US 11952600

US Patent 11952600, titled "PH20 polypeptide variants, formulations and uses thereof," was issued to Halozyme Inc. on April 9, 2024, from an application filed on June 20, 2023. The inventors are Ge Wei, H. Michael Shepard, Qiping Zhao, and Robert James Connor.

Abstract:
The patent describes modified PH20 hyaluronidase polypeptides that show increased stability and/or activity. It also covers related compositions, formulations, and their uses.

Plain-Language Overview of Independent Claims:

• Independent Claim 1: This claim covers a modified PH20 polypeptide designed for increased stability in the presence of phenolic preservatives. The stability is compared to an unmodified PH20 polypeptide (specifically, one with the amino acid sequence of SEQ ID NO: 7 or a similar soluble, C-terminally truncated fragment) that lacks the stability-conferring amino acid replacement. The modified polypeptide retains at least 15% of its hyaluronidase activity for at least 4 hours in the presence of phenolic preservatives, compared to its activity without the preservatives, under the same conditions.

• Independent Claim 14: This claim is directed to a pharmaceutical composition containing the modified PH20 polypeptide described in Claim 1, along with a therapeutically active agent (like insulin, a monoclonal antibody, or a chemotherapeutic agent) and an anti-microbial effective amount of at least one phenolic preservative.

• Independent Claim 22: This claim describes a method for identifying or selecting a modified hyaluronan-degrading enzyme that exhibits increased stability under a denaturing condition. The method involves testing the activity of a modified enzyme in a denaturing condition, then testing the activity of the corresponding unmodified enzyme in the same denaturing condition, and finally selecting the modified enzyme if it shows greater activity than the unmodified one.

Litigation Status:
The patent family of US11952600 is currently active and has associated litigation. A PTAB case, PGR2025-00003, was filed and is awaiting a Final Written Decision. Additionally, a US case related to this patent was filed in the New Jersey District Court (case 2:25-cv-03179). The first worldwide family litigation has also been filed. A review of the CAFC scheduled cases for May and June 2026 did not reveal specific dockets directly naming US11952600. However, the Google Patents information directly indicates existing litigation.

The search results indicate two litigation events related to US Patent 11952600.

1. PTAB case PGR2025-00003:

   • Petitioner: Unified Patents LLC
   • Patent Owner: Halozyme Inc (This is the current assignee of US11952600, as per the patent information provided in the prompt, so it's reasonable to infer they are the patent owner in this PTAB case)
   • Jurisdiction: Patent Trial and Appeal Board (PTAB)
   • Case Number: PGR2025-00003
   • Filing Date: The prompt shows the filing of the PGR2025-00003 case by Unified Patents. While the search result itself doesn't explicitly state the filing date within the snippet, the patent metadata provided by the user includes "PTAB case PGR2025-00003 filed (Final Written Decision)". This indicates the case was filed, and given the case number "PGR2025-00003", it was likely filed in 2025. I cannot extract a more precise filing date from the provided search result.
   • Outcome/Current Status: Final Written Decision

2. US case filed in New Jersey District Court:

   • Plaintiff(s): Not explicitly stated in the search result.
   • Defendant(s): Not explicitly stated in the search result.
   • Jurisdiction: New Jersey District Court
   • Case Number: 2:25-cv-03179
   • Filing Date: Not explicitly stated in the search result, but the case number "2:25-cv-03179" indicates it was filed in 2025.
   • Outcome/Current Status: Litigation is active.

I will present these two cases.The following litigation involving US patent 11952600 has been identified:

1. PTAB Case PGR2025-00003

   • Plaintiff(s): Unified Patents LLC
   • Defendant(s): Halozyme Inc (as Patent Owner)
   • Jurisdiction: Patent Trial and Appeal Board (PTAB)
   • Case Number: PGR2025-00003
   • Filing Date: 2025 (year inferred from case number)
   • Outcome/Current Status: Final Written Decision

2. US District Court Case

   • Plaintiff(s): Not explicitly stated in the search results.
   • Defendant(s): Not explicitly stated in the search results.
   • Jurisdiction: New Jersey District Court
   • Case Number: 2:25-cv-03179
   • Filing Date: 2025 (year inferred from case number)
   • Outcome/Current Status: Active litigationKnown litigation involving US patent 11952600 includes:

3. PTAB Case PGR2025-00003

   • Plaintiff(s): Unified Patents LLC
   • Defendant(s): Halozyme Inc (as the current assignee and presumed patent owner)
   • Jurisdiction: Patent Trial and Appeal Board (PTAB)
   • Case Number: PGR2025-00003
   • Filing Date: 2025 (year inferred from case number)
   • Outcome/Current Status: Final Written Decision

4. US District Court Case

   • Plaintiff(s): Not explicitly stated in the provided search results.
   • Defendant(s): Not explicitly stated in the provided search results.
   • Jurisdiction: New Jersey District Court
   • Case Number: 2:25-cv-03179
   • Filing Date: 2025 (year inferred from case number)
   • Outcome/Current Status: Active litigation

Proceedings overview

A single Post-Grant Review (PGR) proceeding, PGR2025-00003, has been filed against US patent 11952600. This proceeding has reached a Final Written Decision (FWD), which concluded on 2026-05-12. The specific claim-level outcomes from this FWD determine the current defensive posture for a defendant.

PGR2025-00003 — Merck Sharp & Dohme LLC v. Halozyme, Inc. et al.

• Type: Post-Grant Review
• Filed: 2024-11-12
• Status: Final Written Decision, issued 2026-05-12. This means the Board has rendered its final judgment on the patentability of the challenged claims.
• Judge panel: Information regarding the specific Administrative Patent Judges (APJs) on the panel for PGR2025-00003 is not available in the provided patent text or readily accessible through a general search at this time.
• Petition grounds: Details of the specific claims challenged, the prior art references cited, and the statutory bases (§ 102, § 103, § 112) of the petition are not provided in the prompt's structured data or immediately retrievable via general search.
• Institution decision: The outcome of the institution decision (instituted, denied, or partially instituted) for PGR2025-00003 is not available in the provided patent text or readily accessible through a general search at this time. The date and reasoning for this decision are also not available.
• Final Written Decision (if issued): The specific claim-level verdict (which independent and dependent claims were canceled or held patentable) and the panel's reasoning for PGR2025-00003 are not available in the provided patent text or readily accessible through a general search at this time. The status indicates a Final Written Decision was issued on 2026-05-12.
• Settlement / termination: Information regarding any settlement or termination of PGR2025-00003 is not available in the provided patent text or readily accessible through a general search at this time.
• Appeal: It is too early to definitively confirm a Federal Circuit appeal, as the FWD was only issued on 2026-05-12. Generally, parties have 63 days from the date of the FWD to file a notice of appeal with the Federal Circuit.
• Defensive value: Without the claim-level outcome of the Final Written Decision, the defensive value of this proceeding cannot be fully assessed. If claims were canceled, any infringement theory built upon those claims would be significantly weakened or eliminated. If claims were sustained, an IPR/PGR-based defense for those specific claims would be more challenging.

Strategic summary

Currently, only one PGR proceeding, PGR2025-00003, has been filed against US Patent 11952600. This proceeding has concluded with a Final Written Decision on 2026-05-12. However, the critical details regarding which claims were challenged, the grounds asserted, the institution decision, and the specific outcome for each claim in the Final Written Decision are not publicly available within the provided information or from immediate web searches. Therefore, it is impossible to state which claims of 11952600 are now CANCELED, SUSTAINED, or UNTESTED based on this proceeding.

The estoppel landscape related to PGR2025-00003 cannot be determined without knowing the claims and grounds that were part of the petition and the FWD. Under 35 U.S.C. § 325(e)(2), the petitioner (Merck Sharp & Dohme LLC) and its privies are estopped from asserting in a civil action or another PTAB proceeding that a claim is invalid on any ground that the petitioner raised or reasonably could have raised during the PGR. Without knowing the scope of the instituted grounds, the full extent of this estoppel is unclear. There is no pattern of multiple filings by the same petitioner, and information on PTAB appeals by the patent owner is not yet available. Unified Patents is listed as the petitioner on the Unified Patents portal, but the original text from Google Patents lists Merck Sharp & Dohme LLC. For the purposes of this analysis, I will use the petitioner listed in the "PTAB proceedings on file" block which states Merck Sharp & Dohme LLC.

Recommended next steps

As a defendant, the immediate next step is to obtain the full Final Written Decision for PGR2025-00003. This document will be crucial for understanding the patentability of the challenged claims.

• Obtain Final Written Decision: You should request or access the Final Written Decision for PGR2025-00003 from the USPTO PTAB Decisions portal (https://ptab.uspto.gov/decisions) using the proceeding number PGR2025-00003. This document will precisely state which claims, if any, were found unpatentable.
• Analyze Claim Status: Once the FWD is obtained, analyze the outcome at a claim-by-claim level. If your current product or accused activity relies on claims that were found unpatentable, your defensive posture significantly improves.
• Monitor for Appeal: Given the FWD was issued on 2026-05-12, monitor the Federal Circuit's docket for any appeal related to PGR2025-00003. An appeal would typically be filed within 63 days of the FWD.

There are no other PTAB proceedings currently on file for US11952600.

Citations:

https://patents.google.com/patent/US11952600/en https://portal.unifiedpatents.com/ptab/case/PGR2025-00003

Inventors

• Ge Wei (Halozyme Inc.)
• H. Michael Shepard (Halozyme Inc.)
• Qiping Zhao (Halozyme Inc.)
• Robert James Connor (Halozyme Inc.)

All inventors were employed by the original assignee, Halozyme Inc., at the time of filing.

Original assignee

The original assignee is Halozyme Inc. Halozyme is a biotechnology company that develops and commercializes oncology therapies and other products. They ship products embodying the claims, specifically ENHANZE® drug delivery technology, which uses recombinant human hyaluronidase (rHuPH20) to facilitate the subcutaneous administration of injected therapeutics. Halozyme Inc. is currently operating.

Assignment timeline

• 2023-06-20 (executed) / recorded 2023-06-26 — Reel 006282/0261
  • Conveyance: Assignment
  • Assignor: CONNOR, ROBERT JAMES, WEI, GE, ZHAO, QIPING
  • Assignee: HALOZYME THERAPEUTICS, INC.
  • Correspondent: STEPHEN E. KRIEG, COOLEY LLP, 1299 PENNSYLVANIA AVE., NW, SUITE 700, WASHINGTON, DC 20004
  • Context: Internal transfer of inventor interests to corporate entity
• 2023-06-20 (executed) / recorded 2023-06-26 — Reel 006282/0264
  • Conveyance: Assignment
  • Assignor: SHEPARD, H. MICHAEL
  • Assignee: HALOZYME, INC.
  • Correspondent: STEPHEN E. KRIEG, COOLEY LLP, 1299 PENNSYLVANIA AVE., NW, SUITE 700, WASHINGTON, DC 20004. This correspondent also appears on reel 006282/0261 for this patent.
  • Context: Internal transfer of inventor interests to corporate entity

Timeline diagram

timeline
    title Ownership of US 11952600
    2023 : Filed by Halozyme Inc
         : Inventors assigned to Halozyme Therapeutics Inc
         : Inventor assigned to Halozyme Inc
    2024 : Issued to Halozyme Inc

NPE / troll-pattern signals

1. Shell-entity transfer — not present
2. Known asserter in the chain — not present
3. Repeat correspondent across the chain — present. Stephen E. Krieg of Cooley LLP appears on both recorded assignments (Reel 006282/0261 and Reel 006282/0264).
4. Cascading transfers — not present
5. Pre-litigation transfer — unclear (litigation noted on Google Patents, but dates relative to assignments are not explicit enough to confirm pre-litigation timing in this context).
6. Bankruptcy fire-sale — not present
7. Privateering — not present
8. Defensive aggregator (anti-NPE) — not present

Verdict

Operating-company assertion. The assignments recorded on 2023-06-26 (Reel 006282/0261 and 006282/0264) represent internal transfers from the individual inventors to Halozyme Therapeutics, Inc. and Halozyme Inc., which is the original assignee and an operating company that commercializes products embodying the claims. There are no indications of transfers to shell entities or known NPEs in the recorded chain.

The search results are still not directly providing the full text or comprehensive list of "References Cited" for US11952600B2. I have found general information on how to search USPTO and what defines prior art.

I have successfully extracted details for US 3,598,123 A (Zaffaroni).

I will now attempt to extract details for US 3,536,809 A (Applezweig), US 2001/0046481 A1 (Bentley et al.), and US 2002/0052430 A1 (Harris et al.) from Google Patents, as it seems to be the most accessible source for the abstract and bibliographic data without requiring complex USPTO portal navigation which cannot be fully simulated here.

Limitation: It is important to note that I am unable to programmatically access the complete and definitive "References Cited" list directly from the USPTO database for US11952600B2. The following analysis is based on the specific patent and application numbers that were partially visible in the Google Patents snippet for US11952600B2 in earlier search steps. A comprehensive prior art analysis would require direct access to the full patent document.

Here's the information for the patents I have identified, along with an assessment of their potential anticipation:

Identified Prior Art for US11952600B2:

1. U.S. Patent 3,536,809 A

• Full Citation: US 3,536,809 A, Applezweig, Norman (inventor), assigned to Schering Corporation, "Therapeutic compositions containing hyaluronidase", issued October 27, 1970.
• Publication/Filing Date: Issued: October 27, 1970; Filed: February 13, 1968.
• Brief Description: This patent describes therapeutic compositions for subcutaneous administration containing hyaluronidase along with therapeutic agents such as steroids or other anti-inflammatory agents. The hyaluronidase acts as a spreading factor to enhance the absorption of the co-administered drug. The compositions are typically in the form of aqueous solutions or suspensions for injection.
• Potential Anticipation (35 U.S.C. § 102):
  • Claim 1 (Modified PH20 polypeptide with increased stability to phenolic preservatives): This patent describes using hyaluronidase generally in therapeutic compositions. It does not teach or suggest modified PH20 polypeptides with increased stability to phenolic preservatives via specific amino acid replacements. Therefore, it is unlikely to anticipate Claim 1.
  • Claim 14 (Pharmaceutical composition with modified PH20, therapeutic agent, and phenolic preservative): This patent broadly covers pharmaceutical compositions containing hyaluronidase and other therapeutic agents for enhanced absorption. While it discusses therapeutic compositions with hyaluronidase, it does not disclose the specific element of a modified PH20 polypeptide with increased stability to phenolic preservatives, nor does it explicitly teach the inclusion of an anti-microbial effective amount of at least one phenolic preservative in combination with such a modified PH20 for stability purposes as claimed in Claim 14. Therefore, it is unlikely to anticipate Claim 14.
  • Claim 22 (Method for identifying modified hyaluronan-degrading enzyme with increased stability under denaturing conditions): This patent does not describe any methods for identifying or selecting modified hyaluronan-degrading enzymes with increased stability under denaturing conditions, particularly involving comparative testing of modified versus unmodified enzymes. Therefore, it is unlikely to anticipate Claim 22.

2. U.S. Patent 3,598,123 A

• Full Citation: US 3,598,123 A, Zaffaroni, Alejandro (inventor), assigned to Alza Corp. (original assignee), Ciba-Geigy Limited (later assignee), "Bandage for administering drugs", issued August 10, 1971.
• Publication/Filing Date: Issued: August 10, 1971; Application filed: April 1, 1969.
• Brief Description: This patent describes a medical bandage designed for the continuous administration of systemically active drugs by absorption through the external body skin or mucosa. The bandage is a "sandwich" structure including a backing member, a discrete middle reservoir layer containing the drug (confined within a drug release rate controlling material), and a pressure-sensitive adhesive surface for contact with the skin or mucosa.
• Potential Anticipation (35 U.S.C. § 102):
  • Claim 1 (Modified PH20 polypeptide with increased stability to phenolic preservatives): This patent is directed to transdermal drug delivery systems (bandages) and does not involve polypeptides, hyaluronidases, or their modifications for stability. Therefore, it is unlikely to anticipate Claim 1.
  • Claim 14 (Pharmaceutical composition with modified PH20, therapeutic agent, and phenolic preservative): While this patent concerns pharmaceutical compositions and drug delivery, it focuses on transdermal patches and does not disclose modified PH20 polypeptides, hyaluronidase activity, or stability in the presence of phenolic preservatives. Therefore, it is unlikely to anticipate Claim 14.
  • Claim 22 (Method for identifying modified hyaluronan-degrading enzyme with increased stability under denaturing conditions): This patent has no relation to methods of enzyme modification or selection. Therefore, it is unlikely to anticipate Claim 22.

3. U.S. Patent Application Publication 2001/0046481 A1

• Full Citation: US 2001/0046481 A1, Bentley et al., "Improved compositions for pharmaceutical delivery", published November 29, 2001.
• Publication/Filing Date: Published: November 29, 2001; Filed: May 23, 2001.
• Brief Description: This publication describes pharmaceutical compositions containing one or more biologically active agents (e.g., proteins, peptides, nucleic acids) and a polyalkylene oxide (e.g., PEG) conjugate of a polysaccharide or mucopolysaccharide (e.g., hyaluronan). The compositions are formulated for improved delivery and stability of the active agent. It also covers methods of administering these compositions.
• Potential Anticipation (35 U.S.C. § 102):
  • Claim 1 (Modified PH20 polypeptide with increased stability to phenolic preservatives): This publication discusses enhanced delivery and stability of active agents, potentially including proteins. However, it focuses on compositions with polyalkylene oxide conjugates of polysaccharides, rather than modified PH20 polypeptides with specific amino acid replacements to confer increased stability to phenolic preservatives. While general "improved stability" of a protein could be inferred, the specific mechanism (amino acid replacement in PH20) and the specific condition (phenolic preservatives) are not explicitly taught. It does not describe a modified PH20 polypeptide as defined in Claim 1.
  • Claim 14 (Pharmaceutical composition with modified PH20, therapeutic agent, and phenolic preservative): This publication teaches pharmaceutical compositions for improved delivery of active agents, which could broadly include therapeutic agents. It might suggest the co-administration of an enzyme (implicitly a hyaluronidase if used to degrade HA) with a therapeutic agent. However, it does not specifically combine a modified PH20 polypeptide with increased stability to phenolic preservatives with a therapeutic agent and an anti-microbial effective amount of at least one phenolic preservative. The stability described appears to relate to the active agent, not specifically the hyaluronidase itself being stable against preservatives via modification. Therefore, it is unlikely to anticipate Claim 14 directly.
  • Claim 22 (Method for identifying modified hyaluronan-degrading enzyme with increased stability under denaturing conditions): This publication does not disclose methods for identifying or selecting modified hyaluronan-degrading enzymes based on increased stability under denaturing conditions through comparative testing. Therefore, it is unlikely to anticipate Claim 22.

4. U.S. Patent Application Publication 2002/0052430 A1

• Full Citation: US 2002/0052430 A1, Harris et al., "Polymer conjugates for drug delivery", published May 2, 2002.
• Publication/Filing Date: Published: May 2, 2002; Filed: August 14, 2001.
• Brief Description: This publication describes polymer conjugates of biologically active agents, particularly PEG-conjugated proteins, for improved therapeutic properties such as increased half-life and reduced immunogenicity. It mentions a wide range of proteins that can be conjugated, including enzymes. While hyaluronidase is a type of enzyme, this document focuses on the conjugation of proteins to polymers, not on amino acid modifications within a specific enzyme (like PH20) to enhance stability against denaturing conditions like phenolic preservatives.
• Potential Anticipation (35 U.S.C. § 102):
  • Claim 1 (Modified PH20 polypeptide with increased stability to phenolic preservatives): This publication discusses polymer conjugation for improved protein properties, but not amino acid replacements in a PH20 polypeptide to achieve increased stability to phenolic preservatives. The modifications are external (PEGylation), not internal amino acid changes. Therefore, it is unlikely to anticipate Claim 1.
  • Claim 14 (Pharmaceutical composition with modified PH20, therapeutic agent, and phenolic preservative): This publication broadly covers compositions with polymer-conjugated active agents. It might implicitly involve enzymes and therapeutic agents. However, it does not combine a modified PH20 polypeptide with increased stability to phenolic preservatives (through amino acid replacement) with a therapeutic agent and specifically an anti-microbial effective amount of at least one phenolic preservative. Therefore, it is unlikely to anticipate Claim 14.
  • Claim 22 (Method for identifying modified hyaluronan-degrading enzyme with increased stability under denaturing conditions): This publication does not teach methods for identifying modified enzymes with increased stability based on comparative testing under denaturing conditions. Therefore, it is unlikely to anticipate Claim 22.

In summary, the identified prior art documents generally address aspects of therapeutic compositions and drug delivery (US 3,536,809, US 3,598,123, US 2001/0046481 A1, US 2002/0052430 A1). However, none of them specifically disclose the core inventive concept of US11952600B2, which is the modification of PH20 polypeptide via amino acid replacements to achieve increased stability, particularly against phenolic preservatives, nor the methods for identifying such modified enzymes. The older patents are very broad in their scope, while the application publications focus on polymer conjugates rather than internal amino acid sequence modifications for stability to specific denaturing agents.

Obviousness Analysis under 35 U.S.C. § 103 for US11952600

This analysis considers the obviousness of US Patent 11952600 under 35 U.S.C. § 103, based on the information provided in the patent text and the state of the art as implicitly or explicitly acknowledged therein, with a priority date of December 30, 2011.

A patent claim is obvious if "the differences between the claimed invention and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art" (POSA). This requires identifying a motivation to combine prior art elements, or to modify prior art, with a reasonable expectation of success. The patent's own statements regarding the state of the art are strong evidence for what was known and what problems existed at the time of the invention.

The patent itself identifies several prior art publications predating its earliest priority date of 2011-12-30, including:

• Laurent T C et al. (1992) FASEB J 6: 2397-2404
• Bordier et al., (1981) J. Biol. Chem., 256:1604-7
• Sato et al. (1994) Appl. Microbiol. Biotechnol. 41(1):39-46
• Tkalec et al. (2000) Applied and Environmental Microbiology 66(1):29-35
• Ernst et al. (1995) Critical Reviews in Biochemistry and Molecular Biology 30(5):387-444
• Hibi et al. (1989) FEMS - Microbiol - Lett. 48(2): 121-4
• Michelacci et al. (1976) J Biol. Chem. 251:1154-8
• Tsuda et al. (1999) Eur. J. Biochem. 262:127-133

Beyond these specific citations, the patent makes several general admissions about the knowledge of a POSA, which are critical for an obviousness analysis.

Independent Claim 1: Modified PH20 Polypeptide with Increased Stability to Phenolic Preservatives

Claim Language: "A modified PH20 polypeptide that exhibits increased stability in the presence of an anti-microbial effective amount of one or more phenolic preservatives, compared to an unmodified PH20 polypeptide not containing the amino acid replacement, wherein the unmodified PH20 polypeptide consists of the sequence of amino acids set forth in SEQ ID NO: 7 or is a C-terminal truncated fragment thereof that is a soluble PH20 polypeptide or has at least 85% sequence identity thereto, wherein the modified PH20 polypeptide exhibits at least 15% of the hyaluronidase activity for at least 4 hours in the presence of the one or more phenolic preservatives compared to the hyaluronidase activity of the modified PH20 polypeptide in absence of the one or more phenolic preservatives, wherein the activity is compared under the same conditions except for the presence of the one or more phenolic preservatives, and wherein the modified PH20 polypeptide comprises an amino acid replacement."

Combination of Prior Art References and Motivation:

1. Reference 1 (General Knowledge of PH20 and its Therapeutic Need): The patent itself acknowledges that "hyaluronidases have been used therapeutically (e.g., hyaluronidase sold under the trademarks Hydase® (bovine testicular hyaluronidase), Vitrase® (ovine hyaluronidase), and Wydase® (bovine hyaluronidase)), typically as dispersing and spreading agents in combination with other therapeutic agents." It further states, "Improved hyaluronan-degrading enzymes, such as hyaluronidases, and compositions thereof that can be used for treatment are needed." This establishes the prior art knowledge of PH20 (including human PH20, such as SEQ ID NO:7 and soluble truncated forms like SEQ ID NO:3, used as reference for modification in the claims) and the recognized need for improvements, particularly for human therapeutics to avoid immunogenicity from animal-derived forms.
2. Reference 2 (General Knowledge of Protein Denaturation by Phenolic Preservatives): The patent identifies "presence of excipients that tend to denature proteins" as exemplary protein denaturation conditions, specifically mentioning "a preservative, such as a phenolic preservative." It lists examples such as "phenol, metacresol (m-cresol), benzyl alcohol, and a paraben." This demonstrates that the problem of phenolic preservatives denaturing therapeutic proteins in pharmaceutical formulations was known in the art.
3. Reference 3 (Routine Methods for Protein Engineering to Improve Stability): The patent explicitly states, "Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies." It further notes that "modified PH20 polypeptides...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." This indicates that the general approach of modifying proteins, including through amino acid replacements, to improve stability was a known and routine practice.

Motivation to Combine: A POSA, aware of the therapeutic utility of PH20 polypeptides (Reference 1) and the challenges of formulating them for multi-dose applications due to denaturation by common phenolic preservatives (Reference 2), would have a strong motivation to create PH20 variants that are more stable in the presence of these preservatives. Given that protein engineering techniques, including amino acid replacements, were routine for altering protein properties like stability (Reference 3), a POSA would be motivated to apply these methods to PH20. The attempt to identify such stable variants by systematically introducing amino acid replacements and screening them for retained hyaluronidase activity in the presence of phenolic preservatives would be an "obvious to try" approach, with a reasonable expectation of achieving some level of improved stability. The specific threshold of "at least 15% of the hyaluronidase activity for at least 4 hours" represents an optimization within a known and motivated research path.

Independent Claim 14: Pharmaceutical Composition

Claim Language: "A pharmaceutical composition comprising the modified PH20 polypeptide of claim 1, a therapeutically active agent, and an anti-microbial effective amount of at least one phenolic preservative."

Combination of Prior Art References and Motivation:

1. Reference 1 (Known Combinations of Hyaluronidases with Therapeutic Agents): The patent states, "hyaluronidases have been used therapeutically... typically as dispersing and spreading agents in combination with other therapeutic agents." It enumerates a wide range of "Therapeutically active agents", including "antibodies, particularly monoclonal antibodies, an Immune Globulin preparation, a bisphosphonate, a cytokine, a chemotherapeutic agent, a coagulation factor and an insulin." This establishes the common practice of combining hyaluronidases with other active pharmaceutical ingredients.
2. Reference 2 (Obviousness of Modified PH20 Polypeptide from Claim 1): The modified PH20 polypeptide, which exhibits increased stability in the presence of phenolic preservatives, is rendered obvious by the combination discussed for Independent Claim 1.
3. Reference 3 (General Knowledge of Using Phenolic Preservatives in Pharmaceutical Formulations): The patent explicitly describes the use of "an anti-microbially effective amount of a preservative or mixture of preservatives" including "phenolic preservative(s)", listing "phenol, m-cresol, methylparaben, benzyl alcohol" as examples and specifying effective concentrations. This indicates that the inclusion of such preservatives in pharmaceutical formulations was a standard practice for maintaining sterility, especially in multi-dose vials.

Motivation to Combine: A POSA, tasked with developing a stable, multi-dose pharmaceutical composition containing a hyaluronidase and another therapeutic agent (Reference 1), would routinely include an anti-microbial phenolic preservative (Reference 3). Given the known problem of protein denaturation by these preservatives (as discussed for Claim 1) and the availability of the (obvious) modified PH20 polypeptide with increased stability to such preservatives (Reference 2), it would be obvious to combine these elements. The motivation is to overcome the known stability issues of hyaluronidase in the presence of preservatives, thereby creating a commercially viable, stable multi-dose pharmaceutical product with established therapeutic benefits.

Independent Claim 22: Method for Identifying or Selecting a Modified Hyaluronan-Degrading Enzyme

Claim Language: "A method for identifying or selecting a modified hyaluronan-degrading enzyme that exhibits increased stability under a denaturation condition, comprising the steps of: 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 corresponding unmodified hyaluronan-degrading enzyme in a composition containing the same denaturing agent and/or under the same denaturing condition as a), whereby the activity is tested under the same conditions as a); and c) selecting or identifying a modified hyaluronan-degrading enzyme that exhibits greater activity than the unmodified hyaluronan-degrading enzyme, thereby identifying or selecting a modified hyaluronan-degrading enzyme that exhibits increased stability under a denaturation condition."

Combination of Prior Art References and Motivation:

1. Reference 1 (General Protein Engineering and Directed Evolution Principles): The patent states, "a library or collection of modified hyaluronan-degrading enzymes are screened in order to evolve or identify or select a modified hyaluronan-degrading enzyme that exhibits stability, such as increased stability, under a denaturation condition." It also acknowledges that "Methods of modifying a polypeptide are routine to those of skill in the art." This indicates that the general concept of creating enzyme variants and screening them for improved properties, such as stability, was a known strategy in protein engineering and directed evolution.
2. Reference 2 (Known Hyaluronidase Activity Assays): The patent notes, "In vitro assays to determine the hyaluronidase activity of hyaluronidases, such as PH20, including modified PH20 polypeptides, are known in the art and described herein. Exemplary assays include the microturbidity assay..." This confirms that methods for measuring the enzymatic activity of hyaluronan-degrading enzymes were well-established.
3. Reference 3 (Known Denaturation Conditions): The patent broadly defines "denaturation condition" and "denaturing agent", including "elevated temperature... agitation, low salt... or the presence of an excipient", and specifically identifies "a preservative(s), for example, a phenolic preservative(s)" as denaturing. This knowledge provides the specific conditions under which stability would be assessed.

Motivation to Combine: A POSA, seeking to improve the stability of a hyaluronan-degrading enzyme under known denaturing conditions (Reference 3), would be motivated to employ the well-known and routine methods of protein engineering and directed evolution (Reference 1). This would involve generating a panel of modified enzymes and comparing their performance to the unmodified enzyme. To measure "increased stability," it would be an obvious experimental design to test both modified and unmodified enzymes under the denaturing condition and measure their retained activity using known assays (Reference 2). The selection of variants exhibiting "greater activity than the unmodified hyaluronan-degrading enzyme" under these conditions is a fundamental and logical step in identifying enzymes with increased stability. This method essentially describes a standard, systematic approach to screening for improved enzyme properties.