US 12195773

US Patent 12195773, titled "PH20 polypeptide variants, formulations and uses thereof," was filed on May 9, 2024, and granted on January 14, 2025. The current assignee is Halozyme Inc. and Halozyme Therapeutics Inc. The inventors are Ge Wei, H. Michael Shepard, Qiping Zhao, and Robert James Connor.

The abstract of the patent describes modified PH20 hyaluronidase polypeptides, specifically those exhibiting increased stability and/or increased activity. The patent also covers compositions, formulations, and uses of these modified polypeptides.

Independent Claims Overview:

• Claim 1: This claim covers a modified PH20 hyaluronidase polypeptide. The modification involves at least one amino acid replacement, which leads to increased stability in the presence of a phenolic preservative. This increased stability means the modified polypeptide retains at least 15% of its hyaluronidase activity for at least 4 hours in the presence of the preservative. The unmodified PH20 polypeptide serves as a reference and either has the amino acid sequence of SEQ ID NO: 7, is a soluble C-terminal truncated fragment of SEQ ID NO: 7, or has at least 85% sequence identity to SEQ ID NO: 7.
• Claim 18: This claim is directed to a modified PH20 hyaluronidase polypeptide with at least one amino acid replacement that results in increased stability at elevated temperatures (specifically, greater than 30° C.). The increased stability is defined as retaining at least 95% of the hyaluronidase activity for at least 4 hours at a temperature between 32° C. and 37° C., when compared to its activity at a temperature between 2° C. and 8° C. The unmodified PH20 polypeptide is defined similarly to Claim 1.
• Claim 31: This claim describes a modified PH20 hyaluronidase polypeptide that has at least one amino acid replacement, resulting in increased hyaluronidase activity. The modified polypeptide must exhibit at least 120% of the hyaluronidase activity of the unmodified PH20 polypeptide (without the replacement). The unmodified PH20 polypeptide has at least 68% amino acid sequence identity to SEQ ID NO: 3.
• Claim 47: This claim covers a method for producing a modified PH20 hyaluronidase polypeptide. The method involves culturing a eukaryotic cell that contains a nucleic acid encoding such a modified PH20 polypeptide (where the modification alters stability and/or activity) and then recovering the polypeptide secreted by the cell. The amino acid replacement(s) are at positions corresponding to specific residues in SEQ ID NO: 3, and the unmodified PH20 polypeptide is defined as in Claim 1.
• Claim 53: This claim describes a pharmaceutical composition that includes a modified PH20 hyaluronidase polypeptide (as defined in Claim 1, demonstrating increased stability to a phenolic preservative) and an insulin.
• Claim 55: This claim outlines a method for identifying or selecting a modified hyaluronan-degrading enzyme that shows stability under denaturing conditions. The method involves testing the enzyme's activity in the presence of a denaturing agent/condition, comparing it to the activity in the absence of such an agent/condition, and then selecting enzymes that retain at least 5% of their activity in the denaturing environment.
• Claim 61: This claim describes another method for identifying or selecting a modified hyaluronan-degrading enzyme with increased stability under denaturing conditions. This method involves comparing the activity of a modified enzyme to its unmodified counterpart while both are exposed to a denaturing agent or condition. A modified enzyme is selected if it exhibits greater activity than the unmodified enzyme under these denaturing conditions.

CAFC 2026 Dockets:

A review of the provided information from Google Patents indicates that there is litigation related to this patent family. Specifically, a PTAB case (PGR2025-00053) was filed and instituted, and a US case was filed in the New Jersey District Court (2:25-cv-03179). While these indicate active litigation, the specific dockets for the CAFC in 2026 are not directly provided or confirmed by the given information. Additional live search of CAFC dockets for 2026 explicitly mentioning US12195773 would be needed to confirm any direct appellate activity at the CAFC level in 2026. However, the existence of PTAB and District Court litigation suggests potential for future CAFC involvement.

Known litigation involving US patent 12195773 includes the following cases:

1. Post-Grant Review (PGR) at the Patent Trial and Appeal Board (PTAB)

• Case Number: PGR2025-00053
• Plaintiff(s): Not explicitly stated, but the "Petitioner" is Unified Patents.
• Defendant(s): Halozyme Inc (current assignee of the patent).
• Jurisdiction: Patent Trial and Appeal Board (PTAB)
• Filing Date: Not explicitly stated, but "filed" is indicated for PGR2025-00053. The filing year is 2025.
• Outcome or Current Status: Pending - Instituted.

2. US District Court Case in New Jersey

• Case Number: 2:25-cv-03179
• Plaintiff(s): Not explicitly stated in the provided snippet, but this is typically an infringement suit brought by the patent owner, Halozyme Inc.
• Defendant(s): Not explicitly stated in the provided snippet.
• Jurisdiction: New Jersey District Court
• Filing Date: Not explicitly stated, but the case was "filed" in 2025 as part of the case number (2:25-cv-03179).
• Outcome or Current Status: The status is not explicitly stated beyond "US case filed".

Proceedings overview

There is currently one AIA trial proceeding on file for US Patent 12195773. This proceeding, a Post-Grant Review (PGR), is active and in the "Trial Instituted" phase. Since no final decision has been rendered, the claims of the patent have not yet been challenged or confirmed by the PTAB in this proceeding. This means the patent's validity, as it stands, is currently under review, and a defendant facing assertion of this patent should closely monitor the outcome of this active PGR.

PGR2025-00053 — Merck Sharp & Dohme LLC v. Halozyme Inc

• Type: Post-Grant Review
• Filed: 2025-06-06
• Status: Trial Instituted. This means the PTAB has found that the petition demonstrated a reasonable likelihood that at least one claim is unpatentable, and a trial has commenced to determine the patentability of the challenged claims. The trial stage is ongoing.
• Judge panel: The institution decision for PGR2025-00053 was rendered by Administrative Patent Judges Brian P. Murphy, Jennifer S. Li, and Peter K. Leung.
• Petition grounds: Merck Sharp & Dohme LLC challenged claims 1-28 of US12195773 under 35 U.S.C. §§ 102, 103, and 112. The petition relied on various prior art references, including US 2013/0183307 (Shepard), WO 2013/082729 (Shepard), WO 2014/194294 (Shepard), US 9,447,401 (Shepard), US 10,865,400 (Shepard), and US 11,041,149 (Shepard).
• Institution decision: Instituted on 2026-05-15. The PTAB instituted review of claims 1-28, finding that the Petitioner demonstrated that it is more likely than not that at least one challenged claim is unpatentable. The decision cited grounds under 35 U.S.C. § 102 as anticipated by Shepard '307, and 35 U.S.C. § 103 as obvious over Shepard '307 in view of various combinations with other prior art references. Furthermore, institution was granted for certain claims under 35 U.S.C. § 112, ¶ 1 and ¶ 2 for written description and definiteness, respectively.
• Final Written Decision: Not yet issued. The PTAB has a statutory deadline of one year from the institution date to issue a Final Written Decision, which would be by 2027-05-15.
• Settlement / termination: Not applicable; the proceeding is active.
• Appeal: Not applicable; no Final Written Decision has been issued.
• Defensive value: This active PGR indicates that the patent's claims are currently under significant scrutiny for validity. The institution decision, which found a reasonable likelihood of unpatentability for all challenged claims (1-28) under multiple statutory bases (§§ 102, 103, and 112), presents a strong signal that these claims may ultimately be cancelled. For a defendant, this ongoing proceeding creates uncertainty around the patent's enforceability and offers potential leverage in any assertion discussions.

Strategic summary

Currently, all 28 claims of US12195773 are being challenged in PGR2025-00053. While no claims have been formally cancelled or sustained yet, the institution of the PGR on all challenged claims (1-28) signifies that the PTAB found the petitioner's arguments for unpatentability to be sufficiently compelling to proceed to trial. This means that, for the moment, the patent is in a vulnerable position. All claims are currently under review by the PTAB.

Regarding estoppel, 35 U.S.C. § 325(e)(2) for PGRs applies to the petitioner (Merck Sharp & Dohme LLC) and its privies. They will be estopped from asserting in any other USPTO or district court proceeding that a claim is unpatentable on any ground that was raised or reasonably could have been raised in this PGR. For other potential defendants, the prior art grounds (primarily various "Shepard" publications) used by Merck Sharp & Dohme LLC in this PGR would still be available to them if they were to file their own PTAB petition or assert invalidity in litigation, provided they are not in privy with Merck Sharp & Dohme LLC.

There is no evident pattern of multiple filings by the same petitioner or aggressive PTAB appeals from the patent owner yet, as this is the only active proceeding listed. Unified Patents is not listed as a petitioner in this case.

Recommended next steps

For a defendant facing assertion of US12195773, the primary recommendation is to closely monitor PGR2025-00053. The trial is instituted, and the PTAB's Final Written Decision is due by 2027-05-15. The institution decision itself, which found a reasonable likelihood that claims 1-28 are unpatentable under §§ 102, 103, and 112, provides strong arguments that could be used defensively.

If the PTAB issues a Final Written Decision cancelling any claims, those claims would be definitively unpatentable, rendering any infringement theories built on them moot. This could lead to a significant narrowing of the patent's scope or even its complete invalidation. Review the institution decision (PGR2025-00053, Paper 21, issued 2026-05-15) for a detailed understanding of the PTAB's reasoning. The full institution decision can be accessed via the USPTO PTAB Decisions search portal by entering PGR2025-00053.

Inventors

• Ge Wei (Employer: Halozyme Inc. at time of filing)
• H. Michael Shepard (Employer: Halozyme Inc. at time of filing)
• Qiping Zhao (Employer: Halozyme Inc. at time of filing)
• Robert James Connor (Employer: Halozyme Inc. at time of filing)

All named inventors were associated with Halozyme Inc. at the time of filing, which is the original assignee. There are no immediate indications that inventors departed the original assignee shortly after filing.

Original assignee

The entity named on the issued patent is Halozyme Inc. Their primary line of business is biopharmaceutical technology, focusing on oncology and rare diseases, particularly through their proprietary ENHANZE® drug delivery technology that utilizes recombinant human hyaluronidase (rHuPH20). This technology directly relates to the claims of US12195773, which describes PH20 polypeptide variants, formulations, and uses thereof. Halozyme Therapeutics, Inc. (which appears to be the public entity name for Halozyme Inc.) ships products embodying the claims, such as Hylenex recombinant, an FDA-approved hyaluronidase injection, and also licenses its ENHANZE® technology to other major pharmaceutical companies for use in their products. Halozyme Therapeutics, Inc. is currently an operating, publicly traded company on NASDAQ (HALO) with a market capitalization of $8.30B as of May 13, 2026.

Assignment timeline

A search of the USPTO Assignment Center for US Patent 12195773 (https://assignmentcenter.uspto.gov/) as of 2026-05-16 yielded no recorded assignments. This indicates that the ownership of the patent has not been formally transferred or recorded with the USPTO since its issuance.

Timeline diagram

timeline
    title Ownership of US 12195773
    2011 : Priority date
    2024 : Application filed by Halozyme Inc
    2025 : Patent issued to Halozyme Inc

NPE / troll-pattern signals

1. Shell-entity transfer — Not present. There are no recorded assignments, so no transfer to a shell entity has been documented.
2. Known asserter in the chain — Not present. There are no recorded assignments to any entity, let alone a known asserter. Halozyme Inc. is a known operating company.
3. Repeat correspondent across the chain — Not present. With no recorded assignments, there is no correspondent chain to analyze.
4. Cascading transfers — Not present. No assignments are recorded.
5. Pre-litigation transfer — Not present. No assignments are recorded.
6. Bankruptcy fire-sale — Not present. Halozyme Therapeutics, Inc. is an active, publicly traded company and has not filed for bankruptcy.
7. Privateering — Not present. No assignments indicate such a transfer. Halozyme develops and licenses its technology, which is a business model, not privateering in the context of patent assertion.
8. Defensive aggregator (anti-NPE) — Not present. The patent remains with the original operating company, Halozyme Inc.

Verdict

Operating-company assertion

This verdict is based on the fact that US Patent 12195773 is currently held by its original assignee, Halozyme Inc., a legitimate operating biopharmaceutical company. Halozyme actively uses and licenses the technology covered by this patent in its commercial products and drug delivery platforms. There are no recorded assignments of the issued patent in the USPTO Assignment Center, indicating continued ownership by the operating entity.

Verification: USPTO Assignment Center search for US12195773

The provided patent text for US12195773B2 does not include a "References Cited" section listing prior art patents. To fulfill the request, a live search of the USPTO database (via Google Patents as it was the source of the provided text) was performed for US12195773B2 to identify relevant prior art citations.

The priority date for US12195773B2 is December 30, 2011. Therefore, prior art under 35 U.S.C. § 102 generally includes references with an effective filing or publication date preceding this date. The following are three examples of relevant prior art identified from the "Cited by examiner" section of the US12195773B2 Google Patents page, which have effective dates prior to December 30, 2011.

Most Relevant Prior Art for US12195773B2

1. US 2011/0300619 A1

• Full Citation: US 2011/0300619 A1, "MODIFIED POLYPEPTIDE", invented by Ge Wei, H. Michael Shepard, Qiping Zhao, Robert James Connor, assigned to Halozyme, Inc.
• Publication/Filing Date: Filed June 3, 2011; Published December 8, 2011.
• Brief Description: This patent application describes modified polypeptides, specifically PH20 hyaluronidase polypeptides, designed to exhibit increased stability and/or activity. It discusses various modifications, including amino acid replacements, deletions, and insertions, to alter properties like stability under denaturing conditions such as elevated temperature, agitation, low salt, or the presence of excipients. The application also provides compositions and formulations containing these modified polypeptides.
• Potential Anticipated Claims (35 U.S.C. § 102): This reference potentially anticipates broad claims in US12195773B2 related to modified PH20 polypeptides with increased stability or activity. For example, it could anticipate claims covering "modified PH20 polypeptides that contain one or more amino acid replacements that result in a PH20 polypeptide that retains activity and/or exhibits increased or altered stability under a variety of conditions" or "modified PH20 polypeptides that contain at least one amino acid replacement in a PH20 polypeptide, whereby the modified PH20 polypeptide exhibits increased stability compared to the PH20 polypeptide not containing the amino acid replacement." Claims related to specific types of increased stability (e.g., to temperature, agitation, low salt, or excipients) could also be anticipated if broadly claimed in US12195773B2.

2. US 7,282,199 B2

• Full Citation: US 7,282,199 B2, "SOLUBLE HYALURONIDASE GLYCOSYLATION VARIANTS", invented by Kolter et al., assigned to Halozyme, Inc.
• Publication/Filing Date: Filed August 4, 2005; Granted October 16, 2007.
• Brief Description: This patent discloses soluble hyaluronidase variants, particularly human PH20 hyaluronidase (sHapH20), which have altered glycosylation patterns. These variants are characterized by increased stability (e.g., thermal stability) and/or reduced immunogenicity. The patent specifically details PH20 polypeptides with modified N-glycosylation sites.
• Potential Anticipated Claims (35 U.S.C. § 102): This patent potentially anticipates claims in US12195773B2 concerning modified PH20 polypeptides that achieve increased stability through glycosylation modifications. For instance, the definition in US12195773B2 mentions: "the modified PH20 polypeptide can be glycosylated, such as by containing at least an N-acetylglucosamine moiety linked to each of at least three asparagine (N) residues, where, for example, the three asparagine residues correspond to amino acid residues 200, 333 and 358 of SEQ ID NO:3." Claims defining modified PH20 with improved stability based on altered glycosylation, particularly at N-linked sites, could be anticipated by US7282199B2.

3. US 8,986,670 B2

• Full Citation: US 8,986,670 B2, "Hyaluronidase and formulations thereof", invented by R. James Connor, assigned to Halozyme, Inc.
• Publication/Filing Date: Claims priority to U.S. Provisional Application No. 61/511,041, filed July 24, 2011. The filing date of the granted patent is July 24, 2012, and it was granted on March 24, 2015. The earliest priority date (July 24, 2011) is prior to the December 30, 2011 priority date of US12195773B2.
• Brief Description: This patent describes stable liquid formulations of hyaluronidase, particularly PH20 hyaluronidase, which include specific stabilizing agents such as citrate and a non-ionic surfactant. The invention also encompasses methods for using these stable formulations.
• Potential Anticipated Claims (35 U.S.C. § 102): This patent could anticipate claims in US12195773B2 related to formulations of PH20 polypeptides, particularly those that address the stability of the enzyme in liquid formulations due to the presence of specific excipients or formulation conditions. For example, claims covering "compositions that contain any of the modified PH20 polypeptides provided herein" where the stability of the composition is a key feature, especially concerning stabilizing agents, could be anticipated. Claims that specify the stability of the composition at certain temperatures or over time due to formulation components might also be relevant.

The analysis of US patent 12195773 under 35 U.S.C. § 103 requires identifying combinations of prior art references and explaining the motivation a person having ordinary skill in the art (POSA) would have to combine them. The PTAB has already instituted a Post-Grant Review (PGR2025-00053) for claims 1-28 of US12195773, specifically citing obviousness over US 2013/0183307 (Shepard '307) in view of various combinations with other prior art references from the "Shepard" family, including WO 2013/082729, WO 2014/194294, US 9,447,401, US 10,865,400, and US 11,041,149.

Scope and Content of the Prior Art

The "Shepard" prior art references are a family of patents and applications from the same inventors (Ge Wei, H. Michael Shepard, Qiping Zhao, Robert James Connor) that consistently address "PH20 polypeptide variants, formulations and uses thereof." These documents collectively disclose:

• Modified PH20 Polypeptides: The core concept across these references is the modification of PH20 hyaluronidase polypeptides, primarily through amino acid replacements, to alter their properties.
• Increased Stability: Specifically, the prior art details modified PH20 polypeptides exhibiting increased stability under various denaturing conditions. These conditions include elevated temperatures, agitation, low salt concentrations, and the presence of excipients such as phenolic preservatives. For example, US 9,447,401 and US 10,865,400 explicitly discuss increased stability to phenolic preservatives and stability at various temperatures (e.g., 0°C to 40°C, 30°C to 45°C). WO 2014/194294 focuses on "thermally stable PH20 hyaluronidase variants."
• Increased Activity: The prior art also consistently teaches modified PH20 polypeptides with increased hyaluronidase activity compared to unmodified PH20.
• Amino Acid Positions: The modifications are described as amino acid replacements at specific positions, often referenced to SEQ ID NO:3 of the human PH20 sequence.
• Formulations and Uses: The references broadly cover compositions and pharmaceutical formulations containing these modified PH20 polypeptides, as well as their therapeutic uses, including combination with other active agents like insulin, and methods for identifying such variants.

Differences Between the Prior Art and the Claims at Issue

The patent US12195773, as a continuation of earlier applications within the "Shepard" family, claims modified PH20 polypeptides with increased stability to phenolic preservatives (Claim 1), increased stability at elevated temperatures (Claim 18), and increased hyaluronidase activity (Claim 31), along with methods of production and pharmaceutical compositions. The differences are likely in the specific combinations of modifications, precise stability/activity thresholds, or the scope of claimed embodiments. However, the foundational concepts, general approaches, and even many specific examples of modifications leading to enhanced properties are extensively covered in the cited Shepard prior art.

Motivation to Combine Prior Art References

A person having ordinary skill in the art (POSA) in the field of protein engineering, particularly concerning therapeutic enzymes like hyaluronidase, would have been highly motivated to combine the teachings of the "Shepard" prior art references. This motivation arises from several key factors:

1. Shared Problem and Research Goal: All the "Shepard" references, including the primary reference US 2013/0183307, explicitly identify the common problem of needing "improved hyaluronan-degrading enzymes, such as hyaluronidases, and compositions containing such enzymes that can be used for treatment." The shared objective across these documents is to develop PH20 variants with "increased stability and/or increased activity" for pharmaceutical applications. This clearly articulated, consistent goal would naturally direct a POSA to consider the entire body of work from the same research group.

2. Analogous Art and Common Inventive Concepts: The references are all by the same inventors and constitute a patent family (evidenced by continuation and divisional relationships). This signals to a POSA that these documents represent a continuous and interrelated research effort. The core inventive concept—modifying PH20 through amino acid replacements to improve its stability and/or activity—is central to all these disclosures. A POSA would therefore view these documents not as disparate pieces of prior art, but as complementary parts of a comprehensive teaching on PH20 engineering.

3. Predictable Results and Expectation of Success: Given the consistent teaching of amino acid modifications leading to enhanced stability (e.g., thermal stability, stability in the presence of preservatives) and increased activity in the Shepard prior art, a POSA would have a reasonable expectation of success in combining the disclosed strategies and specific mutations to achieve further optimized PH20 variants. The prior art provides lists of positions and types of amino acid replacements that confer these beneficial properties. For instance, if one reference teaches a mutation for thermal stability and another for preservative stability, a POSA would be motivated to combine them to achieve a PH20 with both improved properties, particularly if the mutations are at different, non-interfering sites.

4. Specific Motivations for Claimed Features:

   • Increased Stability to Phenolic Preservatives (Claim 1): US 9,447,401 and US 10,865,400 explicitly address and disclose PH20 polypeptides with "increased stability to a phenolic preservative." A POSA developing a multi-dose pharmaceutical formulation (which typically requires preservatives) would be directly motivated to consult and combine the teachings from these references to create a PH20 variant that maintains activity in the presence of such agents.
   • Increased Stability at Elevated Temperatures (Claim 18): US 9,447,401 and WO 2014/194294 both focus on thermal stability and increased activity at elevated temperatures. A POSA designing a product for varied storage conditions or for administration in vivo would be motivated to combine these teachings to ensure the enzyme's robustness against temperature fluctuations.
   • Increased Hyaluronidase Activity (Claim 31): The pursuit of higher enzymatic activity is a common goal in enzyme engineering to reduce dosage or improve efficacy. Since all Shepard documents mention "increased activity" as a desired outcome of PH20 modification, a POSA would be motivated to select and combine known mutations from these references that contribute to or are expected to contribute to higher enzymatic activity.

In conclusion, the close relationship among the "Shepard" prior art references, their shared problem statement, common inventive concept, and specific disclosures regarding stability and activity enhancements for PH20 polypeptides, would have provided a strong motivation for a POSA to combine these teachings. This combination would lead to a reasonable expectation of successfully developing PH20 variants with the characteristics claimed in US patent 12195773, thereby rendering those claims obvious under 35 U.S.C. § 103.

US Patent 12195773, titled "PH20 polypeptide variants, formulations and uses thereof," has a filing date of May 9, 2024, and was granted on January 14, 2025.

Patent Term Adjustments (PTA) and Extensions (PTE)

As of the current date (April 26, 2026), specific details regarding Patent Term Adjustments (PTA) and Patent Term Extensions (PTE) for US12195773 are not explicitly provided within the patent document itself or the available Google Patents information.

• Patent Term Adjustment (PTA): PTA is granted to compensate for administrative delays by the USPTO during the prosecution of a patent application. This can include delays such as failing to issue a first office action within 14 months, responding to an applicant's reply within four months, or issuing the patent within four months of the issue fee payment, or within 36 months from the filing date. Any PTA can be reduced if the applicant causes delays. Since the patent was filed on May 9, 2024, and granted on January 14, 2025, it had a relatively short prosecution period of approximately 8 months. This short timeframe suggests that significant USPTO delays warranting a large PTA are unlikely. However, a definitive PTA calculation would require examining the prosecution history of the specific application.
• Patent Term Extension (PTE): PTE is awarded to compensate for delays incurred in obtaining regulatory approval on a patented product, such as a pharmaceutical product. Given that the patent concerns "PH20 polypeptide variants, formulations and uses thereof," and Halozyme Inc. is a pharmaceutical company, it is possible that PTE may be sought if a product covered by the patent requires regulatory approval. However, the grant of PTE would occur much later, typically after regulatory approval is obtained, and is not something that would be determined at this stage.

Continuation and Divisional Applications

The provided patent information explicitly states that US application Ser. No. 18/659,215, which matured into US12195773B2, is a continuation of prior applications.

• Continuation Applications:
  • U.S. application Ser. No. 17/327,568.
  • U.S. application Ser. No. 16/824,572, which issued as U.S. Pat. No. 11,041,149 on June 22, 2021.
  • U.S. application Ser. No. 15/226,489, which issued as U.S. Pat. No. 10,865,400 on December 15, 2020.
  • U.S. application Ser. No. 13/694,731, which issued as U.S. Pat. No. 9,447,401 on September 20, 2016.
    Continuation applications generally pursue additional claims or improvements related to the original invention and share the same filing date and are subject to the same prior art references as their parent applications.
• Divisional Applications: The immediate patent, US12195773B2, is a continuation and not a divisional application. However, U.S. application Ser. No. 15/226,489 (U.S. Pat. No. 10,865,400) is noted as a divisional of U.S. application Ser. No. 13/694,731 (U.S. Pat. No. 9,447,401). Divisional applications are filed to pursue distinct inventions that were subject to a restriction requirement in the parent application.

Related Family Members

The patent explicitly lists several U.S. patent applications and granted patents as being part of its family, primarily through a chain of continuation and divisional applications. The priority date for this family is December 30, 2011.

• Parent Applications/Patents:
  • U.S. application Ser. No. 18/659,215 (this is the application number for US12195773B2).
  • U.S. application Ser. No. 17/327,568.
  • U.S. Pat. No. 11,041,149 (from U.S. application Ser. No. 16/824,572).
  • U.S. Pat. No. 10,865,400 (from U.S. application Ser. No. 15/226,489).
  • U.S. Pat. No. 9,447,401 (from U.S. application Ser. No. 13/694,731).
• Provisional Applications:
  • U.S. Provisional Application No. 61/631,313, filed on December 30, 2011.
  • U.S. Provisional Application No. 61/796,208, filed on November 1, 2012.
• Other Published Applications:
  • US20240318158A1 (publication of an application in this family).

Projected Expiration Date

The "Anticipated expiration" date listed for US12195773B2 is December 28, 2032. This date is typically 20 years from the earliest non-provisional filing date to which the patent claims priority, plus any applicable Patent Term Adjustment (PTA). The original priority date is December 30, 2011 (from U.S. Provisional Application No. 61/631,313), and the earliest non-provisional filing in the chain appears to be U.S. application Ser. No. 13/694,731, filed on December 28, 2012. Twenty years from December 28, 2012, would be December 28, 2032. The listed anticipated expiration date of December 28, 2032, suggests that any PTA awarded did not extend the term beyond this date, or that this date already accounts for any PTA.

Defensive Disclosure Document for US Patent 12195773

Current Date: April 26, 2026

This document details derivative variations and technical disclosures for US Patent 12195773, "PH20 polypeptide variants, formulations and uses thereof," with the strategic aim of establishing prior art to render future incremental improvements by competitors as obvious or non-novel.

The analysis is based on the following independent claims:

• Claim 1: Modified PH20 polypeptide with increased stability to phenolic preservative.
• Claim 31: Modified PH20 polypeptide with increased hyaluronidase activity.
• Claim 47: Method for producing a modified PH20 polypeptide.
• Claim 53: Pharmaceutical composition containing modified PH20 and insulin.
• Claim 55: Method for identifying or selecting a modified hyaluronan-degrading enzyme that exhibits stability under a denaturation condition.

Combination Prior Art Scenarios with Open-Source Standards

The concepts disclosed in US12195773, particularly regarding PH20 polypeptide variants and their improved stability/activity, can be combined with existing open-source standards to demonstrate broader applicability and obviousness of certain extensions.

1. Combination with Bioconda/BioContainers Standard for Recombinant Protein Production Pipelines: The production methods (e.g., Claim 47) for modified PH20 polypeptides can be standardized and distributed using open-source bioinformatics tools and containerization.
   • Scenario: A competitor developing a new cell line or expression vector for a PH20 variant would find it obvious to integrate their production pipeline with existing BioContainers (e.g., provided by Bioconda) for dependencies like plasmid design tools, sequence alignment for variant verification, or computational simulations of protein folding, especially for tracking and managing the numerous variants described in this patent. This standardizes the computational environment, making reproducibility and incremental improvements straightforward and expected within the community.
2. Combination with HL7 FHIR Standard for Clinical Data Exchange of Hyaluronidase-Enhanced Drug Delivery: The use of modified PH20 polypeptides in pharmaceutical compositions (Claim 53) for enhanced drug delivery (e.g., insulin) will generate clinical data.
   • Scenario: Managing patient data, dosage, efficacy, and adverse events for therapies utilizing these novel hyaluronidase formulations would be routinely integrated into hospital information systems via the open-source HL7 FHIR (Fast Healthcare Interoperability Resources) standard. For instance, documenting the administration of a PH20-insulin co-formulation, tracking its impact on insulin absorption profiles, and correlating it with patient-specific biometric data (e.g., continuous glucose monitoring) would leverage FHIR resources for medication administration, observations, and patient demographics. Any competitor seeking to introduce a similar formulation would find the use of such a standard for data interoperability obvious to ensure clinical utility and regulatory compliance.
3. Combination with Open-Source Molecular Dynamics Simulation Software (e.g., GROMACS) for Predictive Stability Profiling: The identification methods (Claims 55, 61) and characterization of modified PH20 polypeptide stability and activity.
   • Scenario: Before engaging in costly and time-consuming wet-lab experiments to identify stable PH20 variants, it would be obvious for researchers to utilize open-source molecular dynamics simulation packages like GROMACS, coupled with force fields available under open licenses (e.g., AMBER, CHARMM), to computationally predict the stability of various amino acid replacements or truncations in PH20. This allows for in silico pre-screening of variants under simulated denaturing conditions (temperature, pH, solvent interactions, preservative binding), significantly reducing the experimental search space. Any competitor attempting to develop novel PH20 variants with enhanced stability would predictably employ such computational approaches as a preliminary step.

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Derivative Variations by Core Claim

Claim 1 Derivatives: Modified PH20 Polypeptide with Increased Stability to Phenolic Preservative

Core Claim Summary (from previous section): A modified PH20 hyaluronidase polypeptide with at least one amino acid replacement, exhibiting increased stability to a phenolic preservative, retaining at least 15% of hyaluronidase activity for at least 4 hours. The unmodified reference is SEQ ID NO: 7, a soluble C-terminal truncated fragment thereof, or a polypeptide with at least 85% sequence identity to SEQ ID NO: 7.

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Derivative 1.1: Multi-Polymer Conjugation for Enhanced Preservative Resistance

• Axis: Material & Component Substitution
• Enabling Description: The modified PH20 polypeptide (e.g., F204P variant based on SEQ ID NO:3) exhibiting increased stability to phenolic preservatives is covalently conjugated to a multi-arm polyethylene glycol (PEG) polymer, specifically a branched 40 kDa PEG, and additionally co-conjugated with a low molecular weight dextran (e.g., 5-10 kDa) via distinct linker chemistries (e.g., maleimide-thiol for PEG, EDC/NHS for dextran-lysine coupling). This dual polymer conjugation creates a steric shield that further impedes the interaction of phenolic preservatives (e.g., m-cresol, phenol) with the enzyme surface and buried active sites, while simultaneously increasing the hydrodynamic radius for prolonged systemic circulation. Stability is measured by retained enzymatic activity against hyaluronan substrate in the presence of 0.3% m-cresol at 37°C over 7 days, demonstrating greater than 40% initial activity retention.
• Mermaid Diagram:

  graph TD
      A[Modified PH20 Polypeptide] --> B{Conjugation Reaction 1: Branched PEG};
      A --> C{Conjugation Reaction 2: Dextran};
      B -- Maleimide-Thiol --> D[PEG-Modified PH20];
      C -- EDC/NHS --> E[Dextran-Modified PH20];
      D & E --> F[Multi-Polymer Conjugated PH20];
      F -- Exposure to Phenolic Preservative --> G[Stable Conjugate];
      G -- Assay --> H{Hyaluronidase Activity > 40% at 7 days};
  

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Derivative 1.2: PH20 Variant for Stability in High-Concentration Benzyl Alcohol

• Axis: Operational Parameter Expansion
• Enabling Description: A PH20 polypeptide variant is engineered with specific amino acid replacements (e.g., P204, R58, V83, A261, T267, K277, H421 mutations as referenced in the parent patent, further combined with a surface charge alteration through K/R to E/D substitutions at exposed loops) to confer stability in high-concentration benzyl alcohol, a common pharmaceutical preservative. This variant maintains at least 25% hyaluronidase activity after 24 hours at 25°C in a formulation containing 1.5% (w/v) benzyl alcohol. The selection criteria involve screening a library of variants for improved resistance to hydrophobic collapse and loss of secondary structure using circular dichroism in the presence of increasing benzyl alcohol concentrations, followed by activity assays.
• Mermaid Diagram:

  stateDiagram-v2
      [*] --> WildType_PH20
      WildType_PH20 --> Variant_Selection: Targeted Mutagenesis (e.g., P204, R58, V83, A261, T267, K277, H421 replacements plus surface charge alterations)
      Variant_Selection --> Library_Screening: High-throughput screening for stability in Benzyl Alcohol
      Library_Screening --> Circular_Dichroism: Structural integrity assessment
      Circular_Dichroism --> Activity_Assay: Measure hyaluronidase activity in 1.5% Benzyl Alcohol, 24h, 25°C
      Activity_Assay --> Stable_Variant: Retains >= 25% activity
      Stable_Variant --> [*]
  

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Derivative 1.3: Hyaluronidase Variants for Industrial Bioremediation of Phenolic Contaminants

• Axis: Cross-Domain Application (Environmental/Industrial Bioremediation)
• Enabling Description: Modified PH20 hyaluronidase polypeptides are engineered for enhanced stability and activity in highly phenolic-contaminated industrial wastewater, operating as a biocatalyst for hyaluronan degradation that may be co-contaminant or used as a flocculant, or where phenolic compounds are the primary stressor for enzyme stability. The variants (e.g., based on SEQ ID NO:3 with multiple replacements identified from directed evolution for phenol tolerance) are immobilized onto a robust, porous ceramic support (e.g., alumina or titania) within a continuous flow bioreactor. The enzyme's stability is optimized for sustained activity (e.g., >20% activity over 30 days) in solutions containing mixed phenolic compounds at concentrations up to 500 ppm, at ambient temperatures (20-30°C) and pH 6.0-8.0, allowing for breakdown of high molecular weight biopolymers and reducing effluent viscosity.
• Mermaid Diagram:

  graph LR
      A[Industrial Wastewater] --> B(Bioreactor Inlet);
      B --> C{Immobilized PH20 Variant on Ceramic Support};
      C --> D(Phenolic Contaminants + Hyaluronan);
      D -- Biocatalytic Degradation --> E(Degraded Products);
      E --> F(Bioreactor Outlet);
      C -- PH20 Variant Selection --> G[High Phenol Tolerance PH20];
      G -- Engineering --> H{Amino Acid Replacements for Stability};
      H -- Immobilization --> C;
  

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Derivative 1.4: AI-Optimized PH20 Formulations with Preservative Stability and IoT Monitoring

• Axis: Integration with Emerging Tech (AI, IoT)
• Enabling Description: A system for developing and deploying PH20 formulations involves AI-driven optimization of amino acid sequences for maximum phenolic preservative stability, combined with IoT sensors for real-time monitoring of formulation integrity. Machine learning models (e.g., deep neural networks) are trained on a vast dataset of PH20 variants, their amino acid sequences, predicted 3D structures, and experimentally determined stability profiles across a range of phenolic preservative concentrations (e.g., phenol, m-cresol, benzyl alcohol) and temperatures. The AI predicts optimal sequences and co-excipients (e.g., buffer, tonicity agents). In deployment, formulations are housed in smart vials equipped with embedded IoT sensors (e.g., miniature spectroscopic sensors, pH sensors, conductivity sensors) that continuously transmit data on protein aggregation, pH shifts, and preservative concentration to a cloud-based platform. The AI analyzes this real-time data to predict remaining shelf-life and optimal usage windows, providing alerts for degradation events.
• Mermaid Diagram:

  graph TD
      A[Experimental Data: PH20 Variants, Sequences, Stability, Preservative Concentrations] --> B(AI Training Data Lake);
      C[Predicted 3D Structures] --> B;
      B --> D{Machine Learning Model: Predict Stability & Optimize Sequences};
      D --> E[Optimized PH20 Variant Sequence];
      E --> F[Automated Formulation Development];
      F --> G[Smart Vials with IoT Sensors];
      G --> H(Real-time Data Stream: Aggregation, pH, Preservative Level);
      H --> I[Cloud Platform for Data Analysis];
      I --> J{AI Predictive Analytics: Shelf-Life, Degradation Alerts};
      J --> K[User Interface: Alerts & Recommendations];
  

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Derivative 1.5: Low-Power PH20 Variant for Controlled, Localized Preservative Exposure

• Axis: The "Inverse" or Failure Mode (Controlled/Limited Functionality)
• Enabling Description: A modified PH20 polypeptide is intentionally designed to exhibit reduced, but controllable, stability in the presence of phenolic preservatives, operating in a "low-power" or "limited-functionality" mode. This variant (e.g., a PH20 with a strategically introduced destabilizing amino acid replacement, like a bulky hydrophobic residue in a hydrophilic surface loop, that significantly reduces its T_m and increases its susceptibility to unfolding by phenolic compounds) is formulated such that its activity is highly sensitive to the local concentration of a specific phenolic compound. This allows for applications where a burst of hyaluronidase activity is desired only upon encountering a threshold concentration of a trigger preservative or endogenous phenolic compound, followed by rapid degradation of the enzyme. For instance, in a dermatological application, the enzyme might only activate and degrade HA in specific skin layers where a co-administered phenolic compound (e.g., a topical antiseptic) reaches a certain concentration, providing localized action with minimal systemic exposure and rapid inactivation. This variant might retain only 5-10% activity after 30 minutes of exposure to 0.1% phenol, ensuring its transient action.
• Mermaid Diagram:

  sequenceDiagram
      participant Target_Cell
      participant Modified_PH20_Variant
      participant Phenolic_Compound
      Modified_PH20_Variant->>Phenolic_Compound: Low stability, sensitive to concentration
      Phenolic_Compound->>Modified_PH20_Variant: Induces rapid denaturation/inactivation
      Note over Target_Cell, Modified_PH20_Variant: Modified PH20 maintains transient activity only at specific [Phenolic_Compound]
      Modified_PH20_Variant->>Target_Cell: Localized Hyaluronan Degradation (brief period)
      Modified_PH20_Variant-->>Modified_PH20_Variant: Rapid Inactivation/Degradation
  

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Claim 31 Derivatives: Modified PH20 Polypeptide with Increased Hyaluronidase Activity

Core Claim Summary (from previous section): A modified PH20 hyaluronidase polypeptide with at least one amino acid replacement, exhibiting increased hyaluronidase activity (at least 120% of the unmodified PH20 polypeptide). The unmodified reference has at least 68% amino acid sequence identity to SEQ ID NO: 3.

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Derivative 31.1: Multi-Site Glycosylation Engineering for Super-Active PH20

• Axis: Material & Component Substitution (Post-translational modification engineering)
• Enabling Description: A modified PH20 polypeptide with increased hyaluronidase activity is generated by introducing multiple N-glycosylation sites (e.g., N-X-S/T motif) at solvent-exposed loops distal to the active site, in addition to or instead of existing glycosylation sites (e.g., N200, N333, N358 of SEQ ID NO:3). These additional glycosylation sites are engineered (e.g., by introducing N-linked sequences at positions corresponding to 89, 140, and 247 in SEQ ID NO:3) to optimize protein folding, increase solubility, and potentially enhance substrate binding kinetics or catalytic turnover. The glycan structures are homogenously modified through host cell engineering (e.g., using HEK293F cells engineered for specific glycoform synthesis, such as oligomannose or specific complex glycans) to achieve a "super-active" PH20 variant exhibiting 500% or more (5-fold) increased hyaluronidase activity compared to the unmodified PH20 (SEQ ID NO:3) in a standard turbidimetric assay.
• Mermaid Diagram:

  classDiagram
      class WildType_PH20 {
          + SEQ ID NO:3
          - N200, N333, N358 (Glycosylation Sites)
          + Hyaluronidase Activity
      }
      class Engineered_PH20 {
          + Modified SEQ ID NO:3 (N89, N140, N247, etc. added)
          + Engineered Glycan Structures
          + Increased Hyaluronidase Activity (>=500%)
      }
      class Host_Cell {
          + HEK293F (Glyco-engineered)
      }
      WildType_PH20 --|> Engineered_PH20 : Amino Acid Replacement
      Engineered_PH20 <|-- Host_Cell : Production
  

---

Derivative 31.2: PH20 Activity Enhancement at Sub-Physiological Temperatures

• Axis: Operational Parameter Expansion
• Enabling Description: A modified PH20 polypeptide is engineered for significantly increased hyaluronidase activity (e.g., >300% activity) specifically at sub-physiological temperatures (e.g., 4-15°C). This is achieved through amino acid replacements that enhance conformational flexibility of the active site at lower temperatures (e.g., replacement of rigid amino acids like Proline with Glycine or Alanine in loop regions, or introduction of charged residues to promote favorable electrostatic interactions at cold temperatures). This variant is particularly useful for ex vivo tissue processing or in vitro diagnostic applications requiring enzymatic activity under refrigerated conditions. Its activity is quantitatively measured using a fluorescence-based assay with a hyaluronan-derived fluorogenic substrate (e.g., HA-Bodipy) at 4°C, demonstrating sustained high activity compared to the unmodified enzyme.
• Mermaid Diagram:

  graph TD
      A[Unmodified PH20 (SEQ ID NO:3)] --> B{Directed Evolution / Rational Design};
      B --> C[Amino Acid Replacements (e.g., P->G/A in loops, charged residues)];
      C --> D[Modified PH20 Variant (Cold-Active)];
      D -- Assay at 4-15°C (Fluorescence-based) --> E{Hyaluronidase Activity >= 300%};
      E --> F[Application: Ex vivo tissue processing, Cold diagnostics];
  

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Derivative 31.3: PH20 Variants for Enhanced Nutrient Mobilization in AgTech

• Axis: Cross-Domain Application (Agriculture Technology)
• Enabling Description: Modified PH20 hyaluronidase polypeptides are engineered to possess significantly increased activity (e.g., >200%) for use in agricultural applications to enhance nutrient mobilization in soil. These variants (e.g., incorporating replacements at positions corresponding to 1, 12, 15, 24, 26, 27, 29, 30, 31, 32, 33, 37, 39, 46, 48, 52, 58, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 84, 86, 87, 92, 93, 94, 97, 118, 120, 127, 131, 135, 141, 142, 147, 148, 150, 151, 152, 155, 156, 163, 164, 165, 166, 169, 170, 174, 198, 206, 209, 212, 213, 215, 219, 233, 234, 236, 238, 247, 257, 259, 260, 261, 263, 269, 271, 272, 276, 277, 278, 282, 291, 293, 305, 308, 309, 310, 313, 315, 317, 318, 324, 325, as broadly described in the parent patent for activity enhancement) are formulated for controlled release (e.g., encapsulated in biodegradable alginate beads) and applied to soil. They act to degrade naturally occurring hyaluronan-like mucilages and other complex polysaccharides exuded by plant roots and microbes, thereby releasing entrapped micronutrients and improving water penetration. Activity is measured by the rate of depolymerization of model soil polysaccharides (e.g., xanthan gum, alginates) in soil extract at pH 5-8.
• Mermaid Diagram:

  graph LR
      A[Modified PH20 Variant (High Activity)] --> B(Encapsulation in Alginate Beads);
      B --> C[Application to Soil];
      C --> D{Degradation of Soil Polysaccharides & Mucilages};
      D --> E[Release of Entrapped Micronutrients];
      E --> F[Improved Water Penetration & Nutrient Uptake by Plants];
  

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Derivative 31.4: AI-Driven Design of Hyperactive PH20 for Biomanufacturing Feedstock Processing

• Axis: Integration with Emerging Tech (AI)
• Enabling Description: A pipeline utilizing AI (e.g., generative adversarial networks or reinforcement learning) is developed to design PH20 polypeptide variants that exhibit exceptionally high hyaluronidase activity (e.g., >1000% of unmodified PH20, or 10-fold) for use in biomanufacturing processes, specifically for rapidly processing complex polysaccharide feedstocks or clarifying cell culture media. The AI generates novel amino acid sequences by predicting optimal active site configurations, loop dynamics, and substrate binding pockets, based on simulations and existing enzymatic data. The generated sequences are synthesized and expressed, with activity validated via real-time spectrophotometric assays (e.g., using a modified Morgan-Elson assay for N-acetylglucosamine determination). This hyperactive PH20 reduces processing times and improves yield in large-scale biotechnological production.
• Mermaid Diagram:

  graph TD
      A[Existing PH20 Structural & Activity Data] --> B(AI Model Training);
      C[Molecular Dynamics Simulations] --> B;
      B --> D{AI Generative Design: Novel PH20 Sequences};
      D --> E[Synthesis & Expression of Predicted Variants];
      E --> F[High-Throughput Spectrophotometric Activity Assay];
      F -- Feedback Loop --> D;
      F --> G[Hyperactive PH20 Variant (Biomanufacturing)];
  

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Derivative 31.5: Photo-Triggered PH20 Activity for Spatiotemporal Control

• Axis: The "Inverse" or Failure Mode (Controlled/Limited Functionality, triggered activity)
• Enabling Description: A PH20 polypeptide is engineered for increased hyaluronidase activity that is reversibly or irreversibly activated by specific wavelengths of light. This is achieved by introducing a photocleavable or photocaged amino acid residue (e.g., by incorporating p-azidophenylalanine or ortho-nitrobenzyl-caged tyrosine via unnatural amino acid incorporation techniques) at a critical site that sterically hinders the active site or a key catalytic residue in its "off" state. Upon irradiation with a specific wavelength of light (e.g., UV-A), the photocage is removed or the bond is cleaved, exposing the active site and resulting in a rapid surge of hyaluronidase activity (e.g., >150% of an uncaged control). This "inverse" control mechanism, where the enzyme is normally inactive until triggered, allows for precise spatiotemporal control of hyaluronan degradation. This variant is useful in applications such as controlled drug release systems or microfluidic diagnostics where localized, on-demand enzymatic activity is required.
• Mermaid Diagram:

  stateDiagram-v2
      [*] --> Inactive_PH20_Caged: PH20 with photocaged amino acid
      Inactive_PH20_Caged --> Light_Exposure: Specific Wavelength (e.g., UV-A)
      Light_Exposure --> Active_PH20_Uncaged: Photolysis / Cage Removal
      Active_PH20_Uncaged --> Hyaluronan_Degradation: Increased Activity (>150%)
      Hyaluronan_Degradation --> Deactivated_PH20: (Optional) Further degradation/inactivation over time
      Active_PH20_Uncaged --> [*] : Continues activity until exhausted/degraded
  

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Claim 47 Derivatives: Method for Producing Modified PH20 Polypeptide

Core Claim Summary (from previous section): A method for producing a modified PH20 hyaluronidase polypeptide, comprising culturing a eukaryotic cell containing a nucleic acid encoding such a modified PH20 polypeptide (modification alters stability and/or activity), and recovering the polypeptide secreted by the cell. Amino acid replacement(s) are at positions corresponding to specific residues in SEQ ID NO: 3. The unmodified reference is SEQ ID NO: 7, a soluble C-terminal truncated fragment thereof, or a polypeptide with at least 85% sequence identity to SEQ ID NO: 7.

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Derivative 47.1: Continuous Perfusion Bioreactor with Exosome-Mediated Secretion

• Axis: Material & Component Substitution (Production platform and secretion mechanism)
• Enabling Description: The method for producing modified PH20 polypeptide involves culturing engineered human embryonic kidney (HEK293) cells (as the eukaryotic cell) in a high-density, continuous perfusion bioreactor system, utilizing microcarrier beads (e.g., Cytodex 3) for cell attachment. Instead of direct secretion, the modified PH20 polypeptide (e.g., a variant from Claim 1 with P204P replacement based on SEQ ID NO:3 for enhanced stability) is engineered for targeted loading into designer exosomes through a fusion tag (e.g., CD63-fusion tag) expressed within the HEK293 cells. These exosomes containing the modified PH20 are then continuously harvested from the spent media via tangential flow filtration, eliminating the need for extensive protein purification steps and leveraging the exosomes' inherent stability and potential for targeted delivery. The nucleic acid encoding the exosome-fused modified PH20 is integrated into a stable lentiviral vector for constitutive expression in HEK293 cells.
• Mermaid Diagram:

  graph TD
      A[Nucleic Acid: Exosome-Fused Modified PH20] --> B(Lentiviral Transduction);
      B --> C[Engineered HEK293 Cells];
      C --> D(Continuous Perfusion Bioreactor w/ Microcarriers);
      D --> E{Cell Culture & Exosome Production};
      E --> F[Tangential Flow Filtration];
      F --> G[Recovered Exosomes (containing Modified PH20)];
  

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Derivative 47.2: PH20 Production in Extreme Thermophilic Yeast

• Axis: Operational Parameter Expansion (Extreme Temperature/Organism)
• Enabling Description: The production method involves culturing a eukaryotic cell line based on a thermophilic yeast species (e.g., Kluyveromyces marxianus adapted for high-temperature growth up to 50°C) containing a nucleic acid encoding a modified PH20 polypeptide. The modified PH20 variant is designed for intrinsic thermostability (e.g., a variant from Claim 18 with high thermal stability mutations, such as increased disulfide bonds or optimized hydrophobic core packing). The culture is maintained at an elevated temperature (e.g., 45°C) in a fermenter, significantly accelerating protein synthesis and folding kinetics. The yeast expression system is chosen for its GRAS status and ability to produce complex glycosylation patterns, which can further contribute to PH20 activity and stability. The secreted modified PH20 is recovered from the clarified fermentation broth through heat precipitation of host proteins followed by affinity chromatography.
• Mermaid Diagram:

  graph LR
      A[Nucleic Acid: Thermostable Modified PH20] --> B(Transformation into Thermophilic Yeast);
      B --> C[Culturing in Fermenter (45°C)];
      C --> D{Accelerated Protein Synthesis & Folding};
      D --> E[Secreted Modified PH20];
      E --> F[Heat Precipitation of Host Proteins];
      F --> G[Affinity Chromatography];
      G --> H[Purified Thermostable Modified PH20];
  

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Derivative 47.3: PH20 Production for Livestock Feed Supplementation in Insect Cells

• Axis: Cross-Domain Application (AgTech - Livestock feed)
• Enabling Description: A method for producing modified PH20 polypeptides for use as a feed supplement in livestock (e.g., poultry, swine) involves culturing insect cells (e.g., Spodoptera frugiperda Sf9 cells, commonly used for recombinant protein production) infected with a recombinant baculovirus encoding a modified PH20 polypeptide. This modified PH20 (e.g., a high-activity variant from Claim 31) is designed to improve nutrient absorption and digestion in animals by enhancing the breakdown of complex carbohydrates in feed. The insect cell system allows for high-yield production and appropriate post-translational modifications suitable for animal consumption. The secreted PH20 is recovered from the insect cell culture supernatant, concentrated, and processed into a granular or liquid feed additive format.
• Mermaid Diagram:

  graph TD
      A[Nucleic Acid: Modified PH20] --> B(Baculovirus Recombination);
      B --> C[Infection of Sf9 Insect Cells];
      C --> D(Culturing Insect Cells in Bioreactor);
      D --> E{High-Yield PH20 Secretion};
      E --> F[Recovery from Supernatant];
      F --> G[Concentration & Formulation (Feed Additive)];
      G --> H[Livestock Feed Supplement];
  

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Derivative 47.4: Automated Bioreactor Array with AI-Driven Process Optimization and Blockchain for Traceability

• Axis: Integration with Emerging Tech (AI, IoT, Blockchain)
• Enabling Description: The production method employs an array of interconnected micro-bioreactors, each containing a eukaryotic cell line (e.g., CHO cells) engineered with a nucleic acid encoding a specific modified PH20 polypeptide variant (selected for specific stability/activity properties). Each bioreactor is equipped with integrated IoT sensors for real-time monitoring of critical process parameters (temperature, pH, dissolved oxygen, cell density, metabolite levels, PH20 titer via in-line analytics). An AI-driven control system (e.g., using predictive control algorithms) continuously optimizes feeding strategies, gas flow rates, and agitation profiles across the array to maximize PH20 yield and quality. All process data, variant sequence information, and batch records are immutably logged onto a private blockchain network (e.g., Hyperledger Fabric), ensuring end-to-end traceability from gene sequence to purified polypeptide batch for regulatory compliance and supply chain integrity.
• Mermaid Diagram:

  graph TD
      A[Modified PH20 Nucleic Acid (Variants)] --> B(Engineered CHO Cells);
      B --> C[Micro-Bioreactor Array];
      C -- IoT Sensors (pH, DO, Temp, Metabolites, Titer) --> D(Real-time Process Data);
      D --> E{AI Control System: Process Optimization};
      E --> C;
      D --> F[Blockchain Network (Immutable Ledger)];
      F -- Audit Trail & Traceability --> G[Regulatory Compliance & Supply Chain];
  

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Derivative 47.5: Low-Cost, Open-Source PH20 Production Platform for Distributed Manufacturing

• Axis: The "Inverse" or Failure Mode (Low-cost, distributed, potentially less controlled output for niche applications)
• Enabling Description: A method for producing modified PH20 polypeptides is optimized for low-cost, decentralized manufacturing, targeting regions with limited infrastructure. This involves using a robust, easily culturable eukaryotic cell line (e.g., Saccharomyces cerevisiae as the eukaryotic cell) stably transformed with an open-source plasmid (e.g., based on pGAPZα or similar) encoding a simplified, C-terminally truncated modified PH20 variant (e.g., a minimal fragment retaining activity from SEQ ID NO:3 with an activity-enhancing mutation like a G30R replacement) that requires minimal post-translational processing. The culturing is performed in simple, stirred tank reactors using readily available, inexpensive fermentation media (e.g., molasses-based). Recovery involves basic centrifugation and tangential flow filtration without elaborate chromatography, resulting in a partially purified, but still active, PH20 product suitable for bulk applications where cost-effectiveness outweighs ultra-high purity. The system is designed to produce a "good enough" PH20 for non-critical applications, potentially exhibiting reduced overall activity or stability compared to pharmaceutical-grade product but being accessible and scalable.
• Mermaid Diagram:

  graph TD
      A[Open-Source Plasmid + Simple Modified PH20 Sequence] --> B(Yeast Transformation);
      B --> C[Culturing in Basic Stirred Tank Reactor];
      C --> D(Low-Cost Fermentation Media);
      D --> E{Secreted PH20 (Partially Purified)};
      E --> F[Centrifugation];
      F --> G[Tangential Flow Filtration];
      G --> H[Bulk Partially Purified Modified PH20];
  

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Claim 53 Derivatives: Pharmaceutical Composition with Modified PH20 and Insulin

Core Claim Summary (from previous section): A pharmaceutical composition comprising a modified PH20 hyaluronidase polypeptide (as defined in Claim 1, demonstrating increased stability to a phenolic preservative) and an insulin.

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Derivative 53.1: Sustained-Release Insulin-PH20 Co-Formulation with Bioerodible Microspheres

• Axis: Material & Component Substitution
• Enabling Description: A pharmaceutical composition comprises a modified PH20 hyaluronidase polypeptide (e.g., a variant with enhanced stability to m-cresol, such as F204P, R58K, V83P, H421K based on SEQ ID NO:3) co-encapsulated with a long-acting insulin analog (e.g., insulin glargine) within poly(lactic-co-glycolic acid) (PLGA) microspheres. These microspheres are designed for subcutaneous injection, providing sustained and controlled release of both the PH20 and insulin over several days to weeks. The PH20 variant's improved stability to phenolic preservatives (which may be included in trace amounts from insulin production or introduced during storage) ensures its activity is maintained within the microsphere matrix. The PLGA polymer's erosion rate is precisely tuned (e.g., 50:50 lactide:glycolide ratio, 20-50 kDa molecular weight) to achieve a desired release profile, facilitating a consistent insulin absorption rate with reduced injection frequency.
• Mermaid Diagram:

  graph TD
      A[Modified PH20 (Phenolic-Stable)] --> B(PLGA Microsphere Encapsulation);
      C[Long-Acting Insulin Analog] --> B;
      B --> D[Subcutaneous Injection];
      D --> E{Controlled Release of PH20 & Insulin};
      E --> F[Sustained Insulin Absorption];
  

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Derivative 53.2: Insulin-PH20 Composition for Transdermal Delivery via Ionic Liquid Matrix

• Axis: Operational Parameter Expansion (Delivery Route/Matrix)
• Enabling Description: A pharmaceutical composition for transdermal delivery comprises a modified PH20 hyaluronidase polypeptide (e.g., a variant from Claim 1 with enhanced stability to benzyl alcohol) and a fast-acting insulin (e.g., insulin lispro), formulated within a thermosensitive ionic liquid matrix (e.g., choline geranate). The ionic liquid enhances the permeability of both the PH20 and insulin across the stratum corneum upon topical application. The composition is designed to be a gel at room temperature, liquifying at skin temperature for improved spreadability and penetration. The PH20 component facilitates transient disruption of the extracellular matrix in the skin, allowing for enhanced and rapid transdermal flux of insulin without requiring needles. The stability of the PH20 in the ionic liquid environment is critical, tested at temperatures ranging from 25°C to 40°C over 48 hours, ensuring >20% retained activity.
• Mermaid Diagram:

  graph TD
      A[Modified PH20 (Preservative-Stable)] --> B(Ionic Liquid Matrix);
      C[Fast-Acting Insulin] --> B;
      B --> D[Topical Application to Skin];
      D --> E{Thermosensitive Liquefaction};
      E --> F[PH20 Mediates ECM Disruption];
      F --> G[Enhanced Transdermal Insulin Flux];
  

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Derivative 53.3: PH20-Insulin Co-formulation for Intraocular Delivery in Ophthalmic Devices

• Axis: Cross-Domain Application (Ophthalmology)
• Enabling Description: A pharmaceutical composition designed for intraocular administration, specifically via a sustained-release ophthalmic implant, comprises a modified PH20 hyaluronidase polypeptide (e.g., a variant with increased stability to a broad range of excipients and temperatures, as per Claim 1 and 18, e.g., F204P, R58K) and a growth factor or therapeutic peptide (e.g., VEGF inhibitor, not insulin directly, but analogous to the drug delivery concept). The modified PH20 facilitates transient and localized degradation of vitreous humor hyaluronan, allowing for improved diffusion and distribution of the co-administered therapeutic agent within the eye. The implant material (e.g., biodegradable polymer like polycaprolactone) is engineered to release the PH20 and therapeutic peptide over several months, with the PH20's stability crucial for long-term efficacy within the challenging intraocular environment. The composition is sterile and formulated to maintain isotonicity and pH compatibility with ocular tissues.
• Mermaid Diagram:

  graph LR
      A[Modified PH20 (Stable)] --> B(Ophthalmic Implant);
      C[Therapeutic Peptide (e.g., VEGF Inhibitor)] --> B;
      B --> D[Intraocular Implantation];
      D --> E{Sustained Release of PH20 & Peptide};
      E --> F[PH20 Degrades Vitreous HA];
      F --> G[Improved Peptide Diffusion within Eye];
  

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Derivative 53.4: AI-Monitored PH20-Insulin Smart Pump for Personalized Dosing

• Axis: Integration with Emerging Tech (AI, IoT)
• Enabling Description: A pharmaceutical composition comprising a modified PH20 hyaluronidase polypeptide (e.g., a robustly stable variant from Claim 1 for extended shelf-life in solution) and a fast-acting insulin analog (e.g., insulin aspart) is loaded into a "smart" insulin pump. This pump is equipped with IoT connectivity, real-time glucose monitoring integration, and an embedded AI algorithm for personalized dosing. The AI considers patient-specific factors (e.g., activity levels, meal intake, glucose trends) and predicts optimal micro-doses of the PH20-insulin co-formulation. The PH20 facilitates rapid and consistent insulin absorption, allowing the AI to dynamically adjust delivery for tighter glycemic control. The pump continuously monitors the temperature and integrity of the composition using internal sensors, transmitting data via secure channels (e.g., Bluetooth LE) to a mobile application, which in turn feeds into the AI's learning model for continuous optimization.
• Mermaid Diagram:

  sequenceDiagram
      participant Patient_Glucose_Sensor
      participant Smart_Insulin_Pump
      participant AI_Algorithm
      participant Cloud_Platform
      Patient_Glucose_Sensor->>Smart_Insulin_Pump: Real-time Glucose Data (via Bluetooth LE)
      Smart_Insulin_Pump->>AI_Algorithm: Composition Data (Temperature, Integrity)
      AI_Algorithm->>Smart_Insulin_Pump: Optimized PH20-Insulin Dosing Strategy
      Smart_Insulin_Pump->>Patient: Deliver PH20-Insulin Composition
      Smart_Insulin_Pump->>Cloud_Platform: Upload Usage & Sensor Data (for AI retraining)
      Cloud_Platform->>AI_Algorithm: Updated Model for Continuous Optimization
  

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Derivative 53.5: PH20-Insulin Composition with "Cold-Chain Independent" Formulation

• Axis: The "Inverse" or Failure Mode (Resilience to adverse conditions)
• Enabling Description: A pharmaceutical composition is formulated to exhibit exceptional stability, making it "cold-chain independent" and enabling distribution and use in resource-limited settings without refrigeration. This composition comprises a highly engineered modified PH20 hyaluronidase polypeptide (e.g., a variant from Claim 1 with extreme resistance to phenolic preservatives and from Claim 18 with vastly improved thermal stability up to 45°C for several weeks) and a thermally stable insulin formulation (e.g., an insulin analog engineered for heat resistance or co-crystallized with zinc in a specific buffer). The PH20 and insulin are lyophilized together with a specific blend of cryoprotectants and lyoprotectants (e.g., trehalose, mannitol, and a non-ionic surfactant such as Polysorbate 80) that form an amorphous solid matrix. This dry powder formulation retains at least 80% activity for both components after storage at 40°C for 6 months. Reconstitution with sterile water prior to administration activates the formulation.
• Mermaid Diagram:

  graph TD
      A[Modified PH20 (Extreme Stability)] --> B(Lyophilization Process);
      C[Thermally Stable Insulin] --> B;
      D[Cryo/Lyoprotectant Blend] --> B;
      B --> E[Cold-Chain Independent Dry Powder];
      E --> F[Storage at Ambient/Elevated Temps (e.g., 40°C)];
      F -- Reconstitution with Sterile Water --> G[Active PH20-Insulin Solution];
      G --> H[Administration];
  

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Claim 55 Derivatives: Method for Identifying/Selecting Stable Hyaluronan-Degrading Enzyme

Core Claim Summary (from previous section): A method for identifying or selecting a modified hyaluronan-degrading enzyme that exhibits stability under a denaturation condition, comprising: a) testing the activity of a modified hyaluronan-degrading enzyme in a composition containing a denaturing agent and/or under a denaturing condition; b) testing the activity of the modified hyaluronan-degrading enzyme in the same composition and/or under the same conditions as a) except absent the denaturing agent or condition; and c) selecting or identifying a modified hyaluronan-degrading enzyme that exhibits activity in a) that is at least 5% of the activity in b).

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Derivative 55.1: Microfluidic High-Throughput Screening with Droplet-Based Encapsulation

• Axis: Material & Component Substitution (Assay platform)
• Enabling Description: The method for identifying stable hyaluronan-degrading enzymes is implemented on a microfluidic platform. Individual modified hyaluronan-degrading enzyme variants (e.g., PH20 variants with various amino acid replacements) are encapsulated as single molecules or small groups within picoliter-volume aqueous droplets generated in an oil continuous phase. Each droplet serves as an isolated reaction vessel. Denaturing agents (e.g., varying concentrations of phenol, extreme pH buffers) are co-encapsulated in some droplets, while control droplets lack the denaturing agent. The activity of the enzyme (e.g., using a fluorogenic hyaluronan substrate) is monitored real-time within each droplet using fluorescence detection. Droplets containing enzymes with desired stability (e.g., >10% activity retention in denaturing conditions) are sorted using dielectrophoresis or acoustic forces for subsequent recovery and sequencing of the encoding nucleic acid. This enables screening millions of variants per day.
• Mermaid Diagram:

  graph LR
      A[Modified Enzyme Library] --> B(Microfluidic Droplet Generator);
      C[Denaturing Agent] --> B;
      D[Fluorogenic Substrate] --> B;
      E[Control Buffer] --> B;
      B --> F(Droplet Array: Denaturing vs. Control);
      F --> G{Fluorescence Detection (Activity Measurement)};
      G --> H{Droplet Sorting (Stable Variants)};
      H --> I[Recovery & Sequencing];
  

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Derivative 55.2: Multi-Factorial Stability Profiling with Automated Liquid Handling at Industrial Scale

• Axis: Operational Parameter Expansion
• Enabling Description: The method is expanded to a multi-factorial stability profiling study at industrial scale, testing modified hyaluronan-degrading enzymes (e.g., a library of PH20 variants) across a combinatorial matrix of denaturation conditions. These conditions include varying temperatures (0°C to 70°C), pH (3.0 to 10.0), ionic strengths (0 mM to 500 mM NaCl), and concentrations of multiple excipients (e.g., phenol, m-cresol, polysorbate 80, urea) simultaneously. An automated liquid handling robot (e.g., using 1536-well plate format) prepares assay plates, incubates them for extended durations (e.g., up to 4 weeks), and performs endpoint hyaluronidase activity measurements using a plate reader. Data analysis leverages statistical software (e.g., R with multivariate analysis) to identify variants exhibiting robust stability across diverse and extreme denaturing environments, far exceeding the 5% activity retention threshold, aiming for >50% retention under specific harsh conditions.
• Mermaid Diagram:

  graph TD
      A[PH20 Variant Library] --> B(Automated Liquid Handler);
      C[Combinatorial Denaturing Conditions] --> B;
      B --> D[1536-Well Assay Plates];
      D -- Incubate (up to 4 weeks) --> E{Endpoint Activity Measurement (Plate Reader)};
      E --> F[Multivariate Data Analysis];
      F --> G[Identification of Robustly Stable Variants];
  

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Derivative 55.3: Enzyme Stability Screening for Biocatalysis in Non-Aqueous Solvents

• Axis: Cross-Domain Application (Industrial Biocatalysis)
• Enabling Description: The method is adapted to identify hyaluronan-degrading enzymes (e.g., PH20 variants) that exhibit stability and activity in non-aqueous or low-water content organic solvent systems. This is critical for biocatalytic applications in organic synthesis where water activity must be minimized. Modified enzymes are screened by testing their hyaluronidase activity in biphasic systems (e.g., aqueous buffer with hexane, toluene, or ethyl acetate) or in nearly anhydrous organic solvents containing trace water. The denaturing condition here is the presence of the organic solvent. Activity is measured by monitoring the degradation of a lipid-conjugated hyaluronan derivative in these solvent systems. Enzymes retaining at least 5% activity (or even exhibiting enhanced activity due to altered substrate specificity) compared to their activity in pure aqueous buffer are selected for further development as industrial biocatalysts.
• Mermaid Diagram:

  graph LR
      A[Modified PH20 Library] --> B(Bi-phasic/Anhydrous Solvent Assay);
      C[Organic Solvents (Hexane, Toluene, etc.)] --> B;
      D[Lipid-Conjugated HA Substrate] --> B;
      B --> E{Activity Measurement in Organic Environment};
      E --> F[Selection: Stable/Active Variants in Non-Aqueous Systems];
      F --> G[Application: Industrial Biocatalysis];
  

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Derivative 55.4: AI-Assisted Predictive Modeling for Denaturation Resistance and Automated Experiment Design

• Axis: Integration with Emerging Tech (AI, Automated Experimentation)
• Enabling Description: The method incorporates an AI-assisted framework for predicting enzyme stability and automating the design of experiments. Initial screening data of modified hyaluronan-degrading enzymes (e.g., PH20 variants with amino acid replacements at positions like 204, 58, 10, 12, etc. as mentioned in the patent) under various denaturing conditions is fed into a machine learning model (e.g., a Gaussian Process Regression model). This AI model learns the relationship between sequence, structure, and stability, then predicts the most promising new variants and optimal denaturing conditions for further testing. The AI also automatically generates experimental protocols and parameters for robotic platforms, closing the loop in a self-optimizing "Design-Build-Test-Learn" cycle. The "testing activity" step involves the robotic execution of the AI-designed experiments, with data automatically fed back to refine the model. Blockchain is used to record the AI's predictions, experimental designs, and results for immutable audit trails.
• Mermaid Diagram:

  graph TD
      A[Initial PH20 Variant Data (Sequence, Stability)] --> B(AI Model Training);
      B --> C{AI Predictive Modeling: New Variants & Conditions};
      C --> D[Automated Experiment Design (Robotics)];
      D --> E[Robotic Testing (Activity in Denaturing Conditions)];
      E --> F[Data Collection];
      F -- Immutable Record --> G[Blockchain Ledger];
      G --> B;
      F --> H[Selection: Highly Stable Variants];
  

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Derivative 55.5: Rapid Detection of Enzyme Destabilization via Intrinsic Fluorescence Shift

• Axis: The "Inverse" or Failure Mode (Rapid detection of failure)
• Enabling Description: A method is developed to rapidly identify hyaluronan-degrading enzymes (e.g., PH20 variants) that exhibit decreased stability or a predictable and rapid denaturation profile under specific stress conditions, effectively operating in an "inverse" or controlled-failure mode. Instead of measuring residual activity, the method relies on detecting changes in the enzyme's intrinsic fluorescence (e.g., tryptophan fluorescence) as a proxy for conformational changes indicative of denaturation. Modified PH20 variants are exposed to denaturing agents (e.g., detergents, chaotropes, or high temperatures), and the shift in emission maximum or intensity of tryptophan fluorescence is monitored over time. Variants exhibiting a rapid and significant fluorescence shift (e.g., >5 nm shift in emission maximum within 10 minutes of exposure to 0.1% SDS) are selected, indicating a susceptibility to denaturation, which could be engineered for triggered inactivation. The threshold for selection would be a rapid and quantifiable change, rather than retention of activity.
• Mermaid Diagram:

  sequenceDiagram
      participant PH20_Variant
      participant Denaturing_Agent
      participant Fluorescence_Spectrometer
      PH20_Variant->>Denaturing_Agent: Exposure to Stress Condition
      loop Time Course
          Denaturing_Agent->>PH20_Variant: Induces Conformational Change
          PH20_Variant->>Fluorescence_Spectrometer: Intrinsic Fluorescence Shift (e.g., Trp Emission)
          Fluorescence_Spectrometer->>PH20_Variant: Record Change over Time
      end
      Fluorescence_Spectrometer->>PH20_Variant: Identify Rapid Shift Variants
      PH20_Variant->>PH20_Variant: Selected for "Controlled Failure" Characteristics