US 12221638

Here's a concise summary of US Patent 12221638:

Title: Method for producing recombinant hyaluronidase
Assignee: Alteogen Inc.
Inventors: Soon Jae Park, Kyuwan KIM, Sang Hoon YUN, Jeong Soo CHO, Kibum PARK, Minsoo Byun, Hyung-Nam Song, Ji-Sun KIM, Ki Seok NAM
Filing Date: 2021-08-06
Issue Date: 2025-02-11
Abstract: The patent discloses a method for producing hyaluronidase or a variant thereof. This method involves culturing host cells under specific conditions, including controlling the glucose concentration in the culture medium and decreasing the culture temperature for a defined period. These conditions are designed to alter N-glycan levels, resulting in a recombinant hyaluronidase with increased specific activity (by 10% or more) and improved quality and production yield.

Independent Claims Overview:

• Claim 1 (Method Claim): This claim describes a two-step method for producing hyaluronidase PH20 or a variant.

  1. Step (1): Host cells (which express the hyaluronidase) are initially cultured at a temperature between 35° C. and 38° C. until they reach a certain "integral viable cell density" (IVCD) of 20×10⁶ to 120×10⁶ cells×day/mL.
  2. Step (2): The culture temperature is then lowered to a range of 28° C. to 34° C., and the cells are cultured for an additional 2 to 18 days. During this second step, at least one of two conditions must be maintained:
     • (a): The concentration of residual glucose in the culture medium is kept between 0.001 g/L and 4.5 g/L throughout the culture period.
     • (b): The pH of the culture medium is maintained between 6.8 and 7.2.

• Claim 11 (Product-by-Process Claim): This claim covers a hyaluronidase PH20 or a variant thereof that is produced specifically by the method of Claim 1. A key characteristic of this product is that the sialylation content of its N-glycan is between 1% and 38%.

• Claim 13 (Product Claim): This claim covers a hyaluronidase PH20 or a variant thereof, defined directly by its N-glycan characteristic: it must have a sialylation content in its N-glycan of 1% to 38%. This claim does not limit the product to a specific production method.

Litigation/Docket Information:
The patent family for US12221638 is currently involved in litigation. A PTAB (Patent Trial and Appeal Board) case, IPR2026-00176, has been filed and is pending. Additionally, a global patent litigation dataset by Darts-ip indicates that the family has litigation filed. No direct CAFC 2026 dockets were found, but the IPR proceeding is a relevant legal challenge.

Note: The numerical values for IVCD, temperature ranges, glucose concentration, pH, and glycan percentages in the claims are stated with a potential 10% error range, as noted in the patent description, due to variations in experimental conditions and measurement.

Citations:
US12221638B2, Method for producing recombinant hyaluronidase.
https://portal.unifiedpatents.com/ptab/case/IPR2026-00176

As of April 26, 2026, there is known litigation involving US patent 12221638:

1. Case Type: PTAB Inter Partes Review (IPR)
   • Case Number: IPR2026-00176
   • Jurisdiction: Patent Trial and Appeal Board (PTAB) of the USPTO
   • Filing Date: (Not explicitly stated in the provided text for this specific PTAB case, but the patent states it was "filed (Pending)").
   • Plaintiff(s): Petitioner (details not available in the provided patent text, but typically Unified Patents is a petitioner for such cases, as indicated by the link to Unified Patents portal).
   • Defendant(s): Alteogen Inc. (assignee of the patent).
   • Status: Pending.

Additionally, the patent indicates "Family has litigation" with "First worldwide family litigation filed," linking to Darts-ip.com. However, the details of this worldwide family litigation (plaintiff, defendant, jurisdiction, case number, filing date, and outcome/status) are not provided within the given patent text or the search snippets.Known litigation involving US patent 12221638 includes:

1. Case Type: Inter Partes Review (IPR) before the Patent Trial and Appeal Board (PTAB)
   • Case Number: IPR2026-00176
   • Jurisdiction: Patent Trial and Appeal Board (PTAB)
   • Filing Date: Not explicitly stated in the provided text.
   • Plaintiff(s): Petitioner (not specified in the provided text).
   • Defendant(s): Not specified in the provided text, but typically the patent owner (Alteogen Inc.) would be the respondent.
   • Status: Pending.

The patent also indicates that the "Family has litigation" and that the "First worldwide family litigation filed" is linked to Darts-ip.com. However, specific details regarding this worldwide litigation (plaintiff(s), defendant(s), jurisdiction, case number, filing date, and outcome or current status) are not available in the provided text.

Proceedings overview

A single AIA trial proceeding, IPR2026-00176, has been filed against US patent 12221638. This proceeding was denied institution, meaning the PTAB did not proceed to a full trial. This outcome strengthens the patent's defensive posture as its claims have withstood a challenge at the institution phase.

IPR2026-00176 — Halozyme, Inc. v. Alteogen Inc.

• Type: Inter Partes Review
• Filed: 2025-12-10
• Status: Institution Denied. The U.S. Patent Tribunal (PTAB) rejected the petition to initiate inter partes review.
• Judge panel: The specific Administrative Patent Judge (APJ) panel for this proceeding is not publicly available from the provided information. Records indicate a "Pending Judge Assignment" as of 2025-12-16.
• Petition grounds: Halozyme, Inc. challenged US Patent No. 12,221,638, which is a manufacturing patent for a method of producing recombinant hyaluronidase, targeting its novelty and progressiveness based on prior art and public literature. The specific claims challenged and the statutory basis (§ 102 / § 103) were not detailed in the available information, beyond a general challenge to "at least one claim".
• Institution decision: Denied on 2026-05-15. The PTAB determined that Halozyme's petition failed to demonstrate a "reasonable possibility of invalidating at least one claim," concluding that the prior art and public literature presented did not sufficiently show the possibility of destroying the novelty or progressiveness of Alteogen's patent. An Alteogen official stated that this was a substantive review of the prior technology and invalidity claims, not a simple procedural dismissal.
• Final Written Decision: Not applicable, as institution was denied.
• Settlement / termination: Not applicable.
• Appeal: Not applicable, as institution was denied. Decisions denying institution are generally non-appealable.
• Defensive value: This decision provides significant defensive value for Alteogen. The patent owner successfully defended against a direct challenge from a competitor, Halozyme, at the institution stage. This means all claims of US12221638 remain unadjudicated and presumed valid by the PTAB against the specific prior art and arguments raised in this petition, making future IPR challenges on similar grounds significantly more difficult for the same petitioner.

Strategic summary

US patent 12221638 has faced one AIA trial proceeding, IPR2026-00176, filed by Halozyme, Inc. The PTAB denied institution of this IPR on 2026-05-15. Consequently, all claims of US12221638 remain UNTESTED in a full PTAB trial. No claims have been canceled or invalidated through an AIA proceeding, and all claims are considered SUSTAINED as issued by the USPTO.

Regarding estoppel, the denial of institution for IPR2026-00176 generally means that statutory estoppel under 35 U.S.C. § 315(e)(2) does not apply to the petitioner (Halozyme, Inc.) or its privies. This is because a full trial was not instituted, and thus no final written decision on the patentability of claims was issued. Therefore, the specific prior art grounds raised in Halozyme's petition could theoretically still be raised in district court litigation by Halozyme, though the PTAB's finding of "no reasonable likelihood of success" may serve as a practical deterrent. All other prior-art grounds that could have been raised in an IPR petition are also still available to other potential challengers not in privity with Halozyme.

The current PTAB activity on US12221638 shows no discernible pattern of repeated challenges or aggressive appeals by the patent owner. Only one IPR has been filed, and it resulted in a favorable outcome for the patent owner, Alteogen, Inc., with institution being denied. This indicates that Alteogen has successfully defended its patent against an initial validity challenge from a competitor, suggesting the patent is robust against the arguments presented.

Recommended next steps

As a defendant facing assertion of US patent 12221638, it is important to note that all claims of the patent are currently SUSTAINED and have not been invalidated by the PTAB. The denial of institution in IPR2026-00176 means that the specific arguments and prior art presented by Halozyme, Inc. were deemed insufficient to establish a reasonable likelihood of success for invalidating any claim.

Since institution was denied, there is no Final Written Decision to link to. However, the decision denying institution for IPR2026-00176 (likely issued by the Director of the USPTO or a delegated panel on 2026-05-15) would be the key document. It is advisable to review the full institution denial decision to understand the specific reasoning and the prior art found insufficient. This will inform whether a new IPR petition, perhaps on different grounds or with stronger arguments, could be viable. The USPTO's PTAB Decisions portal or the Unified Patents portal (for IPR2026-00176) may contain the full decision.

Inventors

• Soon Jae Park (Alteogen Inc.)
• Kyuwan KIM (Alteogen Inc.)
• Sang Hoon YUN (Alteogen Inc.)
• Jeong Soo CHO (Alteogen Inc.)
• Kibum PARK (Alteogen Inc.)
• Minsoo Byun (Alteogen Inc.)
• Hyung-Nam Song (Alteogen Inc.)
• Ji-Sun KIM (Alteogen Inc.)
• Ki Seok NAM (Alteogen Inc.)

All inventors appear to have been employed by Alteogen Inc. at the time of filing, as indicated by the application being filed by Alteogen Inc. and subsequent assignments of interest from the inventors to Alteogen Inc. There are no unusual patterns suggesting inventor departures or a portfolio fire-sale.

Original assignee

The original assignee on the issued patent is Alteogen Inc..
Alteogen Inc. is a South Korea-based biotechnology company founded in 2008, focused on the research, development, and commercialization of novel biologics, including biobetters and biosimilars, utilizing proprietary platform technologies such as NexP™ Fusion Technology, NexMab™ ADC Technology, and Hybrozyme™ Technology.
Yes, Alteogen Inc. ships products embodying the claims, specifically a recombinant human hyaluronidase (Hybrozyme™ / Tergase®).
Alteogen Inc. is currently operating and active, with a market capitalization of $13 billion as of May 2026.

Assignment timeline

The detailed assignment records including reel/frame and correspondent information from the USPTO Assignment Center were not provided in the prompt and could not be accessed. However, Google Patents legal events indicate the following reassignment events to the original assignee:

• NOT PROVIDED (executed) / recorded 2022-10-15 — Reel NOT PROVIDED/NOT PROVIDED
  • Conveyance: Assignment of Assignors Interest
  • Assignor: BYUN, Minsoo, CHO, Jeong Soo, KIM, JI-SUN, KIM, Kyuwan, NAM, KI SEOK, PARK, Kibum, PARK, SOON JAE, SONG, Hyung Nam, YUN, SANG HOON (inventors)
  • Assignee: ALTEOGEN INC.
  • Correspondent: NOT PROVIDED
  • Context: Internal assignment from inventors to the original assignee.
• NOT PROVIDED (executed) / recorded 2024-09-04 — Reel NOT PROVIDED/NOT PROVIDED
  • Conveyance: Corrective Assignment
  • Assignor: BYUN, Minsoo, CHO, Jeong Soo, KIM, JI-SUN, KIM, Kyuwan, NAM, KI SEOK, PARK, Kibum, PARK, SOON JAE, SONG, Hyung-Nam, YUN, SANG HOON (inventors)
  • Assignee: ALTEOGEN INC.
  • Correspondent: NOT PROVIDED
  • Context: Corrective assignment of inventors' interest to the original assignee.

Timeline diagram

timeline
    title Ownership of US 12221638
    2021 : Application filed by Alteogen Inc
    2022 : Inventors assign to Alteogen
    2024 : Corrective assignment to Alteogen
    2025 : Patent granted

NPE / troll-pattern signals

1. Shell-entity transfer — not present. The assignments are from the inventors to Alteogen Inc., which is an operating company producing and commercializing biopharmaceutical products.
2. Known asserter in the chain — not present. Alteogen Inc. is not identified as a known NPE.
3. Repeat correspondent across the chain — unclear. Correspondent information (name, firm, address) for the recorded assignments was not provided in the available data.
4. Cascading transfers — not present. There are only two assignments recorded, both involving the inventors and the original assignee, Alteogen Inc., and separated by nearly two years. These are not transfers through chained LLCs.
5. Pre-litigation transfer — not present. There is no indication of litigation for this patent in the provided information, and the assignments are internal from inventors to the company.
6. Bankruptcy fire-sale — not present. Alteogen Inc. is an active, operating company.
7. Privateering — not present. There is no evidence of Alteogen Inc. transferring the patent to an NPE for assertion on its behalf.
8. Defensive aggregator (anti-NPE) — not present. The patent is currently assigned to Alteogen Inc., an operating company.

Verdict

Insufficient data.
While Alteogen Inc. appears to be an operating company, the absence of detailed assignment records including reel/frame numbers and correspondent information prevents a full and confident assessment of NPE patterns, particularly those relying on correspondent identities. The only recorded events are initial and corrective assignments from the inventors to the original assignee.

Verification on USPTO Patent Assignment Search

US patent 12221638, titled "Method for producing recombinant hyaluronidase," was filed on August 6, 2021, and published on February 11, 2025. It claims priority from Korean Patent Application No. 10-2020-0099100, filed on August 7, 2020. The patent describes a method for producing hyaluronidase PH20 or variants thereof with improved enzymatic activity and productivity by controlling N-glycosylation levels under specific culture conditions, including controlled glucose concentration and a decreased culture temperature for a specific period.

Here's an analysis of the most relevant prior art cited in US patent 12221638:

1. US Patent No. 9,447,401

• Full Citation: U.S. Pat. No. 9,447,401.
• Publication/Filing Date: The full text of US12221638 mentions this patent in the context of human-derived "hyaluronidase PH20" or "PH20 variant" being exemplified. While the exact filing/publication dates for US9447401 aren't directly provided in the text of US12221638, US12221638's priority date is August 7, 2020. Therefore, US9447401 would have a publication date prior to this.
• Brief Description: US12221638 states that US9447401 exemplifies human-derived hyaluronidase PH20 or PH20 variants, which include substitutions, deletions, and insertions of one or more amino acid residues, and optionally truncations of N-terminal and/or C-terminal amino acid residues, while retaining hyaluronidase enzymatic activity.
• Potential Anticipation: This patent potentially anticipates claims related to the basic structure and function of hyaluronidase PH20 variants. Specifically, it could be relevant to claims that broadly define the hyaluronidase PH20 protein or its variants, such as those that describe the enzyme itself rather than the specific production method. Claims 1, 2, and any other claims that broadly define the recombinant hyaluronidase PH20 or its variants, particularly the sialylation, galactosylation, and mannosylation contents, could be at least partially anticipated if US9447401 discloses hyaluronidase PH20 with N-glycan characteristics falling within the ranges claimed in US12221638.

2. International Patent Publication No. 2020/022791

• Full Citation: International Patent Publication No. 2020/022791.
• Publication/Filing Date: The full text of US12221638 mentions this international publication in the same context as US9447401, referring to it as exemplifying human-derived "hyaluronidase PH20" or "PH20 variant." The publication number itself indicates a publication year of 2020. Since US12221638 claims priority to a Korean application filed on August 7, 2020, International Patent Publication No. 2020/022791 would have a publication date on or before this date to be considered prior art.
• Brief Description: Similar to US9447401, this international publication is cited in US12221638 as exemplifying human-derived hyaluronidase PH20 or variants with amino acid modifications and hyaluronidase activity.
• Potential Anticipation: This international patent publication also potentially anticipates claims related to the composition of hyaluronidase PH20 variants, particularly if it describes such variants with N-glycan profiles or enzymatic activities similar to those claimed in US12221638. Claims related to the recombinant hyaluronidase PH20 or its variants and their N-glycan characteristics (sialylation, galactosylation, mannosylation) could be potentially anticipated.

3. Arming et al., "In vitro mutagenesis of PH-20 hyaluronidase from human sperm", Eur. J. Biochem, 1997, pp. 810-814, vol. 247.

• Full Citation: Arming, S., et al., "In vitro mutagenesis of PH-20 hyaluronidase from human sperm", Eur. J. Biochem, 1997, pp. 810-814, vol. 247.
• Publication Date: 1997.
• Brief Description: This reference is cited in US12221638 as having "revealed that an arginine residue having positive charges in hyaluronidase PH20 is essential for enzymatic activity for binding to hyaluronic acid, which is a substrate having a large amount of negative charges distributed therein".
• Potential Anticipation: This publication provides foundational knowledge about the enzymatic activity of PH20 hyaluronidase and the role of specific amino acid residues. While it focuses on in vitro mutagenesis and the mechanism of activity, it could potentially anticipate claims that generally describe the enzymatic activity of hyaluronidase PH20 or its variants, especially if such claims do not specifically tie the activity to the novel production methods or specific N-glycan profiles of US12221638. It might anticipate aspects of the problem definition or the general understanding of hyaluronidase function, but less likely the specific method claims or the resulting glycan profiles.

4. Bookbinder et al., 2006

• Full Citation: Bookbinder et al., 2006.
• Publication Date: 2006.
• Brief Description: This reference is cited multiple times in US12221638. It discusses hyaluronidases degrading hyaluronic acid to reduce viscosity and increase tissue permeability, their use in enhancing absorption of body fluids, improving diffusion of local anesthetics, and mentions the recombinant human PH20 protein developed by Halozyme Therapeutics Inc. and sold as "Hylenex."
• Potential Anticipation: This reference provides a general overview of hyaluronidase function, therapeutic uses, and the existence of recombinant human PH20 (Hylenex). It establishes the background knowledge in the field. It could potentially anticipate claims that broadly describe the application or general characteristics of recombinant human PH20. However, it is unlikely to anticipate the specific culture methods, N-glycan profiles, or enhanced activity described in the claims of US12221638, as the patent aims to improve upon existing production methods and resulting protein characteristics.

5. Other General References Cited in the Background
The background section of US12221638 also lists several other academic papers that describe the general state of the art regarding hyaluronidase uses, glycosylation processes, and factors affecting N-glycosylation in cell culture:

• Muchmore et al., 2012
• Krantz et al., 2016
• Clement et al., 2003
• Thomas et al., 2009
• Harb et al., 2010
• Wasserman et al., 2014
• Harris R J et al., 2004
• Schilling, et al., 2002
• Tachibana et al., 1994
• Restelli et al., 2006
• Borys et al., 1993
• Borys et al., 1994
• Clark et al., 2004
• Alan Fersht (1977). Enzyme structure and mechanism

These references contribute to the understanding of the general knowledge base at the time of the invention but are less likely to directly anticipate specific claims of US12221638, which focuses on a novel method for producing hyaluronidase with improved characteristics through specific culture condition manipulation and resulting N-glycan profiles. They establish the technical field and the problems US12221638 seeks to address.

It is important to note that a recent development (May 15, 2026) indicates that the USPTO denied an Inter Partes Review (IPR) petition filed by Halozyme against US Patent No. 12,221,638. Halozyme had argued that Alteogen's "temperature-shift cultivation method" was known in the prior art, but the PTAB found that Halozyme failed to demonstrate a "reasonable likelihood of success" on any of the challenged claims. This suggests that the USPTO considered the prior art cited by Halozyme (which would likely include many of the above) and found it insufficient to invalidate the claims of US12221638.

Obviousness Analysis under 35 U.S.C. § 103 for US12221638

This analysis considers the obviousness of US Patent 12221638 under 35 U.S.C. § 103, based on the prior art references explicitly cited within the patent text. The core invention relates to a method for producing recombinant hyaluronidase PH20 or variants with improved enzymatic activity and productivity by controlling N-glycan levels through specific cell culture conditions.

Identification of Key Prior Art Teachings

The patent itself identifies several relevant pieces of prior art and the problems they address:

1. Recombinant Hyaluronidase PH20 and its Importance: Recombinant human PH20 protein, active at neutral pH, was developed by Halozyme Therapeutics Inc. and sold as "Hylenex" (Bookbinder et al., 2006). This establishes the existence and commercial importance of recombinant PH20. Production in animal cells, such as Chinese Hamster Ovary (CHO) cells, is a well-known method for producing biopharmaceuticals, especially when complex and human-like glycosylation is desired. The patent explicitly states that CHO cells are "particularly preferably" used for mass expression.
2. Impact of N-Glycosylation on Protein Activity and Side Effects: The patent acknowledges that recombinant PH20 proteins produced in insect cells and yeast "differ from human PH20 in terms of the pattern of N-glycosylation during post-translational modification, thus affecting the activity thereof and entailing the risk of side effects occurring in the body." [cite: "The recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in terms of the pattern of N-glycosylation during post-translational modification, thus affecting the activity thereof and entailing the risk of side effects occurring in the body."] It also states, "N-glycosylation may greatly affect the folding or activity of proteins". [cite: "N-glycosylation may greatly affect the folding or activity of proteins, and there is a very high possibility that the presence of glycosylation and the structure or form of glycans may vary depending on the host cell type, recombinant manipulation method, and culture conditions (Schilling, et al., 2002) when producing proteins or variants thereof present in nature using genetic engineering methods for industrial application."]
3. Role of Charge and Sialylation in Hyaluronidase Activity: Arming et al. (1997) revealed that "an arginine residue having positive charges in hyaluronidase PH20 is essential for enzymatic activity for binding to hyaluronic acid, which is a substrate having a large amount of negative charges distributed therein". [cite: "Arming et al. revealed that an arginine residue having positive charges in hyaluronidase PH20 is essential for enzymatic activity for binding to hyaluronic acid, which is a substrate having a large amount of negative charges distributed therein (Arming et al. 1997)."] The patent further infers that "the charge distribution of the N-glycan also affects such enzymatic activity." [cite: "Therefore, it can be inferred that the charge distribution of the N-glycan also affects such enzymatic activity."] Crucially, the patent states, "It is important to prove that when hyaluronic acid, which is a substrate having a large amount of negative charges, binds to a hyaluronidase, the level of negatively charged sialic-acid-capping sugars in the N-glycans, that is, the sialylation level, affects the formation of the enzyme-substrate complex or the progress of the enzymatic reaction. In order to limit the sialylation level, the transfer of sialic acid to the galactose residue should be limited, desialylation should be performed, or the galactosylation level should be limited." [cite: "It is important to prove that when hyaluronic acid, which is a substrate having a large amount of negative charges, binds to a hyaluronidase, the level of negatively charged sialic-acid-capping sugars in the N-glycans, that is, the sialylation level, affects the formation of the enzyme-substrate complex or the progress of the enzymatic reaction." and "In order to limit the sialylation level, the transfer of sialic acid to the galactose residue should be limited, desialylation should be performed, or the galactosylation level should be limited."] This provides a clear motivation to limit sialylation.
4. Influence of Culture Conditions on N-Glycosylation: The patent lists several culture conditions known to affect N-glycosylation, including "glucose or glutamine concentration in the culture medium (Tachibana et al. 1994), concentration of dissolved oxygen (DO) (Restelli et al. 2006), culture medium pH (Borys et al. 1993), concentration of ammonia of culture medium (Borys et al. 1994), culture temperature (Clark et al. 2004), and the like." [cite: "Culture conditions affecting N-glycosylation include glucose or glutamine concentration in the culture medium (Tachibana et al. 1994), concentration of dissolved oxygen (DO) (Restelli et al. 2006), culture medium pH (Borys et al. 1993), concentration of ammonia of culture medium (Borys et al. 1994), culture temperature (Clark et al. 2004), and the like."] Schilling et al. (2002) generally supports that glycosylation patterns can vary depending on host cell type, recombinant manipulation, and culture conditions. [cite: "N-glycosylation may greatly affect the folding or activity of proteins, and there is a very high possibility that the presence of glycosylation and the structure or form of glycans may vary depending on the host cell type, recombinant manipulation method, and culture conditions (Schilling, et al., 2002) when producing proteins or variants thereof present in nature using genetic engineering methods for industrial application."]

Obviousness Argument for Claims 1, 11, and 13

A person having ordinary skill in the art (PHOSITA) in the field of biopharmaceutical manufacturing, seeking to optimize recombinant hyaluronidase PH20 (rHuPH20) activity and productivity, would have found the claimed methods and products obvious in light of the cited prior art.

Motivation to Combine References:

The motivation stems from the recognized problems and existing knowledge:

• Need for improved rHuPH20: rHuPH20 is an important therapeutic agent (Bookbinder et al., 2006), but producing it with optimal activity and avoiding side effects due to aberrant glycosylation, especially from non-mammalian hosts, is a known challenge. [cite: 1, 2, 3, 4, "The recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in terms of the pattern of N-glycosylation during post-translational modification, thus affecting the activity thereof and entailing the risk of side effects occurring in the body."] Mammalian cells, particularly CHO cells, are a preferred expression system to achieve desired glycosylation patterns for biopharmaceuticals.
• Controlling N-glycosylation for activity: It was widely known that N-glycosylation significantly affects protein activity and folding (Schilling et al., 2002). [cite: "N-glycosylation may greatly affect the folding or activity of proteins, and there is a very high possibility that the presence of glycosylation and the structure or form of glycans may vary depending on the host cell type, recombinant manipulation method, and culture conditions (Schilling, et al., 2002) when producing proteins or variants thereof present in nature using genetic engineering methods for industrial application."]
• Specific desire to limit sialylation for hyaluronidase activity: Arming et al. (1997) highlighted the critical role of charge in PH20's interaction with the negatively charged hyaluronic acid substrate. [cite: "Arming et al. revealed that an arginine residue having positive charges in hyaluronidase PH20 is essential for enzymatic activity for binding to hyaluronic acid, which is a substrate having a large amount of negative charges distributed therein (Arming et al. 1997)."] The patent itself clearly articulates that the level of negatively charged sialic acid in N-glycans impacts enzyme-substrate complex formation and enzymatic reaction, and therefore, limiting sialylation is a desired goal to improve activity. [cite: "It is important to prove that when hyaluronic acid, which is a substrate having a large amount of negative charges, binds to a hyaluronidase, the level of negatively charged sialic-acid-capping sugars in the N-glycans, that is, the sialylation level, affects the formation of the enzyme-substrate complex or the progress of the enzymatic reaction." and "In order to limit the sialylation level, the transfer of sialic acid to the galactose residue should be limited, desialylation should be performed, or the galactosylation level should be limited."]
• Known methods to modulate N-glycosylation: The prior art unequivocally established that culture conditions, including temperature (Clark et al., 2004), glucose concentration (Tachibana et al., 1994), and pH (Borys et al., 1993), directly influence N-glycosylation patterns. [cite: "Culture conditions affecting N-glycosylation include glucose or glutamine concentration in the culture medium (Tachibana et al. 1994), concentration of dissolved oxygen (DO) (Restelli et al. 2006), culture medium pH (Borys et al. 1993), concentration of ammonia of culture medium (Borys et al. 1994), culture temperature (Clark et al. 2004), and the like."]

Obviousness of Claim 1 (Method Claim):

A PHOSITA, motivated to produce a more active rHuPH20 by limiting its sialylation, would have reasonably looked to manipulate known culture conditions, such as temperature, glucose concentration, and pH, which were already known to influence N-glycosylation. The two-step method described in Claim 1 – an initial culture phase at 35-38°C to a certain cell density, followed by a temperature shift to 28-34°C for an extended period (2-18 days), while simultaneously controlling residual glucose (0.001-4.5 g/L) or pH (6.8-7.2) – represents a combination of known techniques for optimizing recombinant protein production and glycosylation.

Temperature shifts, particularly lowering temperature during the production phase, are a common strategy in mammalian cell culture to reduce metabolic rate, increase cell viability, and improve recombinant protein yield and quality, including glycosylation. Similarly, controlling nutrient levels like glucose and maintaining pH are standard parameters adjusted in bioprocess optimization to impact cell metabolism and post-translational modifications. For instance, Aghamohseni et al. (2014) showed that reducing average culture pH resulted in lower growth but higher sialylation and galactosylation levels for a monoclonal antibody. Conversely, the patent aims for lower sialylation, and adjusting these parameters to achieve that specific outcome is within the realm of routine experimentation given the known effects.

Therefore, combining the teachings that: (1) rHuPH20 activity is linked to charge distribution and sialylation (Arming et al., 1997, and the patent's own problem statement) [cite: "Arming et al. revealed that an arginine residue having positive charges in hyaluronidase PH20 is essential for enzymatic activity for binding to hyaluronic acid, which is a substrate having a large amount of negative charges distributed therein (Arming et al. 1997). Therefore, it can be inferred that the charge distribution of the N-glycan also affects such enzymatic activity." and "It is important to prove that when hyaluronic acid, which is a substrate having a large amount of negative charges, binds to a hyaluronidase, the level of negatively charged sialic-acid-capping sugars in the N-glycans, that is, the sialylation level, affects the formation of the enzyme-substrate complex or the progress of the enzymatic reaction."], and (2) culture conditions like temperature, glucose, and pH modulate N-glycosylation (Tachibana et al., 1994; Borys et al., 1993; Clark et al., 2004) [cite: "Culture conditions affecting N-glycosylation include glucose or glutamine concentration in the culture medium (Tachibana et al. 1994), concentration of dissolved oxygen (DO) (Restelli et al. 2006), culture medium pH (Borys et al. 1993), concentration of ammonia of culture medium (Borys et al. 1994), culture temperature (Clark et al. 2004), and the like."], would have led a PHOSITA to the claimed method through predictable and routine experimentation. The specific numerical ranges for temperature, IVCD, culture duration, glucose concentration, and pH, while presented as optimal, would be discoverable through standard process development efforts, not requiring inventive ingenuity beyond what is taught in the prior art.

Obviousness of Claim 11 (Product-by-Process Claim):

If the method of Claim 1 is deemed obvious, then the hyaluronidase PH20 or variant produced by that method, as claimed in Claim 11, would also be obvious. The product's characteristic of a 1-38% sialylation content is directly achieved by the obvious method.

Obviousness of Claim 13 (Product Claim):

Claim 13 covers a hyaluronidase PH20 or variant defined by a sialylation content in its N-glycan of 1% to 38%. Given the clear motivation in the prior art to limit sialylation for improved hyaluronidase activity, a PHOSITA would have sought to produce such a molecule. The ability to control glycosylation through known culture parameters makes achieving a target sialylation range an expected outcome of optimization, rather than an unpredictable discovery. Therefore, the product itself, characterized by a specific range of sialylation to achieve a desired functional improvement that was motivated by the prior art, would be obvious. The "unexpectedly high levels" of activity claimed by the patent would need to be sufficiently striking and beyond the predictable range of optimization to overcome this obviousness challenge. However, the fundamental concept of modulating sialylation to affect enzyme activity was already understood. [cite: "Arming et al. revealed that an arginine residue having positive charges in hyaluronidase PH20 is essential for enzymatic activity for binding to hyaluronic acid, which is a substrate having a large amount of negative charges distributed therein (Arming et al. 1997). Therefore, it can be inferred that the charge distribution of the N-glycan also affects such enzymatic activity." and "It is important to prove that when hyaluronic acid, which is a substrate having a large amount of negative charges, binds to a hyaluronidase, the level of negatively charged sialic-acid-capping sugars in the N-glycans, that is, the sialylation level, affects the formation of the enzyme-substrate complex or the progress of the enzymatic reaction."]