Invented by Yow-Pin Lim, Edward S. Sirya, Peter Brne, Prothera Biologics Inc

The market for the preparation of inter-alpha inhibiting proteins from blood and their composition is a rapidly growing sector in the biopharmaceutical industry. Inter-alpha inhibiting proteins (IAIPs) are a group of plasma proteins that play a crucial role in various physiological processes, including inflammation, immune response, and tissue repair. The therapeutic potential of IAIPs has garnered significant attention, leading to increased demand for their production and formulation. IAIPs are primarily derived from human blood plasma, making the collection and purification of these proteins a critical step in their preparation. The market for blood plasma collection has witnessed substantial growth in recent years, driven by the rising demand for plasma-derived therapies. Plasma collection centers, both commercial and non-profit, have emerged to meet this demand and ensure a steady supply of blood plasma for IAIP production. Once collected, the blood plasma undergoes a series of purification steps to isolate IAIPs. These purification processes involve techniques such as fractionation, chromatography, and filtration, which separate IAIPs from other plasma proteins and impurities. The market for purification technologies has seen significant advancements, with companies developing innovative methods to improve the efficiency and yield of IAIP purification. The composition of IAIPs is another crucial aspect of their market. IAIPs are composed of multiple protein subunits, including heavy chains, light chains, and glycosaminoglycans (GAGs). The specific composition of IAIPs can vary depending on factors such as age, health condition, and genetic background. Understanding the composition of IAIPs is essential for their formulation and subsequent therapeutic applications. The market for IAIP formulation is witnessing remarkable growth, driven by the increasing demand for IAIP-based therapies. Formulation involves the development of stable and bioactive IAIP products that can be administered through various routes, including intravenous, subcutaneous, and intramuscular. Companies are investing in research and development to optimize IAIP formulations, ensuring their safety, efficacy, and long-term stability. The therapeutic potential of IAIPs is vast, with applications in various medical fields. IAIP-based therapies have shown promise in treating inflammatory diseases, autoimmune disorders, and tissue injuries. Additionally, IAIPs have demonstrated potential as biomarkers for diagnosing certain diseases and monitoring treatment response. The expanding scope of IAIP applications is driving the market growth, attracting investments from pharmaceutical companies and research institutions. The market for the preparation of IAIPs from blood and their composition is highly competitive, with several key players operating in the industry. These companies are focused on expanding their production capacities, improving purification techniques, and developing novel formulations to gain a competitive edge. Collaborations and partnerships between industry players and research institutions are also common, facilitating the exchange of knowledge and resources to accelerate IAIP development. In conclusion, the market for the preparation of inter-alpha inhibiting proteins from blood and their composition is experiencing significant growth due to the increasing demand for IAIP-based therapies. The collection and purification of IAIPs from human blood plasma, along with the formulation of stable and bioactive IAIP products, are key areas of focus for industry players. With the expanding therapeutic potential of IAIPs, this market is poised for further expansion and innovation in the coming years.

The Prothera Biologics Inc invention works as follows

The present invention provides general processes for purification and compositions of Inter-alpha inhibiting proteins (I?Ip), from blood.

Background for Preparation of inter-alpha inhibiting proteins from blood and their composition

Both “Sepsis” and “Systemic Inflammatory Response Syndrome” (SIRS) refer to a severe, biochemical reaction that occurs after exposure to an infectious agent. Anthrax or a bacterial toxin. Septic shock is caused by the systemic response, characterized as a sudden drop in blood pressure and/or cardiovascular collapse. The mortality rate for those diagnosed with septica shock is higher than breast, colon or prostate cancer, despite the introduction of antibiotics more than fifty years ago. In the U.S., there are about 800,000 cases of sepsis per year, which cost $17 billion. The same number of cases occur in other parts of the world. The global incidence of sepsis and SIRS is increasing due to the antibiotic resistance and biological threats. The population ‘at risk’ The?at risk? The mortality rate is expected to be higher in bioterrorism and battlefield exposure. It has been hard to treat sepsis and SIRS with conventional drugs.

The inter-alpha inhibit protein (I-Ip) is a family of serine protease-inhibitors that modulate the systemic inflammation associated with sepsis and other infections, traumas, and injuries. The inter-alpha inhibit protein (IIp) was shown to improve survival and condition in test animals with sepsis, anthrax infection, Ebola virus, Dengue virus, or lung injury caused by toxic chemicals or radiation. I?Ip, a large blood protein, is isolated. “Because inter-alpha inhibiting proteins are used to treat sepsis, SIRS and other diseases, it is urgently necessary to purify or prepare I?Ip.

The present invention is described as follows: “As described herein, the present technology relates to the purification of interalpha inhibitors (I?Ip), and their use in treating disease or symptoms of such diseases including sepsis or acute inflammatory conditions, severe shock or septic-shock, rheumatoid, arthritis, cancer or cancer metastasis or infectious diseases and preterm birth; or reducing mortality risk associated with these diseases.

In one aspect, “the invention provides a purification method for an inter-alpha inhibit protein (IIp), the method including a step in which the IIp is exposed to conditions with a pH of 4.0 or less (e.g. 3.7, 3,5, 3.4, 3,1, 3.0, 2,9, 2.0). The invention, in one aspect provides a composition that contains an I?Ip purified by a method including a step in which the I?Ip is exposed to conditions with a pH of about 4 or less (e.g., 3.7, 3.5 3.4 3.3 3.1 3.0 2.9 2.0).

The invention also provides a pharmaceutical formulation containing a purified I?Ip-protein effective dose, which is obtained by a method that involves exposing the I?Ip-protein to conditions with a pH of 4.0 or less and an excipient acceptable to pharmaceuticals.

In another aspect, the invention provides for a method of treating or preventing disease in a patient or symptom of disease by administering a composition that contains an I?Ip purified protein according to a technique involving a step which exposes the I?Ip to conditions with pH of about 4 or lower.

In a third aspect, the invention provides instructions on how to use a kit that purifies an I?Ip-protein using at least one wash buffer with a pH below 4.0. In one embodiment, the kit contains at least one wash buffer solution with a pH around 4.0 or below. In another embodiment the kit contains at least two wash buffers that have a pH below 4.0. In one embodiment, the first wash buffer is about pH 4.0 while the second wash buffer is about pH 3.3. In a different specific embodiment, the first wash buffer is about pH 4.0 while the second wash buffer is about pH 2.9.

The invention also provides a kit that contains an I?Ip purified by a method in which the I?Ip is exposed to pH levels of 4.0 or below.

The invention also provides a kit that contains an I?Ip purified by a method which involves exposing the I?Ip to conditions with a pH of 4.0 or less.

The invention also provides a method to purify an inter-alpha inhibit protein (I?Ip), which involves placing blood or a blood fraction or intermediate plasma fraction onto a chromatography slant and then washing the slant with a buffer that has a pH below 4.0.

In a related aspect, “the invention provides a purified Inter-alpha Inhibitor Protein (I?Ip) made by a process involving a step in which the I?Ip is exposed to conditions with a pH of about 4.0 and lower. The I?Ip also achieves an increased binding when compared to a standard reference.” In one embodiment, binding of the I-Ip is greater than 1, 1.5, 2, 3, 4, 5, or 10 times that of the reference protein (i.e., I-Ip not treated with low acidity).

In various embodiments, any of the aspects above or any other invention described herein involves a step in which the I?Ip is exposed to conditions with a pH of 3.6 or less. In different embodiments, the I?Ip is exposed to a pH of 3.3 or less. In different embodiments, the I?Ip is exposed to pH conditions between about 3.3 and about 3.1. In different embodiments, a step is included in the method where the I?Ip proteins are exposed to pH conditions between about 3.1 and about 2.9.

In various embodiments, the method includes a solid phase extraction or chromatography step. In different embodiments, chromatography can be liquid chromatography or column chromatography. It could also be anion-exchange, or any combination of these. In some embodiments, chromatography is performed using a monolithic or particle-based substrate. In some embodiments, a monolithic or particle-based support includes an anion exchange ligand immobilized. The immobilized anion exchange ligand can be a diethylaminoethane or a Q.

In various embodiments, any of the above aspects, or any other invention described herein, involves at least a buffer wash step. The wash buffer in at least one of these buffer wash steps has a pH around 4.0 or below. In different embodiments, at least one wash step is used, with a pH around 3.6 or below. In different embodiments, at least one wash step is used, with a pH around 3.3 or less. In different embodiments, at least one wash step is used, and the pH of the wash buffer in at least one wash step must be below 3.1. In some embodiments, at least one wash step is required, with a pH ranging from 3.1 to 2.9. In some embodiments, inter-alpha inhibit protein (I?Ip) binds the column. “In various embodiments, the inter-alpha inhibit protein (I?Ip) is isolated.

In various embodiments, any of the above aspects, or any other invention described herein, the methods involves a step in which the I?Ip is exposed to salt concentrations at or higher than 250 mM (e.g. 260, 275, 280, or 290 mM). The method of various embodiments for any of the above-described aspects includes at least one wash buffer step. This wash buffer contains a salt concentration of about 250 mM or more (e.g. 260, 272, 280, and 290 mM).

In various embodiments, the I-Ip protein can be purified by blood plasma or a blood fraction. The I?Ip is purified in various embodiments from blood plasma, or a fraction of blood plasma. In some embodiments, blood plasma is cryopoor plasma. In some embodiments, intermediate plasma fractions are I?Ip-containing fractions. The blood, blood fraction or intermediate plasma fraction can be human, primate or bovine or equine or porcine or ovine or feline or canine or combinations thereof.

In various embodiments, either of the above aspects of the invention or any other described herein, I?Ip has an apparent mollecular mass of about 60 to 280 kDa. I?Ip or its compositions have a purity that ranges from about 85% up to about 100% in various embodiments. In various embodiments, the I-Ip protein has a yield that ranges from about 85% up to about 100%. In certain embodiments, I?Ip composition or protein can be used as an analytical tool (e.g. to quantify the amount of I-Ip in a unknown I-Ip concentration). In certain embodiments, I?Ip or its composition have biological activity. In some embodiments, biological activity can be cytokine inhibition activity, chemokine inhibiting activity or serine protease inhibiting activity. In some embodiments, a step of viral inactivation or nanofiltration is performed either before or following the chromatography.

In various embodiments, the composition or pharmaceutical formulation is used to treat a subject who needs it. In different embodiments, a subject identified as needing treatment is treated with the composition. In different embodiments, the patient is identified as having a need for treatment of acute inflammation, sepsis or severe shock. Other conditions include cancer, cancer metastasis and preterm labor. The subject that is in need of treatment for any of the above aspects can be human, primate or bovine.

The invention provides methods for preparing or separating inter-alpha inhibiting proteins (I?Ips) from blood plasma. The detailed description and the claims will reveal other features and benefits of the invention.

Definitions

As used herein, ?alteration? “As used herein,?alteration? “An alteration is defined as a change of 10% in yield, activity or purity. Preferably, this change will be 25%, but more preferably 40%. Most preferably, it will be 50% or more.

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