Obviousness

Obviousness Analysis under 35 U.S.C. § 103 for US7704721

Under 35 U.S.C. § 103, a patent claim is obvious if "the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains."

Claim 1 of US7704721 details a method for preventing aggregation of recombinant adeno-associated virus (rAAV) virions in a purified preparation. Key features include:

1. Providing a lysate comprising rAAV virions.
2. Purifying rAAV virions from the lysate using ultracentrifugation and/or chromatography.
3. Adding one or more salts of multivalent ions (citrate, phosphate, sulfate, magnesium) to achieve an ionic strength of at least 200 mM.
4. The preparation has a high concentration of rAAV virions (exceeding 1×10^13 vg/ml up to 6.4×10^13 vg/ml).
5. The pH of the preparation is between 7.5 and 8.0.

An analysis of the prior art cited within US7704721 suggests that the claimed method, particularly Claim 1, would have been obvious to a person having ordinary skill in the art (POSA) by combining existing knowledge and routine experimentation.

Combinations of Prior Art References and Motivation to Combine

A primary combination of references that would render Claim 1 obvious includes:

1. Xie, Q. et al. (2004) J. Virol. Methods 122: 17-27 ("Xie 2004")
2. Huang, J. et al. (2000) Mol. Therapy 1: S286 ("Huang 2000")
3. Qu, G. et al. (2003) Mol. Therapy 7: S238 ("Qu 2003")
4. U.S. Pat. No. 6,593,123 ("US '123")
5. Matsushita, T. et al. (1998) Gene Therapy 5: 938-945 ("Matsushita 1998")

Motivation to Combine:
The overarching motivation for a POSA would be to develop stable, highly concentrated, and physiologically compatible rAAV formulations for gene therapy applications, as AAV aggregation was a known and significant problem impacting purification, storage, potency, and safety.

Detailed Obviousness Argument:

• Steps 1 & 2: Providing and Purifying rAAV Virions

  • The initial steps of providing a lysate comprising rAAV virions and purifying them using ultracentrifugation and/or chromatography were well-established in the art. Matsushita 1998 describes the production of AAV2 vectors by triple transfection of HEK293 cells, which generates a lysate. US '123 teaches large-scale rAAV production and purification using cation exchange chromatography. The patent itself references these and other conventional purification methods (e.g., CsCl gradient ultracentrifugation). A POSA would routinely employ these known methods to obtain purified rAAV virions.

• Step 3: Adding Salts of Multivalent Ions to Achieve High Ionic Strength at High Concentration and Specific pH

  • Problem of Aggregation at High Concentrations: Huang 2000 reported that AAV vectors undergo concentration-dependent aggregation. Xie 2004 similarly reported that at concentrations exceeding 0.1 mg/mL, AAV2 vectors require "elevated concentrations of salt to prevent aggregation." This reference also demonstrated the feasibility of concentrating AAV2 to very high levels (4.4 to 18×10^14 particles/ml) using 25% (w/v) glycerol. Thus, the problem of aggregation in concentrated rAAV preparations and the general solution of using "elevated concentrations of salt" were known.

  • Use of Multivalent Ions: The patent itself acknowledges the general knowledge that "Salt species with multiple charge valencies (e.g. salts of sulfate, citrate, and phosphate) that are commonly used as excipients in human parenteral formulations can provide the level of ionic strength needed to prevent AAV2 vector aggregation when used at isotonic concentrations." Magnesium sulfate, sodium citrate, sodium phosphate, and sodium sulfate are explicitly listed in the patent's Table 1 and discussed in Figure 1A/B as excipients evaluated for preventing aggregation. Given the teaching from Xie 2004 regarding the need for "elevated concentrations of salt," and the common use of these specific multivalent ion salts as excipients for achieving high ionic strength while maintaining isotonicity in parenteral formulations, a POSA would have been motivated to test these known agents to address AAV aggregation.

  • Ionic Strength of at least 200 mM: Building on the general teaching of "elevated concentrations of salt" from Xie 2004, a POSA would engage in routine optimization to determine the effective concentration of various salts required to prevent aggregation in highly concentrated AAV preparations. The patent's own Figure 1B illustrates that "vector aggregation is prevented when ionic strength is ˜200 mM or greater regardless of which salt is used." This indicates that reaching an ionic strength of at least 200 mM was a predictable outcome of increasing salt concentrations to prevent aggregation, rather than a surprising discovery.

  • High Concentration of rAAV Virions: The target concentration range (exceeding 1×10^13 vg/ml up to 6.4×10^13 vg/ml) is encompassed by, and even lower than, the "very high concentrations" (up to 18×10^14 particles/ml) taught as achievable by Xie 2004. The desire to formulate highly concentrated vectors for clinical administration, particularly to sites like the central nervous system, was a recognized need.

  • pH between 7.5 and 8.0: Qu 2003 reported that AAV2 vector aggregation is pH dependent. Optimizing the pH for stability of biological products like viruses is a routine aspect of formulation development. A pH range of 7.5-8.0 is a common physiological or slightly alkaline pH range used in many biological buffers (e.g., Tris buffer pH 8.0 is used in the patent's own TF2 formulation), and a POSA would routinely test within such ranges to enhance stability.

Conclusion:
A POSA, aware of the pervasive problem of AAV aggregation at high concentrations (Huang 2000, Xie 2004), the need for elevated salt concentrations (Xie 2004), the pH-dependent nature of aggregation (Qu 2003), and equipped with standard purification techniques (Matsushita 1998, US '123), would have been motivated to combine these teachings. Furthermore, given the common knowledge (as articulated within US7704721 itself) that multivalent ion salts are effective excipients for achieving high ionic strength in isotonic parenteral formulations, a POSA would have routinely experimented with these salts. Determining the specific ionic strength threshold (at least 200 mM) and an optimal pH range (7.5-8.0) would have been a matter of routine optimization, resulting in the claimed method. Therefore, Claim 1 of US7704721 would have been obvious.