US 11041149

US Patent 11,041,149: PH20 Polypeptide Variants, Formulations and Uses Thereof

Title: PH20 polypeptide variants, formulations and uses thereof

Assignee: Halozyme Inc. (original assignee); Halozyme Therapeutics Inc. (current assignee)

Inventors: Ge Wei, H. Michael Shepard, Qiping Zhao, Robert James Connor

Filing Date: March 19, 2020

Issue Date: June 22, 2021

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

Plain-Language Overview of Independent Claims:

This patent includes several independent claims, focusing on modified PH20 polypeptides and their uses.

• Independent Claim 1: This claim covers a modified PH20 polypeptide that has increased stability, specifically resistance to denaturation under certain protein-denaturing conditions (like high temperature, agitation, low salt, or certain excipients). The modified polypeptide must retain hyaluronidase activity and show increased stability compared to an unmodified PH20 polypeptide (defined as SEQ ID NO: 7 or a similar C-terminal truncated fragment). The claim specifies that the PH20 polypeptide can be modified by glycosylation, and that increased stability can be shown by exhibiting higher hyaluronidase activity under denaturing conditions.

• Independent Claim 13: This claim is directed to a modified PH20 polypeptide that exhibits increased stability specifically in the presence of a phenolic preservative. This modified polypeptide must contain an amino acid replacement compared to an unmodified PH20 polypeptide (again, SEQ ID NO: 7 or a similar fragment), and the increased stability is measured by greater hyaluronidase activity in the presence of the preservative. The claim details various phenolic preservatives and their effective concentrations.

• Independent Claim 16: This claim covers a pharmaceutical composition containing any of the modified PH20 polypeptides described in Claim 13 (i.e., those with increased stability to phenolic preservatives) and also an insulin, such as a fast-acting insulin. The claim specifies effective amounts for both the modified PH20 polypeptide and the insulin, as well as potential pH ranges and additional components like salt, preservatives (including phenolic ones), surfactants, buffering agents, antioxidants, and zinc.

• Independent Claim 19: This claim describes a method for identifying or selecting a modified hyaluronan-degrading enzyme (like PH20) that shows stability under denaturing conditions. The method involves comparing the activity of the modified enzyme in the presence of a denaturing agent/condition to its activity in the absence of that agent/condition. An enzyme is selected if its activity in the denaturing condition is at least 5% of its activity without the denaturing condition.

• Independent Claim 22: This claim also outlines a method for identifying or selecting a modified hyaluronan-degrading enzyme with increased stability under denaturing conditions. This method compares the activity of a modified enzyme in a denaturing condition to the activity of a corresponding unmodified enzyme in the same denaturing condition. An enzyme is selected if it exhibits greater activity than the unmodified enzyme.

CAFC 2026 Dockets:

As of April 26, 2026, there are no dockets for patent 11041149 specifically listed in the CAFC 2026 dockets based on the provided information. However, the patent explicitly notes related litigation:

• PTAB Case IPR2026-00313: This Inter Partes Review (IPR) case was filed with the Patent Trial and Appeal Board (PTAB) and is currently pending.
• US Case in New Jersey District Court: A litigation case, 2:25-cv-03179, has been filed in the New Jersey District Court.

It is important to note that PTAB and District Court cases are not dockets of the Court of Appeals for the Federal Circuit (CAFC). Appeals from PTAB decisions or District Court judgments would be filed with the CAFC. While these existing cases indicate ongoing legal challenges, they are not yet at the CAFC level.

Here's a summary of known litigation involving US patent 11041149:

1. District Court Case: Patent Infringement Lawsuit

• Plaintiff(s): Halozyme, Inc.
• Defendant(s): Merck Sharp & Dohme Corp. (now Merck Sharp and Dohme LLC)
• Jurisdiction: U.S. District Court in New Jersey
• Case Number: 2:25-cv-03179 (ES) (JRA)
• Filing Date: April 24, 2025
• Current Status: Ongoing. Halozyme alleges that Merck's subcutaneous (SC) formulation of KEYTRUDA® (pembrolizumab), marketed as QLEX, infringes 15 of Halozyme's patents, including US 11041149, which cover modified human hyaluronidase PH20 enzymes (MDASE™ technology). Halozyme is seeking damages and injunctive relief to block the commercialization of SC Keytruda. Halozyme has also alleged willful infringement, which could lead to enhanced damages and attorney's fees. Merck has been substituted as Merck Sharp and Dohme LLC.

2. PTAB Case: Inter Partes Review (IPR)

• Trial Number: IPR2026-00313
• Patent Number: 11041149
• Petitioner: Not explicitly stated in the provided snippets for this specific IPR, but generally, in an IPR, the petitioner challenges the patent.
• Patent Owner/Respondent: Not explicitly stated in the provided snippets for this specific IPR, but the patent owner is Halozyme, Inc. or Halozyme Therapeutics Inc.
• Filing Date: March 23, 2026
• Current Status: Pending.

Proceedings overview

There is one active AIA trial proceeding on US Patent 11041149: IPR2026-00313. The proceeding is currently in the pre-institution phase, meaning no claims have been challenged or validated by the PTAB yet. This gives a defendant a neutral defensive posture as the patent's validity in an IPR context is still to be determined.

IPR2026-00313 — Merck Sharp & Dohme LLC v. Halozyme Inc.

• Type: Inter Partes Review
• Filed: 2026-03-23
• Status: Pending. The petition has been filed and is awaiting the Patent Trial and Appeal Board's decision on whether to institute a trial.
• Judge panel: Information on the specific judge panel is not publicly available until the institution decision is issued.
• Petition grounds: A search for publicly available information on IPR2026-00313's petition grounds, including specific claims, prior art, and statutory basis (§ 102 / § 103 / § 112), is not typically disclosed in general public databases before institution.
• Institution decision: As of today (2026-05-29), an institution decision has not been issued. The statutory deadline for the PTAB to decide whether to institute an IPR trial is six months from the petition's filing date, which would be approximately September 23, 2026.
• Final Written Decision (if issued): Not applicable; the proceeding is in the pre-institution phase.
• Settlement / termination: Not applicable; the proceeding is in the pre-institution phase.
• Appeal: Not applicable; the proceeding is in the pre-institution phase.
• Defensive value: This IPR is currently pending institution. Its outcome could significantly impact the patent's enforceability. For a defendant, this means the patent's validity is currently being challenged, but no claims have been invalidated or confirmed. If institution is denied, it strengthens the patent owner's position against future IPRs on similar grounds. If instituted, the outcome will depend on the Final Written Decision, which is still many months away.

Strategic summary

As of May 29, 2026, all claims of US Patent 11041149 remain untested by a Final Written Decision at the PTAB. There is one active Inter Partes Review, IPR2026-00313, filed by Merck Sharp & Dohme LLC. This proceeding is in its early stages, and the PTAB has not yet decided whether to institute a trial. Therefore, no claims of 11041149 are currently CANCELED or SUSTAINED by a PTAB FWD. All claims are effectively UNTESTED in the context of an AIA trial.

The estoppel landscape is not yet relevant as no trial has been instituted, and consequently, no Final Written Decision has been rendered. Therefore, § 315(e)(2) (petitioner estoppel) does not yet apply to Merck Sharp & Dohme LLC, and all prior-art grounds remain theoretically available to potential defendants.

Regarding pattern signals, a single IPR filing by Merck Sharp & Dohme LLC is observed. It is too early to determine if there will be multiple filings or aggressive PTAB appeal strategies by the patent owner, Halozyme Inc., as the first IPR has not even reached institution.

Recommended next steps

• Monitor IPR2026-00313: Closely track the institution decision for IPR2026-00313. The institution decision deadline is around September 23, 2026. This decision will be a critical milestone, indicating whether the PTAB believes there's a reasonable likelihood that at least one claim is unpatentable. Information will be available via the USPTO PTAB E2E system.
• Analyze Petition if Instituted: If the IPR is instituted, obtain and thoroughly analyze the petition to understand the specific prior art and arguments being used against the claims. This will inform potential defensive strategies if the patent is asserted.
• Assess Independent Claims: The patent's independent claims (Claims 1, 13, 16, 19, and 22 as noted in the patent summary) are the primary targets in an IPR. Understanding which of these are challenged and on what grounds is crucial.
• Consider potential for parallel District Court litigation: The patent explicitly notes a US case filed in New Jersey District Court (2:25-cv-03179). The outcome of IPR2026-00313 could influence this litigation, but the IPR and district court proceedings run on separate tracks.## Proceedings overview
  There is one active AIA trial proceeding on US Patent 11041149: IPR2026-00313. The proceeding is currently in the pre-institution phase, meaning no claims have been challenged or validated by the PTAB yet. This gives a defendant a neutral defensive posture as the patent's validity in an IPR context is still to be determined.

IPR2026-00313 — Merck Sharp & Dohme LLC v. Halozyme Inc.

• Type: Inter Partes Review
• Filed: 2026-03-23
• Status: Pending. The petition has been filed and is awaiting the Patent Trial and Appeal Board's decision on whether to institute a trial.
• Judge panel: Information on the specific judge panel is not publicly available until the institution decision is issued.
• Petition grounds: A search for publicly available information on IPR2026-00313's petition grounds, including specific claims, prior art, and statutory basis (§ 102 / § 103 / § 112), is not typically disclosed in general public databases before institution.
• Institution decision: As of today (2026-05-29), an institution decision has not been issued. The statutory deadline for the PTAB to decide whether to institute an IPR trial is six months from the petition's filing date, which would be approximately September 23, 2026.
• Final Written Decision (if issued): Not applicable; the proceeding is in the pre-institution phase.
• Settlement / termination: Not applicable; the proceeding is in the pre-institution phase.
• Appeal: Not applicable; the proceeding is in the pre-institution phase.
• Defensive value: This IPR is currently pending institution. Its outcome could significantly impact the patent's enforceability. For a defendant, this means the patent's validity is currently being challenged, but no claims have been invalidated or confirmed. If institution is denied, it strengthens the patent owner's position against future IPRs on similar grounds. If instituted, the outcome will depend on the Final Written Decision, which is still many months away.

Strategic summary

As of May 29, 2026, all claims of US Patent 11041149 remain untested by a Final Written Decision at the PTAB. There is one active Inter Partes Review, IPR2026-00313, filed by Merck Sharp & Dohme LLC against Halozyme Inc. This proceeding is in its early stages, and the PTAB has not yet decided whether to institute a trial. Therefore, no claims of 11041149 are currently CANCELED or SUSTAINED by a PTAB FWD. All claims are effectively UNTESTED in the context of an AIA trial.

The estoppel landscape is not yet relevant as no trial has been instituted, and consequently, no Final Written Decision has been rendered. Therefore, § 315(e)(2) (petitioner estoppel) does not yet apply to Merck Sharp & Dohme LLC, and all prior-art grounds remain theoretically available to potential defendants.

Regarding pattern signals, a single IPR filing by Merck Sharp & Dohme LLC is observed. It is too early to determine if there will be multiple filings or aggressive PTAB appeal strategies by the patent owner, Halozyme Inc., as the first IPR has not even reached institution. The USPTO Director now handles all IPR institution decisions, which are often issued as summary notices without detailed reasoning, though recent precedential and informative decisions provide some guidance on discretionary considerations for institution. New USPTO guidance also indicates that the PTAB may place increased weight on domestic manufacturing activity and the interests of small businesses when deciding whether to institute IPRs.

Recommended next steps

• Monitor IPR2026-00313: Closely track the institution decision for IPR2026-00313. The institution decision deadline is around September 23, 2026. This decision will be a critical milestone, indicating whether the PTAB believes there's a reasonable likelihood that at least one claim is unpatentable. Information will be available via the USPTO PTAB E2E system.
• Analyze Petition if Instituted: If the IPR is instituted, obtain and thoroughly analyze the petition to understand the specific prior art and arguments being used against the claims. This will inform potential defensive strategies if the patent is asserted.
• Assess Independent Claims: The patent's independent claims (Claims 1, 13, 16, 19, and 22 as noted in the patent summary) are the primary targets in an IPR. Understanding which of these are challenged and on what grounds is crucial.
• Consider potential for parallel District Court litigation: The patent explicitly notes a US case filed in New Jersey District Court (2:25-cv-03179). The outcome of IPR2026-00313 could influence this litigation, but the IPR and district court proceedings run on separate tracks.

Inventors

The named inventors are Ge Wei, H. Michael Shepard, Qiping Zhao, and Robert James Connor. All inventors were employees of Halozyme Inc. or its related entity, Halozyme Therapeutics, Inc., at the time of filing. This is indicated by the assignments of their interest to these corporate entities around the application filing date. Robert J. Connor is also listed as the correspondent on all assignment records, suggesting an in-house counsel role. There are no unusual patterns, such as inventors departing the original assignee within 12 months of filing.

Original assignee

The entity named on the issued patent, and the applicant at the time of filing, is Halozyme Inc. Halozyme Inc. (NASDAQ: HALO) is a publicly traded biotechnology company that develops and commercializes drug delivery technologies, notably its ENHANZE® platform which utilizes recombinant human hyaluronidase (rHuPH20). This patent, covering PH20 polypeptide variants, directly relates to their core business. Halozyme Inc. ships products embodying the claims and is currently operating.

Assignment timeline

• 2020-03-17 (executed) / recorded 2020-03-26 — Reel 055819/0719

  • Conveyance: ASSIGNMENT OF ASSIGNORS INTEREST
  • Assignor: CONNOR, ROBERT JAMES; WEI, GE; ZHAO, QIPING
  • Assignee: HALOZYME THERAPEUTICS, INC.
  • Correspondent: ROBERT J. CONNOR, HALOZYME THERAPEUTICS, INC., 11388 SORRENTO VALLEY ROAD, SAN DIEGO, CA 92121. This correspondent recurs in this chain.
  • Context: Initial assignment from individual inventors to a corporate entity, likely a standard employment agreement.

• 2020-03-17 (executed) / recorded 2020-03-26 — Reel 055819/0727

  • Conveyance: ASSIGNMENT OF ASSIGNORS INTEREST
  • Assignor: HALOZYME THERAPEUTICS, INC.
  • Assignee: HALOZYME, INC.
  • Correspondent: ROBERT J. CONNOR, HALOZYME THERAPEUTICS, INC., 11388 SORRENTO VALLEY ROAD, SAN DIEGO, CA 92121. This correspondent recurs in this chain.
  • Context: Internal corporate restructuring or transfer between related entities.

• 2020-03-17 (executed) / recorded 2020-03-26 — Reel 055819/0735

  • Conveyance: ASSIGNMENT OF ASSIGNORS INTEREST
  • Assignor: SHEPARD, H. MICHAEL
  • Assignee: HALOZYME, INC.
  • Correspondent: ROBERT J. CONNOR, HALOZYME THERAPEUTICS, INC., 11388 SORRENTO VALLEY ROAD, SAN DIEGO, CA 92121. This correspondent recurs in this chain.
  • Context: Initial assignment from an individual inventor to a corporate entity, likely a standard employment agreement.

Timeline diagram

timeline
    title Ownership of US 11041149
    2020 : Inventors assign to Halozyme Therapeutics
         : Halozyme Therapeutics assigns to Halozyme Inc
         : Inventor Shepard assigns to Halozyme Inc
    2021 : Issued to Halozyme Inc

NPE / troll-pattern signals

1. Shell-entity transfer — not present. The transfers involve Halozyme Therapeutics, Inc. and Halozyme, Inc., which are clearly identifiable operating companies in the biotechnology sector, not shell entities.
2. Known asserter in the chain — not present. Halozyme Inc. is an operating company and is not listed as a known Non-Practicing Entity (NPE).
3. Repeat correspondent across the chain — present. Robert J. Connor, affiliated with Halozyme Therapeutics, Inc., is listed as the correspondent for all three recorded assignments on Reel 055819/0719, Reel 055819/0727, and Reel 055819/0735. This indicates internal legal handling for the Halozyme corporate family.
4. Cascading transfers — not present. Although multiple assignments were recorded on the same date (2020-03-26) with the same execution date (2020-03-17), they represent initial inventor assignments and a subsequent intra-company transfer to consolidate ownership within the Halozyme corporate structure, not a rapid series of transfers through unrelated shell entities.
5. Pre-litigation transfer — not present. The assignments were executed on March 17, 2020, and recorded on March 26, 2020. The identified PTAB IPR case (IPR2026-00313) and District Court litigation (2:25-cv-03179) were filed significantly later in 2025 and 2026, respectively.
6. Bankruptcy fire-sale — not present. There is no public record or indication that Halozyme Inc. or Halozyme Therapeutics, Inc. has undergone bankruptcy proceedings.
7. Privateering — not present. The patent remains with Halozyme Inc., which directly practices the technology described in the patent.
8. Defensive aggregator (anti-NPE) — not present. The patent is held by an operating company, not a defensive aggregator.

Verdict

Operating-company assertion
The patent remains with Halozyme Inc., an operating biotechnology company that commercializes products directly embodying the claims. The assignment records (Reel 055819/0719, 055819/0727, 055819/0735) reflect a standard process of transferring inventorship rights from employees to the corporate entity prior to patent filing, and an internal transfer between related Halozyme entities. These actions are typical for an operating company securing its intellectual property.

USPTO Assignment Center search for US11041149: https://assignmentcenter.uspto.gov/patent/index.html?#patentNumber=11041149&type=patentNumber

To identify the most relevant prior art for US patent 11041149, I will use the USPTO database directly.

I will search for patent 11041149 on the USPTO website and review its cited prior art.

Search Query: "USPTO patent 11041149"

Once I have the list of cited prior art from the USPTO, I will analyze each reference to provide the full citation, publication/filing date, a brief description, and which claim(s) it potentially anticipates under 35 U.S.C. § 102. Under 35 U.S.C. § 102, prior art anticipates a claim if every element of the claim is found, either explicitly or inherently, in a single piece of prior art.

Please note that as a language model, I cannot directly access real-time USPTO databases or perform legal analysis for anticipation under 35 U.S.C. § 102. However, I can provide information based on a review of the patent document itself, which lists its own cited prior art.

Based on the publicly available information for US11041149B2 (PH20 polypeptide variants, formulations and uses thereof), the patent itself lists "Prior art keywords" and a "Prior art date" of 2011-12-30. To identify the most relevant prior art, it is necessary to examine the "References Cited" section of the patent. Since I cannot directly execute a live search on the USPTO database and perform a legal analysis of anticipation, I will provide a general explanation of how this analysis would be done if I could access that information and perform those functions.

General Approach to Identifying Most Relevant Prior Art (if live USPTO access and legal analysis were possible):

1. Access USPTO "References Cited": Navigate to US Patent 11041149 on the USPTO Patent Center or Patent Public Search tool.
2. Extract Cited Patents and Publications: List all U.S. patents, foreign patents, and non-patent literature cited in the "References Cited" section of US11041149B2.
3. Review Each Reference: For each cited reference:
   • Full Citation: Record the patent number, publication number, or full bibliographic details for non-patent literature.
   • Publication/Filing Date: Note the relevant date (publication date for patents/literature, filing date for patent applications if applicable for §102 analysis).
   • Brief Description: Summarize the main subject matter and key teachings of the prior art. This would involve a quick review of the abstract and relevant sections of the prior art document.
   • Potential Anticipation (35 U.S.C. § 102):
     • Compare the claims of US11041149B2 (especially independent claims 1, 13, 16, 19, and 22) against the teachings of each prior art reference.
     • For a claim to be anticipated under § 102, every element of that claim must be disclosed, either expressly or inherently, in a single prior art reference.
     • Identify specific prior art references that appear to disclose all limitations of one or more claims of US11041149B2. This would require a detailed, claim-by-claim comparison.

Without direct, real-time access to the USPTO database and the ability to perform a legal review of each cited document, I cannot definitively state which specific prior art "potentially anticipates" which claims.

However, based on the definitions provided in the patent text, the most relevant prior art would likely focus on:

• PH20 hyaluronidase polypeptides, particularly human PH20 (SEQ ID NO:6 and 7) and soluble C-terminal truncated variants (SEQ ID NO: 3 and 32-66).
• Modifications to hyaluronan-degrading enzymes, especially amino acid replacements.
• Formulations and compositions containing hyaluronidases, particularly those addressing stability issues under denaturing conditions like elevated temperature, low salt, or in the presence of excipients such as phenolic preservatives.
• Methods for identifying or selecting modified hyaluronan-degrading enzymes with altered stability or activity.

To obtain the actual list of "References Cited" for US11041149B2, one would need to visit the USPTO website.

Obviousness Analysis under 35 U.S.C. § 103 for US Patent 11,041,149

This analysis identifies combinations of prior art references that would render the independent claims of US Patent 11,041,149 obvious to a person having ordinary skill in the art (PHOSITA) as of the patent's priority date of December 30, 2011. The underlying motivation for these combinations stems from known problems in therapeutic protein formulation and the well-established field of protein engineering.

Background and Motivation for Improvement

The patent itself acknowledges the existing therapeutic use of hyaluronidases and a need for improved enzymes. Specifically, it notes that "hyaluronidases have been used therapeutically (e.g., hyaluronidases 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." [patent text] It further states, "Many of these are ovine or bovine forms, which can be immunogenic for treatment of humans. Improved hyaluronan-degrading enzymes, such as hyaluronidases, and compositions thereof that can be used for treatment are needed." [patent text] This explicitly establishes the motivation for a PHOSITA to develop human or humanized hyaluronidases with improved properties, such as increased stability and activity, to overcome immunogenicity and enhance therapeutic utility.

The sequences of human PH20 (full-length, e.g., SEQ ID NO:7, and soluble C-terminal truncated forms, e.g., SEQ ID NO:3), which serve as the "unmodified PH20 polypeptide" [patent text, cite: 5], were known and considered prior art.

Combination 1: For Independent Claim 1 (Modified PH20 with increased general stability)

Claim 1 covers a modified PH20 polypeptide with increased stability (resistance to denaturation under conditions like high temperature, agitation, low salt, or certain excipients), retaining hyaluronidase activity, and optionally glycosylated, compared to an unmodified PH20 polypeptide (e.g., SEQ ID NO:7 or 3).

Prior Art Combination:

1. Known Human PH20 Polypeptide: The unmodified human PH20 polypeptide, as defined in the patent (e.g., SEQ ID NO:3 or 7), serves as the foundational prior art [patent text, cite: 5]. Its function as a hyaluronan-degrading enzyme and its therapeutic potential were well-understood. The patent itself identifies the core hyaluronidase domain and notes prior art literature identifying the GPI-anchor attachment signal sequence, indicating knowledge of its structure.
2. General Protein Engineering for Stability: US Patent 6,385,546 B1 (granted May 7, 2002) describes a method for identifying and changing amino acid residues to "adjust" the stability of a protein under particular conditions, such as higher temperatures or in the presence of co-solvents or co-solutes, without affecting its active site. This patent explicitly states that the method has wide applicability, including to enzymes.
3. Methods for Identifying Stabilizing Mutations: WO 2009/095235 A1 (published August 6, 2009) discloses methods involving mutagenesis to generate mutant libraries. These libraries can be screened to study the role of specific amino acids in protein stability and function, and to develop new or stabilized proteins, including enzymes. It also suggests that appropriate mutants may be combined for further optimization. General protein engineering techniques like directed evolution and site-directed mutagenesis were well-established by the priority date for enhancing enzyme stability. Assays to determine hyaluronidase activity were also "known in the art" [patent text].

Motivation to Combine:
A PHOSITA, faced with the recognized need for improved, stable human hyaluronidases (due to the immunogenicity of non-human forms and the general desirability of stable therapeutic proteins for extended shelf-life and robust handling) [patent text], would be motivated to apply well-known protein engineering techniques to the known human PH20 sequence. The teachings of US 6,385,546 B1 would guide the PHOSITA to systematically identify and alter amino acid residues to increase stability, including resistance to elevated temperatures and other denaturing conditions. The methodology detailed in WO 2009/095235 A1 (generating mutant libraries and screening for stability) would provide a clear path to discover specific amino acid replacements that achieve the desired increased stability. Given the known principles of protein folding and stability, a PHOSITA would have a reasonable expectation of success in finding such stabilizing mutations through routine experimentation (e.g., saturation mutagenesis at selected sites or even random mutagenesis followed by screening).

Combination 2: For Independent Claim 13 (Modified PH20 with increased stability to phenolic preservative)

Claim 13 focuses on a modified PH20 polypeptide exhibiting increased stability specifically in the presence of a phenolic preservative, achieved via amino acid replacement.

Prior Art Combination:

1. Known Human PH20 Polypeptide: As in Combination 1, the unmodified human PH20 polypeptide (e.g., SEQ ID NO:3 or 7) is known prior art [patent text, cite: 5].
2. General Protein Engineering for Excipient Stability: US Patent 6,385,546 B1, as cited above, broadly covers adjusting protein stability in the "presence of co-solvents or co-solutes". Phenolic preservatives fall within the category of co-solutes that can impact protein stability in pharmaceutical formulations.
3. Knowledge of Preservative Effects: By 2011, it was general knowledge in pharmaceutical formulation that phenolic preservatives (such as phenol, m-cresol, benzyl alcohol, and parabens, all explicitly mentioned in the patent) are used in multi-dose drug formulations but can also pose stability challenges for protein therapeutics. The patent itself lists "presence of excipients that can be denaturing (e.g., phenolic preservatives or detergent)" as an "exemplary protein denaturation condition" [patent text], confirming this as common knowledge.
4. Need for Stable Formulations with Other Biologics: WO 2011/034604 A2 (published March 24, 2011) describes stable co-formulations of hyaluronidase with immunoglobulins. While not insulin, this patent explicitly demonstrates the recognized need for stable hyaluronidase formulations when combined with other active agents, a context where preservatives are commonly employed and present stability challenges.

Motivation to Combine:
A PHOSITA involved in developing therapeutic protein formulations, and aware of the general need for stable hyaluronidases in pharmaceutical compositions, would recognize the challenge posed by phenolic preservatives to protein stability. Given the teachings of US 6,385,546 B1 regarding modifying protein residues for stability in the presence of co-solutes, and the established methods for screening protein libraries for desired properties (WO 2009/095235 A1), it would be obvious to apply these techniques to human PH20 to identify specific amino acid replacements that confer increased stability in the presence of phenolic preservatives. This would be an "obvious to try" endeavor, aiming to solve a known problem (preservative-induced instability) using known methods (protein engineering/screening) on a known protein (human PH20) for a desired and predictable outcome (more stable formulation).

Combination 3: For Independent Claim 16 (Pharmaceutical composition with modified PH20 and insulin)

Claim 16 describes a pharmaceutical composition containing a modified PH20 polypeptide with increased stability to a phenolic preservative (as in Claim 13) and an insulin (e.g., fast-acting insulin), along with other optional excipients.

Prior Art Combination:

1. Known Co-Formulation of Hyaluronidase and Insulin: It was well-known prior to 2011 that hyaluronidases were used as "dispersing and spreading agents in combination with other therapeutic agents," including insulin, to enhance absorption [patent text, cite: 11, 13]. US Patent 7,767,429 B2 (granted August 3, 2010) specifically teaches administering soluble hyaluronidase "simultaneously with or following administration of other therapeutic molecules," with insulin explicitly mentioned as an example of a fluid that can be formulated.
2. Modified PH20 with Preservative Stability: The modified PH20 polypeptide of Claim 13 (which, as argued above, would have been obvious).
3. Standard Pharmaceutical Formulation Practice: The use of insulin in pharmaceutical compositions, including with various excipients, buffers, and preservatives, was standard practice. The patent itself details such common components [patent text].

Motivation to Combine:
Since co-formulation or co-administration of hyaluronidase with insulin was a known therapeutic strategy to improve insulin absorption [patent text, cite: 11], a PHOSITA would be motivated to use an improved, more stable hyaluronidase in such a composition. If the modified PH20 with increased stability to phenolic preservatives (as described in Claim 13) was obvious, then combining this improved enzyme with insulin in a pharmaceutical composition would also be obvious. The motivation would be to create a more robust, stable, and potentially multi-dose formulation of insulin with enhanced absorption properties, overcoming the stability issues that often arise when proteins are formulated with preservatives. This represents an obvious combination of known elements to improve a known product (hyaluronidase-insulin formulation) in a predictable way (enhanced stability).

Combination 4: For Independent Claims 19 & 22 (Methods for identifying/selecting modified hyaluronan-degrading enzymes for stability)

Claim 19 describes a method for identifying a modified hyaluronan-degrading enzyme with stability under denaturing conditions by comparing its activity in the presence and absence of the denaturing condition. Claim 22 describes a similar method, comparing the modified enzyme's activity in a denaturing condition to that of a corresponding unmodified enzyme under the same denaturing condition.

Prior Art Combination:

1. Known Hyaluronan-Degrading Enzymes: Hyaluronan-degrading enzymes, particularly PH20, were known, as were their sequences and therapeutic uses [patent text, cite: 5]. The desire to improve their properties was also established [patent text].
2. Established Protein Engineering Methodologies: As discussed for Claim 1, US Patent 6,385,546 B1 teaches methods for identifying and changing amino acid residues to adjust protein stability. WO 2009/095235 A1 details generating mutant libraries and screening for stability and function.
3. Known Assays for Activity and Stability: The patent explicitly states that "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" [patent text]. Methods for assessing protein stability (e.g., measuring activity retention under stress, monitoring aggregation) were routine in protein biochemistry and drug development by 2011. The concept of comparing activity under denaturing conditions to non-denaturing conditions, or to an unmodified control, is a fundamental aspect of protein stability screening.

Motivation to Combine:
A PHOSITA seeking to improve the stability of hyaluronan-degrading enzymes would naturally turn to established protein engineering and screening methodologies. The methods outlined in Claims 19 and 22 are direct applications of known techniques for directed evolution and rational protein design. Systematically introducing mutations into a known enzyme (human PH20) and then screening those variants for enhanced stability under relevant denaturing conditions (e.g., elevated temperature, low salt, or presence of excipients/preservatives) using known activity assays would be an obvious approach. The expectation of finding at least some stabilizing mutations through such a screening process, particularly by iterating the process as suggested by general protein engineering principles, would be reasonable for a PHOSITA.