide primer, a reverse oligonucleotide primer, and a combination thereof.
13. The PAC plasmid of claim 1 , further comprising binding sequences for one or more M13 oligonucleotide primers.
14. The PAC plasmid of claim 13 , wherein said one or more M13 oligonucleotide primers are selected from the group consisting of a forward oligonucleotide primer, a reverse oligonucleotide primer, and a combination thereof.
15. The PAC plasmid of claim 1 , wherein said NS-1 sequence is selected from the group consisting of minute virus of mice prototype strain (MVMp), minute virus of mouse immunosuppressive strain (MVMi), minute virus of mouse Cutter strain (MVMc); mouse parvovirus 1b (MPV-1b), mouse parvovirus 1a (MPV-1a), mouse parvovirus 1c (MPV-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a, rat minute virus, and Umass strain of Rat virus L (RV-Umass).
16. The PAC plasmid of claim 1 , comprising the sequence of SEQ ID NO: 11.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Dec 6, 2022
From: MONPOEHO, SERGE; MINK, SHELDON; VESCIO, PAUL
To: REGENERON PHARMACEUTICALS, INC.
Reel/Frame 061988/0915 →
Continuity (4)
Provisional Application
62139321
· Mar 27, 2015
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