A packing apparatus for improving the stability of peg-hemoglobin

Abstract

The present invention describes a packaging apparatus for storing hemoglobin composition that provides long term storage stability and minimizes subsequent degradation of hemoglobin in the pharmaceutical composition. Furthermore, the present invention also provides a stable pharmaceutical composition of PEG-Hb-CO with metHb less than about 5% for at least 6 months wherein the PEG-Hb-CO is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

Description

Attorney Docket No.: PLG-028WOA PACKING APPARATUS FOR IMPROVING THE STABILITY OF PEG-HEMOGLOBINFIELD

[0001] The present disclosure describes a packaging apparatus for storing a pharmaceutical composition comprising hemoglobin that provides long term storage stability and minimizes subsequent degradation of hemoglobin in the pharmaceutical composition.

[0002] The present disclosure also describes a packaging process for storing hemoglobin composition in a primary container that is further enclosed by a gas impermeable secondary container. More particularly, the secondary container maintains less than about 5% oxygen in the headspace to provide long term storage stability and to minimize subsequent degradation of hemoglobin.

[0003] Furthermore, the present disclosure also provides a stable pharmaceutical composition of PEG-Hb-CO with metHb less than about 5% for at least 6 months wherein the PEG-Hb-CO is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.BACKGROUND

[0004] Blood products, particularly those containing hemoglobin (i.e. Hemoglobin based oxygen carriers), are highly prone to oxidation when exposed to oxygen. Hemoglobin, the primary oxygen-carrying protein in blood, can readily oxidize, forming methemoglobin (metHb), a non-functional form of hemoglobin that cannot effectively carry oxygen. This oxidation not only reduces the efficacy of blood products but also can lead to increased toxicity and degradation over time. Preventing or controlling oxidation is crucial to maintaining the functional integrity of blood products during storage and use. Hemoglobin bound to oxygen (i.e. Oxyglobin) has the highest susceptibility to spontaneous oxidation and metHb formation. Many blood substitute products aim to tackle this by storing the final product in deoxy or CO bound manner. However, the IV bags that are primarily used for these types of products are gas permeable in nature which leads to slow gas exchange across the primary container and subsequent oxidation of the hemoglobin.

[0005] The disclosure described herein introduces an innovative secondary packaging solution designed to enhance the stability of blood products in IV bags. This packaging creates an additional protective barrier around the IV bag, significantly reducing oxygen exposure and thus minimizing oxidation. By limiting the formation of methemoglobin, this solution maintains the functional quality of the blood product, enabling safe, effective, and prolonged1IPTS / 200205494.1Attorney Docket No.: PLG-028WOstorage, making it particularly valuable in medical settings where blood products must remain stable over extended periods.SUMMARY

[0006] The embodiment of the present invention provides a packaging apparatus suitable to store the pharmaceutical composition of hemoglobin.

[0007] In an embodiment, the packaging apparatus of the present invention is capable to effectively store the pharmaceutical composition of hemoglobin at varied temperature ranges selected from 2°C to 8°C, room temperature of about 25°C ±2°Cand at 40°C.

[0008] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at varied temperature range selected from 2°C to 8°C, room temperature of about 25 °C ±2°C and at 40°C, by maintaining the % oxygen in headspace of the packaging apparatus.

[0009] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at varied temperature range selected from 2°C to 8°C, room temperature of about 25°C ±2°C and at 40°C, by maintaining the % oxygen of about less than 5% in the headspace of the packaging apparatus.

[0010] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at temperature 2°C to 8°C, by maintaining the % oxygen in headspace of the packaging apparatus for a period of at least 3 months to 54 months.

[0011] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at room temperature about 25°C ±2°Cby maintaining the % oxygen in headspace of the packaging apparatus for a period of at least 3 months to 24 months.

[0012] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at 40 °C temperature, by maintaining the % oxygen in headspace of the packaging apparatus for a period of at least 1 month to 9 months.

[0013] In an embodiment, the present invention discloses a packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a pri mary packaging apparatus;b. a secondary packaging apparatus;wherein the primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and suitable excipients;wherein, the secondary packaging apparatus envelops the primary packaging apparatus thereby forming the packaging apparatus;2IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein, the pharmaceutical composition of hemoglobin stored in primary packaging apparatus remains physiologically active.

[0014] In an embodiment, the % oxygen in the headspace is maintained in the secondary packaging apparatus such that the % oxygen in the secondary packaging apparatus is maintained below 5%.

[0015] In an embodiment, the present invention provides a packaging apparatus, wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%, below 4%, below 3%, below 2%, and below 1 % analysed by oximetry.

[0016] In an embodiment, the present invention provides a packaging apparatus, wherein the stored pharmaceutical composition of hemoglobin has a purity of more than 90%, more than 95% and about 99%.

[0017] In another embodiment, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0018] In another embodiment, the secondary packaging apparatus maintains less than about 4% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0019] In another embodiment, the secondary packaging apparatus maintains less than about 3% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0020] In another embodiment, the secondary packaging apparatus maintains less than about 2% oxygen in the headspace; wherein the primary packaging comprising physiologically active 3IPTS / 200205494.1Attorney Docket No.: PLG-028WOhemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0021] In another embodiment, the secondary packaging apparatus maintains less than about 1% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0022] In another embodiment, the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0023] In another embodiment, the secondary packaging apparatus maintains less than about 0% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a urity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0024] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 4%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.4IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0025] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 3%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0026] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 2%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0027] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 1%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0028] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3% below 2% below 1%; wherein the physiologically active hemoglobin has a purity more than 95%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.5IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0029] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3% below 2% below 1%; wherein the physiologically active hemoglobin has a purity about 99%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0030] In another embodiment, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0031] In another embodiment, the secondary packaging apparatus maintains less than about 4% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0032] In another embodiment, the secondary packaging apparatus maintains less than about 3% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0033] In another embodiment, the secondary packaging apparatus maintains less than about 2% oxygen in the headspace; wherein the primary7packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the 6IPTS / 200205494.1Attorney Docket No.: PLG-028WOrange of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0034] In another embodiment, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 1%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 1 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0035] In another embodiment, the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0036] In another embodiment, the secondary packaging apparatus maintains 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25OC±2°C.

[0037] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary' packaging comprising physiologically active hemoglobin wherein the metHb is below 4%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0038] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and 7IPTS / 200205494.1Attorney Docket No.: PLG-028WOless than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 3%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0039] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 2%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0040] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 1%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0041] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3%, below 2%, below 1%; wherein the physiologically active hemoglobin has a purity' more than 95%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0042] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3%,8IPTS / 200205494.1Attorney Docket No.: PLG-028WObelow 2%, below 1%; wherein the physiologically active hemoglobin has a purity about 99%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0043] In another embodiment, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0044] In another embodiment, the secondary packaging apparatus maintains less than about 4% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0045] In another embodiment, the secondary packaging apparatus maintains less than about 3% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0046] In another embodiment, the secondary packaging apparatus maintains less than about 2% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0047] In another embodiment, the secondary packaging apparatus maintains less than about 1% oxygen in the headspace; wherein the primary packaging comprising physiologically active 9IPTS / 200205494.1Attorney Docket No.: PLG-028WOhemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0048] In another embodiment, the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0049] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0050] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 4%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0051] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 3%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is 10IPTS / 200205494.1Attorney Docket No.: PLG-028WOstable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0052] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary7packaging comprising physiologically active hemoglobin wherein the metHb is below 2%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0053] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 1%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0054] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3% below 2% below 1% ; wherein the physiologically active hemoglobin has a purity- more than 95%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0055] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3% below 2% below 1% ; wherein the physiologically active hemoglobin has a about 99%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about11IPTS / 200205494.1Attorney Docket No.: PLG-028WO16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0056] In an embodiment, the present invention provides a packaging apparatus, wherein the hemoglobin in the pharmaceutical composition is conjugated with polyethylene glycol (PEG).

[0057] In an embodiment, hemoglobin in the pharmaceutical composition is PEGylated carboxyhemoglobin or PEG-Hb-CO.

[0058] In an embodiment, the present invention discloses a packaging apparatus, wherein the primary packaging apparatus is an intravenous bag (IV Bag).

[0059] In an embodiment, the present invention discloses a packaging apparatus, comprising a primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and other excipients; and a secondary packaging apparatus enveloping the primary packaging apparatus to form the packaging apparatus, wherein the packaging apparatus provides mechanical and environmental protection to pharmaceutical composition.

[0060] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin, wherein the hemoglobin is biologically active and isolated from animal; wherein the hemoglobin is PEGylated; wherein the hemoglobin is carboxylated; wherein the hemoglobin is reoxygenated; wherein the hemoglobin is free of viruses and other pathogens; wherein the hemoglobin is de-oxygenated and heated at high temperature more than 60°C for about more than 5 hours; thereafter the deoxygenated hemoglobin is re-oxygenated; wherein the hemoglobin is non -natural.

[0061] In an embodiment, the present invention discloses a packaging apparatus, wherein the primary packaging apparatus is enveloped within the secondary packaging apparatus to form a packaging apparatus.

[0062] In an embodiment, the present invention provides a primary packaging apparatus, wherein the primary apparatus comprises two or more ports.

[0063] In an embodiment, the present invention provides a primary packaging apparatus, wherein the primary apparatus comprises a filling port, a sampling port and a spike port.

[0064] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus has an inward layer and an outward layer.

[0065] In an embodiment, the inward layer of the primary packaging apparatus holds the pharmaceutical composition.12IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0066] In an embodiment, the outward layer of the primary packaging apparatus is in contact with the secondary packaging apparatus.

[0067] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is a multi-layered metallized polymer bag.

[0068] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is Mylar pouches.

[0069] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is gas impermeable.

[0070] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is oxygen impermeable.

[0071] In yet another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is configured to shield the primary packaging from gaseous exchange, humidity, and light, preventing oxygenation and degradation of the hemoglobin composition, thereby preserving the integrity and stability of the pharmaceutical composition.

[0072] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains gaseous mixture of an inert gas and oxygen in the headspace; wherein, oxygen in the headspace is maintained less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5% and 0% in the headspace.

[0073] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus comprises inert gas; wherein the headspace of secondary packaging is substantially free of oxygen.

[0074] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains the oxygen in the headspace through inert gas sparging.

[0075] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein 13IPTS / 200205494.1Attorney Docket No.: PLG-028WOsparged inert gas in the secondary packaging apparatus is selected from nitrogen, carbon dioxide, helium, neon, argon, krypton, xenon, radon, and oganesson.

[0076] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is configured to shield the primary packaging apparatus containing the pharmaceutical composition from one or more condition selected from fluctuations, fluctuation in oxygen, fluctuation in humidity, effect of light, thereby preserving the integrity and stability of the pharmaceutical composition.

[0077] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition maintains p50 value in the range of about 7mmHg to about 16 mmHg.

[0078] In an embodiment, the present invention discloses a packaging apparatus that is cost effective.

[0079] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition is stable for at least about 3 months to at least about 54 months at 2-8°C, and for even longer periods, which is very difficult as the level of metHb in hemoglobin gradually increases, even when the composition is not in contact of oxygen.

[0080] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition is stable for at least about 1 month to at least about 24 months at 25°C±2°C, and for even longer periods, which is very difficult as the level of metHb in hemoglobin gradually increases, even when the composition is not in contact of oxygen.

[0081] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition is stable for at least about 1 month to about 9 months at 40°C, and for even longer periods, which is very difficult as the level of metHb in hemoglobin gradually increases, even when the composition is not in contact of oxygen.14IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0082] In an embodiment, the primary packaging apparatus alone is not sufficient because the primary packaging permits oxygen ingress, leading to hemoglobin oxygenation and degradation during storage. Therefore, a secondary, gas-impermeable packaging apparatus is required to prevent oxygen exposure, ensuring the stability and efficacy of the hemoglobin composition.

[0083] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of PEGylated hemoglobin, in a packaging apparatus, the method comprising:a. filling the pharmaceutical composition in a primary7packaging apparatus;b. enveloping the primary packaging apparatus with a secondary packaging apparatus to form the packaging apparatusc. sealing the secondary packaging;d. storing the packaging apparatus containing the pharmaceutical composition; wherein the primary packaging apparatus comprises a pharmaceutical composition of PEGy lated hemoglobin and other excipients;wherein, the secondary packaging apparatus maintains oxygen less than about 5% in the headspace;wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein the stored pharmaceutical composition maintains metHb below 5% analyzed by oximetry.

[0084] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is sealed at seal dwell of about 1 second, to about 3.0 second.

[0085] In an embodiment, the secondary packaging apparatus is sealed at takt time selected from about 1.5 second to about 3.5 second.

[0086] In an embodiment, the secondary packaging apparatus sealed at seal pressure selected from about 25 psi to about 75 psi.

[0087] In an embodiment, the secondary packaging apparatus sealed at temperature selected from about 330°F to about 400°F.15IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0088] In another embodiment, the present invention discloses a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, comprising, storing the pharmaceutical composition of hemoglobin in a suitable packaging apparatus consisting of:a. a primary' packaging apparatus;b. a secondary packaging apparatus;wherein the primary packaging apparatus comprises a pharmaceutical composition of hemoglobin and other excipients;wherein, the secondary' packaging apparatus envelops the primary' packaging apparatus; wherein the secondary packaging apparatus maintains less than about 5% Oxygen in the headspace through inert gas sparging;wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein the hemoglobin composition remains physiologically active;wherein, the stored hemoglobin composition purity is more than about 90%; wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%.

[0089] In an embodiment, the present invention discloses a method for prevention of metHb fonnation during the storage of pharmaceutical composition of hemoglobin, wherein the hemoglobin composition has a purity of more than about 90%, more than about 91%. More than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, and more than about 99%.

[0090] In an embodiment, the present invention discloses a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, wherein the hemoglobin composition has a purity of more than about 90%.

[0091] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, comprising hemoglobin molecules covalently conjugated with PEG polymers, and other suitable excipients selected from buffer, stabilizers, tonicity modifier, and rehydrating salts;wherein, the packaging apparatus comprising, a primary packaging apparatus and a secondary packaging apparatus;wherein, the secondary packaging apparatus envelops the primary packaging apparatus; wherein, the pharmaceutical composition of hemoglobin is contained in the primary packaging apparatus;16IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein, the pharmaceutical composition has stability for at least three months at room temperature;wherein, the pharmaceutical composition has stability for at least 24 months at refrigerated conditions.

[0092] In another embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein, the packaging apparatus comprising, a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains oxygen below 5%, enabling consistency in the pharmaceutical composition product quality across batches.

[0093] In an embodiment, the pharmaceutical composition, comprises PEGylated hemoglobin molecules and other excipients selected from buffer, stabilizers, tonicity modifier, and rehydrating salts.

[0094] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises buffer selected from citrate, Tris, MOPS, HEPES, phosphate buffer, and sodium acetate and ammonia buffers in an amount of about 1 mM to about 5 mM.

[0095] In an embodiment, the buffer is phosphate buffer in an amount of about 1 mM to about 5 mM.

[0096] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises stabilizer selected from amine stabilizers, sodium metabisulfite, polyethylene glycol, EDTA and polysorbates.

[0097] In an embodiment, the stabilizer is an amine stabilizer preferably cysteine In an embodiment, the cysteine is maintained in an amount more than 5 mM.

[0098] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises tonicity modifier selected from potassium chloride, glycerin, lactose, sodium chloride, dextrose, and mannitol in an amount of about 100 mM to about 200 mM.

[0099] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises tonicity modifier is sodium chloride in an amount of about 150 mM.

[0100] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises rehydrating salts are selected from sodium chloride, sodium bicarbonate, magnesium sulfate hetpahydrate, calcium chloride dihydrate, and potassium chloride in an amount of about 0.5 mM to about 5 mM.17IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0101] In certain embodiment, the present invention discloses a method of treating a disease condition in a subject in need thereof, by administering to the subject a therapeutically effective amount of pharmaceutical composition, wherein the pharmaceutical composition comprising hemoglobin and suitable excipients; wherein, the hemoglobin composition is stored in a suitable packaging apparatus, the packaging apparatus consisting of:a. a primary packaging apparatus;b. a secondary packaging apparatus;wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein the hemoglobin composition remains physiologically active;wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%.

[0102] In an embodiment, the present invention discloses a method of treating a disease condition, wherein, the disease condition is selected from cancer, hemorrhagic shock, intracranial hemorrhage, hypoxia, necrosis, myocardial injury, acute myocardial infarction, hypertension, pulmonary hypertension.

[0103] In another embodiment, the present invention discloses a method of administering physiologically active hemoglobin composition stored in a suitable packaging apparatus to a subject in need thereof, wherein the packaging apparatus consists of a primary packaging apparatus and a secondary packaging apparatus, the method comprising:a. opening the secondary packaging apparatus to access the primary packaging apparatus comprising pharmaceutical composition;b. administering to the subject a therapeutically effective amount of pharmaceutical composition stored in the primary packaging;wherein the pharmaceutical composition comprising PEGylated hemoglobin and suitable excipients:wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein the hemoglobin composition in the packaging apparatus remains physiologically active;wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C.18IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0104] In an embodiment, the present invention discloses a method of administering physiologically active hemoglobin composition stored in a suitable packaging apparatus to a subject in need thereof, wherein the subject in need is administered with a therapeutically effective amount of pharmaceutical composition; wherein the therapeutic effective amount of PEGylated hemoglobin is selected from about 100 mg / kg to about 1500 mg / kg. In an embodiment, the therapeutic effective amount of PEG-HB-CO is selected from about 500 mg / kg to about 1200 mg / kg. In an embodiment, the therapeutic effective amount of PEG-HB-CO is selected from about 800 mg / kg to about 1000 mg / kg.

[0105] In an embodiment, the therapeutic effective amount of PEGylated hemoglobin is administered in at least two cycles in a subject in need thereof.

[0106] In an embodiment, the therapeutic effective amount of PEGylated hemoglobin is administered in at least two cycles in a subject in need thereof, wherein the first infusion cycle has higher flow rate than the second cycle.

[0107] In an embodiment, the first infusion cycle delivers a lower amount of therapeutic effective amount of PEGylated hemoglobin than the second cycle.

[0108] In an embodiment, the first infusion cycle maintains a flow rate of about 3 ml / min, to about 8 ml / min based on the weight of subject and effective amount of pegylated hemoglobin needs to be administrated.

[0109] In an embodiment, the first infusion cycle maintains a flow rate of about 6.7 ml / min, based on the weight of subject and effective amount of pegylated hemoglobin needs to be administrated.

[0110] In an embodiment, the first infusion cycle delivers PEG-Hb-CO in the range of about 100 ml, to about 200 ml wherein the total dose volume is at least 500ml.

[0111] In certain embodiment, the first cycle last for least 5 min, to about 35 min.

[0112] In an embodiment, the second infusion cycle maintains flow rate of about 1 ml / min, to about 5ml / min based on the weight of subject and effective amount of pegylated hemoglobin needs to be administrated.

[0113] In an embodiment, the second infusion cycle maintains flow rate of about 3.3 ml / min based on the weight of subject and effective amount of pegylated hemoglobin needs to be administrated.

[0114] In an embodiment, the second cycle delivers PEG-HB-CO in the range of about 275 ml, to about 500 ml.

[0115] In certain embodiment, the first cycle last for least about 10 min, to about 90 min.19IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0116] In an embodiment, the present invention discloses a process for storing a physiologically active hemoglobin composition stored in a suitable packaging apparatus; wherein the packaging apparatus consists of a primary packaging apparatus and a secondary packaging apparatus, the process comprising:a. washing fresh whole blood collected from animal sources to produce washed RBCs; b. extracting hemoglobin from the RBCs;c. performing filtration of the extracted hemoglobin:d. performing ultrafiltration and concentration of hemoglobin;e. performing deoxygenation of ultrafiltered and concentrated hemoglobin;f. performing heat inactivation of deoxygenated hemoglobin for reduction of vims and / or prion by heat treatment at suitable temperature;g. performing reoxygenation of heat-treated deoxygenated hemoglobin to produce oxygenated hemoglobin composition;h. optionally performing PEGylation of the oxygenated hemoglobin composition; i. optionally performing filtration of the PEGylated hemoglobinj. optionally performing carboxylation process to produce carboxy lated PEGylated hemoglobin composition;k. filling the pharmaceutical composition of carboxylated PEGylated hemoglobin obtained from (i) in the primary packaging apparatus;l. enveloping the primary packaging apparatus with a secondary packaging apparatus to form a packaging apparatus;m. sealing the packaging apparatus; and,n. storing the packaging apparatus containing the pharmaceutical composition; wherein the viral inactivation of deoxygenated hemoglobin is performed by heat treatment and maintains L-cysteine concentration in deoxygenated hemoglobin during step f) for more than 5 mM;wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein the hemoglobin composition stored in the packaging apparatus remains physiologically active;wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C.

[0117] In an embodiment, the present invention discloses a process for storing a physiologically active hemoglobin stored in a suitable packaging apparatus, wherein the 20IPTS / 200205494.1Attorney Docket No.: PLG-028WOhemoglobin composition comprises impurities selected from charge variants, Low molecular weight impurities, high molecular weight impurities, HCPs and HC DNA.

[0118] In an embodiment, the charge variants are below 25%.

[0119] In an embodiment, the charge variants are selected from acidic variants and basic variants.

[0120] In another embodiment, the acidic variants are less than 15%.

[0121] In another embodiment, the basic variants are less than 15%.

[0122] In an embodiment, the present invention discloses a process for storing a physiologically active hemoglobin stored in a suitable packaging apparatus, wherein the hemoglobin obtained from step (f) is re -oxygenated.

[0123] In an embodiment, the L-cysteine concentration in deoxygenated hemoglobin is maintained more than 5 mM.

[0124] Disclosed herein, in some embodiments, is a pharmaceutical composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability for at least three months at 25°C±2°C.

[0125] Disclosed herein, in some embodiments, is an improved packaging apparatus suitable to store physiologically active hemoglobin comprising:a. Primary packaging apparatus;b. Secondary packaging apparatus;wherein the primary packaging apparatus comprising a pharmaceutical composition of hemoglobin;wherein the secondary packaging apparatus enveloped the primary packaging apparatus;wherein the secondary packaging apparatus is opaque metallized plastic barrier that is gas and moisture impermeable;wherein the secondary packaging apparatus maintains less than about 5% Oxygen in headspace;wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2°C to 8°C and the hemoglobin remains physiologically active;wherein the pharmaceutical composition of hemoglobin maintains metHb below 5%; wherein the pharmacal composition of hemoglobin maintains p50 value in between 7 to 16 mmHg analysed by Hemox analyser;21IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein the hemoglobin is conjugated with polyethylene glycol (PEG). In an embodiment, the packaging apparatus stores the hemoglobin at least for 18 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0126] Disclosed herein, in some embodiments, is an improved packaging apparatus suitable to store physiologically active hemoglobin comprising:a. Primary packaging apparatus;b. Secondary packaging apparatus;wherein the primary packaging apparatus comprising hemoglobin, buffer, stabilizer; wherein the secondary packaging apparatus enveloped the primary packaging apparatus;wherein the secondary packaging apparatus is gas impermeable;wherein the secondary packaging apparatus maintains gaseous mixture comprising predominantly higher argon and less than about 5% Oxygen;wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0127] Disclosed herein, in some embodiments, is a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, comprising: storing the pharmaceutical composition of hemoglobin in suitable packaging consisting of primary packaging and secondary packaging,wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin;wherein the secondary packaging apparatus enveloped the primary packaging apparatus;wherein the secondary packaging apparatus is sparged with argon;wherein the secondary packaging apparatus maintains less than about 5% Oxygen in the headspace;wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0128] In an embodiment, the present disclosure provides a pharmaceutical composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability for at least three months at 25°C±2°C.22IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0129] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 6 months at 25°C±2°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0130] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 12 months at 25°C±2°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0131] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 24 months at 25°C±2°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0132] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 30 months at 2°C to 8°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0133] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 36 months at 2°C to 8°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0134] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 42 months at 2°C to 8°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0135] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 54 months at 2°C to 8°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0136] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 12 months at 25°C ± 2°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace; wherein the composition improved the stability of hemoglobin for at least 12 months at 2°C to 8°C compared to the composition stored in suitable packaging and maintains more than 5% oxygen.23IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0137] In an embodiment, the present disclosure provides a pharmaceutical composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability for at least three months at 25°C±2°C and at least for two years at 2°C to 8°C.

[0138] In an embodiment, the present disclosure provides a pharmaceutical composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability for at least three months at room temperature and at least for two years at 2°C to 8°C; wherein the pharmaceutical composition comprises less than 5% metHb formation.

[0139] In an embodiment, the primary container is an intravenous bag (IV Bag) and the secondary container is a multi-layered metallized polymer bag that is gas impermeable and opaque (e.g. Mylar pouches).

[0140] In an embodiment, the present disclosure provides a pharmaceutical composition contained in a primary container comprising PEG polymers covalently conjugated with a hemoglobin molecule; wherein the primary container is enclosed in a secondary container; wherein the secondare' container is gas impermeable; wherein the headspace of the secondary container is controlled to contain less than about 5% Oxygen; wherein the pharmaceutical composition has stability for at least three months at room temperature and at least for two years at 2°C to 8°C; wherein the pharmaceutical composition comprises less than 5% metHb formation.

[0141] In an embodiment, the present disclosure provides a pharmaceutical composition contained in a primary container comprising PEG polymers covalently conjugated with a hemoglobin molecule; wherein the primary container is enclosed in a secondary container; wherein the secondary container is a multi-layered metallized polymer bag that is gas impermeable and opaque (e.g. Mylar pouches); wherein the headspace of the secondary' container is controlled to contain less than about 5% oxygen by flushing an inert gas and followed by heat sealing the secondary container; wherein the pharmaceutical composition has stability for at least three months at room temperature and at least for two years at 2°C to 8°C; wherein the pharmaceutical composition comprises less than 5% metHb formation.

[0142] In an embodiment, the present disclosure provides an improved packaging apparatus suitable to store physiologically active hemoglobin comprising:a) Primary packaging apparatus;24IPTS / 200205494.1Attorney Docket No.: PLG-028WOb) Secondary' packaging apparatus;wherein the primary packaging apparatus comprising a pharmaceutical composition of hemoglobin; wherein the secondary packaging apparatus enveloped the primary packaging apparatus; wherein the secondary packaging apparatus is opaque metallized plastic barrier that is gas and moisture impermeable; wherein the secondary' packaging apparatus maintains less than about 5% Oxygen in headspace; wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2°C to 8°C and the hemoglobin remains physiologically active; wherein the pharmaceutical composition of hemoglobin maintains metHb below 5%; wherein the pharmaceutical composition of hemoglobin maintains p50 value in between 7 to 16 mmHg analysed by Hemox analyser; wherein the hemoglobin is conjugated with polyethylene glycol (PEG). In an embodiment, the packaging apparatus stores the hemoglobin at least for 18 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0143] In an embodiment, the packaging apparatus stores the hemoglobin at least for 24 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0144] In an embodiment, the packaging apparatus stores the hemoglobin at least for 30 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0145] In an embodiment, the packaging apparatus stores the hemoglobin at least for 36 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0146] In an embodiment, the packaging apparatus stores the hemoglobin at least for 40 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0147] In an embodiment, the present disclosure provides an improved packaging apparatus suitable to store physiologically active hemoglobin comprising:a) Primary packaging apparatus;b) Secondary packaging apparatus;wherein the primary packaging apparatus comprising hemoglobin, buffer, stabilizer; wherein the secondary packaging apparatus enveloped the primary packaging apparatus; wherein the secondary' packaging apparatus is gas impermeable; wherein the secondary packaging apparatus maintains gaseous mixture comprising predominantly higher argon and less than about 5% Oxygen; wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0148] In an embodiment, the present disclosure provides a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, comprising, storing the pharmaceutical composition of hemoglobin in suitable packaging consisting of primary packaging and secondary packaging wherein the primary packaging 25IPTS / 200205494.1Attorney Docket No.: PLG-028WOapparatus comprising pharmaceutical composition of hemoglobin; wherein the secondary¬ packaging apparatus enveloped the primary packaging apparatus; wherein the secondary packaging apparatus is sparged with argon; wherein the secondary packaging apparatus maintains less than about 5% Oxygen in the headspace; wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0149] In an embodiment, the present disclosure provides a packaging and composition system comprising a primary packaging with a hemoglobin composition and a secondary gas-impermeable packaging, wherein the packaging system is configured to maintain less than 5% oxygen in the headspace and preserves hemoglobin stability for at least 24 months.

[0150] In an embodiment, the present disclosure provides a process for preparing a pharmaceutical composition for storage, comprising:a) Primary' packaging apparatus;b) Secondary' packaging apparatus;wherein the pharmaceutical composition is enclosed in a primary packaging apparatus; wherein the primary packaging apparatus is further enclosed in the secondary packaging apparatus;wherein, sparging the secondary packaging apparatus with an inert gas to displace oxygen; and sealing the secondary packaging apparatus with controlled oxygen content in the headspace;wherein secondary container that maintains less than about 5% oxygen in its headspace; wherein the pharmaceutical composition is Pegylated hemoglobin.

[0151] In an embodiment, the present disclosure provides a method for enhancing the stability of a hemoglobin pharmaceutical composition, comprising controlling the oxygen level in the headspace to below 5% through argon sparging; wherein the hemoglobin pharmaceutical composition maintains metHb below 5%; wherein the stability7of the hemoglobin pharmaceutical composition at least for 18 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0152] In an embodiment, the present disclosure provides a pharmaceutical composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability for at least three months at room temperature and at least for two years at 2°C to 8°C; wherein the pharmaceutical composition comprises less than 5% metHb formation.26IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0153] The present disclosure describes a heat-sealing recipe that produces a desired secondary packaging seal, free of visual imperfections as well as maintains the seal integrity and subsequently the headspace oxygen content for at least 5 years at refrigerated condition and 2 years at room temperature storage.

[0154] In an embodiment, the secondary bag is sealed at seal dwell of about 1.5 Sec, Takt Time of about 2.0 Sec, Seal pressure at about 50 psi and temperature at about 370 °F.BRIEF DESCRIPTION OF FIGURES:

[0155] FIG. 1 : shows % HbCO content and %metHb over time (18 months) across different oxygen levels. The x-axis represents the time points and oxygen levels, while the y-axis shows the content percentages for %HbCO and %metHb.

[0156] FIG. 2 : shows effect on % HbCO content and % metHb over time (18 months) when the %oxygen level is maintained <2% O2 and 16% O2.

[0157] FIG. 3: shows the % metHb, p50, and Headspace Oxygen % over various time points (till 54 Months) stored at 2-8°C. The dashed lines indicate the acceptance criteria: % metHb acceptance criterion is set at 5.0 and p50 has an acceptance range of 7 to 16 mmHg.

[0158] FIG. 4 : shows stability of %HbCO over time (1 month-60 months) across different temperatures (2-8°C, 22-28°C and 40°C).

[0159] FIG. 5: showing the % metHb, p50 (mmHg), Headspace % Oxygen at 24-month at room temperature (25°C ± 2°C).

[0160] FIG. 6: shows the effect of less than 2% O2 in the headspace content on MetHb % in a pharmaceutical composition over period of 7 days at 40°C.

[0161] FIG. 7A: shows the primary packaging apparatus filled with Hemoglobin composition.

[0162] FIG. 7B: shows the Secondary packaging apparatus.

[0163] FIG. 7C: shows the components of the packaging apparatus DETAILED DESCRIPTIONDefinitions

[0164] The term “comprises” or “comprising” is used in the present description, it does not exclude other elements or steps. For the purpose of the present disclosure, the term “consisting of’ is considered to be an optional embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which optionally consists only of these embodiments.27IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0165] As used throughout the specification and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0166] The term “about”, as used herein, is intended to refer to ranges of approximately 10-20% greater than or less than the referenced value. In certain circumstances, one skill in the art will recognize that, due to the nature of the referenced value, the term “about” can mean more or less than a 10-20% deviation from that value.

[0167] The term “hemoglobin” or “Hb” as used herein refers generally to the protein within red blood cells that transports oxygen. Hb by itself refers both to native unmodified Hb as well as modified Hb. Each molecule of Hb has 4 subunits, 2 alpha-chain Subunits and 2 beta-chain subunits, which are arranged in a tetrameric structure. Each subunit also contains one heme group, which is the iron-containing center that binds oxygen. Therefore, each Hb molecule can bind 4 molecules of oxygen. Hemoglobin of use in the present disclosure is derived from substantially any mammalian source. Exemplary sources of hemoglobin include common livestock animals, e.g., cows, bovine, pigs, sheep and the like. The present disclosure is not limited by the source of the hemoglobin. In various embodiments, the hemoglobin is bovine hemoglobin.

[0168] The term “effective amount” or “an amount effective to” or a “therapeutically effective amount” or “therapeutic effective amount” or “therapeutic effective dose” any grammatically equivalent term means the amount that, when administered to a subject for treating a disease, condition or injury, is sufficient to effect treatment for that disease. In exemplary embodiments, this term refers to any amount of a conjugate of the invention (or a formulation including a conjugate of the invention) sufficient to repay at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or up to about 100% of tissue or organ oxygen debt attributable to disease, insult or injury. When used in the context of delivery of CO to a tissue, this term refers to an amount administered sufficient to derive a detectable therapeutic effect from the delivery of CO to a tissue.

[0169] In an embodiment, the therapeutic effective amount of PEG-HB-CO is selected from about 300 mg / kg to about 1500 mg / kg. In an embodiment, the therapeutic effective amount of PEG-HB-CO is selected from about 500 mg / kg to about 1200 mg / kg. In an embodiment, the therapeutic effective amount of PEG-HB-CO is selected from about 800 mg / kg to about 1000 mg / kg.28IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0170] The term “takt time” herein refers maximum amount of time allowed to complete the sealing process. For example, if there are 480 minutes of production time and demand is 240 units, and then the takt time is 2 minutes per unit.

[0171] The term “seal pressure” refer to the force exerted to seal the bag / apparatus.

[0172] The term “seal dwell” herein refers to the duration of contact between the sealing jaws and the material in processes like heat sealing. This dwell time is a critical factor, along with temperature and pressure, in creating a quality seal. A longer dwell time allows more heat to penetrate and melt the sealant, leading to a stronger bond, but too long can cause thermal damage.

[0173] The term “first dose” refers to intravenous infusion of PEGylated hemoglobin or PEGylated hemoglobin containing carbon monoxide to a subject administrate in a first cycle.

[0174] The term “first cycle” or “first infusion cycle” refers to the administration of first dose of PEG-Hb or PEGylated hemoglobin by intravenous infusion in effective amount at adequate flow rate for the treatment of diseases and prevention, amelioration, or treatment of any side effect associated with improper perfusion of said pegylated Hb. First cycle maintains higher flow rate than second or subsequent cycle at least by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 95% and about 100% than the second cycle.

[0175] In certain embodiment, the first cycle has lower amount of pegylated hemoglobin in first cycle at least about 100ml, about 150 ml, about 200ml, about 225ml wherein the total blood volume is at least 500ml.

[0176] In certain embodiment, the first cycle last for least 5 min, about 10 min, aboutl5 min, about 20 min, about 25 min, about 30 min, about 35 min.

[0177] In certain embodiment, the flow rate is maintained during first cycle is at least by 3 ml / min, about 4 ml / min, about 5 ml / min, about 6ml / min, about 6.5ml / min, about 7ml / min, 8 ml / min based on the weight of subject and effective amount of pegylated hemoglobin needs to be administrated.

[0178] The term “second dose” refers to intravenous infusion of PEG-Hb or PEGylated hemoglobin to a subject administrates in a second cycle.

[0179] The term “second cycle” or “second infusion cycle” or “second dose” refers to the administration of PEG-Hb or PEGylated hemoglobin by intravenous infusion with low flow rate or infusion rate and has higher amount of pegylated hemoglobin in second cycle than first 29IPTS / 200205494.1Attorney Docket No.: PLG-028WOcycle. In certain embodiment the second cycle last for least 5 min, about 10 min, about 15 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, about 60 min, about 65 min, about 70 min, about 75 min, about 80 min, about 85 min and about 90 min.

[0180] In certain embodiment, the flow rate is maintained during second cycle is at least by 1 ml / min, about 2 ml / min, about 2.5 ml / min, about 3ml / min, about 3.3ml / min, about 4ml / min, about 4.5 ml / min based on the weight of subject and effective amount of pegylated hemoglobin needs to be administrated.

[0181] The term “at least two cycles” refers to intravenous infusion of PEGylated hemoglobin by first cycle or second cycle and can be optionally increase to subsequent third cycle and forth cycle.

[0182] The term “subsequent dose” refers to intravenous infusion of PEG-Hb or PEGylated hemoglobin to a subject after the completion of second dose and second cycle. It can be administrated as third or fourth dose based the subject need.

[0183] The term “subsequent cycle” refers to intravenous infusion of PEG-Hb or PEGylated hemoglobin to a subject after the completion of second cycle. It includes third or fourth dose based the subject need. In certain embodiment the subsequent cycle comprises the same dose and / or flow rate identical to second cycle.

[0184] The term “Sanguinate™” or “Sg” or “PP-007” or “PEGylated bovine hemoglobin” or “PEGylated carboxyhemoglobin” as used herein are interchangeable and refers to a PEG-Hb-CO composition of the invention.

[0185] The term “PEG-hemoglobin” or “PEG-Hb” are interchangeable and refers to a hemoglobin which is attached with the PEG molecule to improve the half-life of oxygenated hemoglobin. PEGylation process is the conjugation of eight to ten chains of 5 kD activated polyethylene glycol (SC-PEG-5K) molecules to the purified hemoglobin. The PEG-Hb is capable to deliver oxygen to tissues. Alternatively, PEG-Hb can be bound to carbon monoxide (CO) instead of oxygen binding refers to PEG-Hb-CO. In some embodiments, the p50 value of PEG-hemoglobin or PEG-Hb-CO is 7-16 mmHg analyzed by Hemox Analyzer.

[0186] The term “refrigerated temperatures” or “refrigerated conditions” refers to the temperatures between 2-8°C. And 2-8°C and “refrigerated temperatures” are interchangeable herein.

[0187] The term “room temperatures” refers to the temperatures between 25°C ± 2°C. And 25°C ± 2°C and “room temperatures” are interchangeable herein.30IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0188] The term “carboxylated” refers to gas bound hemoglobin. In some embodiments, the gas bound hemoglobin has CO attached to hemoglobin.

[0189] The term “CO” refers to carbon monoxide.

[0190] The term “methemoglobin' or “MetHb” as used herein refers to an oxidized form of Hb that contains iron in the ferric state. MetHb does not function as an oxygen carrier. The term “methemoglobin %” as used herein refers to the percentage of oxidized Hb or MetHb to total Hb. metHb is determined by any art -recognized method of analysis. In the present disclosure, the metHb impurity is determined by co-oximetry.

[0191] The term “contained” or “contain” herein refers to holding or storing the PEGylated hemoglobin in the packaging apparatus for future therapeutic use.

[0192] The term “buffer” herein refers to substance that maintains a stable pH, which is critical for the drug’s efficacy, stability, and safety. In an embodiment, the buffer is selected from citrate, Tris, MOPS, HEPES, ammonia buffers, phosphate buffer solution, and sodium acetate.

[0193] The term “stabilizers” or “stabilizing agent” herein refers to a species that prevents or retards the reoxygenation of deoxygenated hemoglobin. An exemplary stabilizing agent is an amine-containing compound, conveniently, though not exclusively, an amino acid. Any amine-containing compound can serve as a stabilizing agent in the formulations of the invention. An additional exemplary stabilizing agent has one or more structural elements that reacts with oxygen preferentially to the hemoglobin reacting with the oxygen. An exemplary structural element found on stabilizing agents of the invention is a thiol moiety. Exemplary sulfhydryl compounds of use as stabilizing agents include, but are not limited to, N-acetyl-L-cysteine (NAC) D,L-cysteine, y-glutamyl-cysteine, glutathione, 2,3-dimercapto-l -propanol, 1,4-butanedithiol, and other biologically compatible sulfhydryl compounds. It is generally preferred that the stabilizing agent is bio-compatible and is non-toxic in the amounts in which it is included in the compositions and formulations of the invention. In an exemplary embodiment, the PEG is itself a stabilizing reagent. Thus, in various embodiments, the PEG conjugated to the Hb obviates the need for a separate stabilizing agent or a separate water-soluble stabilizing fraction. Accordingly, the invention provides formulations equivalent to those set forth herein including a water soluble stabilizing fraction, which, in fact, do not include this fraction. In an embodiment, the stabilizer is cysteine.

[0194] The term “0%” or “negligent amount of oxygen” or “negligent oxygen amount” in the headspace of the secondary packaging herein refers to amount of oxygen in the headspace that does not affect the stability of the stored pharmaceutical composition. It also refers to the 31IPTS / 200205494.1Attorney Docket No.: PLG-028WOamount of oxygen at which the pharmaceutical composition remains physiologically active. In an embodiment, it can also mean the amount of oxygen that helps in prevention of oxygenation of hemoglobin. In an embodiment, it can also mean the amount of oxygen that prevents metHb formation. In an embodiment, it refers to substantially oxygen free headspace.

[0195] In another embodiment, the present invention discloses a packaging apparatus, wherein the secondary packaging apparatus maintains 0% or negligent oxygen amount in the headspace due to which the hemoglobin composition remains physiologically active.

[0196] The term “substantially free from oxygen” or “substantially oxygen free” are interchangeable terms and herein refer to condition wherein the headspace of the secondary packaging apparatus is maintained less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, to maintain the stability of the stored pharmaceutical composition.

[0197] The term “co-oximetry” or “oximetry” used herein refers to the measurement of various forms of hemoglobin by dedicated multiwavelength spectrophotometry. It is a measure of the potential oxygen-carrying capacity of the blood. CO-oximeters are multiwavelength spectrophotometers that measure the optical absorbance of blood at different wavelengths and automatically calculate the fractional concentration of the four major Hb species (oxy-, deoxy-, carboxy-, and methemoglobin) from a total Hb concentration.

[0198] The term “pharmaceutical composition” used herein refers to a composition of a pharmaceutical active which renders the biological activity of the active ingredient therapeutically effective, but which does not include other ingredients which are obviously toxic to a subject to which the compositions are intended to be administered.

[0199] The term “purity of hemoglobin” or “purity of pharmaceutical composition” or “HbCO purity” are interchangeable herein and refers to the purity of hemoglobin composition post the composition is oxygenated and the metHb in pharmaceutical composition increases.

[0200] The term “stable” or “stability” generally refers to the physical stability and / or chemical stability and / or biological stability of a component, typically an active or composition thereof, during preservation / storage.

[0201] The term “Headspace" refers to the air or empty space left above the contents in a sealed container.

[0202] The term “Primary container” or “Primary packaging” or “Primary packaging apparatus” refers to the IV Bag that uses for Intravenous therapy containing the drug composition. In an embodiment, the primary packaging apparatus comprises “a sampling port”, “a spike port” and “fill port”.32IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0203] The term “filling port” herein refers to the port and / or opening in the primary packaging apparatus from wherein the pharmaceutical composition is filled in the primary bag.

[0204] The term “sampling port” herein refers to the port and / or opening in the primary packaging apparatus from wherein the samples of the composition are drawn for testing.

[0205] The term “Spike port” or “infusion port” are interchangeable herein and refers to the port or the connection that connects the IV administration tubing set to the IV bag.

[0206] The term “Secondary container” or “secondary bag” or “secondary packaging” or “Secondary packaging apparatus” or “additional outer packaging” refers to the bag that covered the primary bag. The headspace of the secondary container is controlled to contain less than about 5% oxygen by flushing an inert gas and followed by heat sealing the secondary container. The secondary container is a multi-layered metallized polymer bag that is gas impermeable and opaque (e.g. Mylar pouches).

[0207] The term “packaging apparatus” or “single packaging unit” or “packaging bag” are interchangeable herein and contains a primary packaging apparatus enveloped with a secondary packaging apparatus. The packaging apparatus comprises of a primary packaging apparatus and a secondary packaging apparatus.

[0208] The term “enveloped” or “enclosed” used herein refers to the plain meaning of English which means to enclose or enfold completely with or as if with a covering. Here in the present disclosure, the pharmaceutical composition is firstly stored at the IV Bag (Intravenous Bag) wherein the IV bag is further stored or enveloped by another Bag i.e., Secondary Packaging Apparatus.

[0209] The term “physiologically active” refers to the active pharmaceutical ingredient (API), which is a chemical compound that produces a therapeutic effect in the body. Physiologically active hemoglobin may also exist as dimer or in equilibrium of dimer and tetramer.

[0210] The term “charge variants” includes product related impurity. In certain embodiment, the charge variants include acidic variants and / or basic variants.

[0211] The term “acidic variants” or “acidic species” and “AV” used herein refer to the variants of a protein, which are characterized by an overall acidic charge.

[0212] The term used “basic variants” or “basic species” refers to variants can result from the presence of C-terminal lysine additional positive charges or removal of negative charges.33IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0213] The term used “Capillary isoelectric focusing (cIEF)” is a high-resolution separation technique that separates proteins and peptides based on their isoelectric point (pl) using a pH gradient within a capillary.

[0214] The meaning of other terminology used herein should be easily understood by someone of reasonable skill in the art.Apparatus

[0215] The embodiment of the present invention provides a packaging apparatus suitable to store the pharmaceutical composition of hemoglobin.

[0216] In an embodiment, the packaging apparatus of the present invention is capable to effectively store the pharmaceutical composition of hemoglobin at varied temperature ranges selected from 2°C to 8 °C, room temperature of about 25°C±2°C and at 40°C.

[0217] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at varied temperature range selected from 2°C to 8 °C, room temperature of about 25°C±2°C and at 40°C, by maintaining the % oxygen in headspace of the packaging apparatus.

[0218] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at varied temperature range selected from 2°C to 8 °C, room temperature of about 25°C±2°C and at 40°C, by maintaining the % oxygen of about less than 5% in the headspace of the packaging apparatus.

[0219] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at temperature 2°C to 8 °C, by maintaining the % oxygen in headspace of the packaging apparatus for a period of at least 3 months to 54 months.

[0220] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at room temperature about 25°C±2°C by maintaining the % oxygen in headspace of the packaging apparatus for a period of at least 3 months to 24 months.

[0221] In an embodiment, the present invention effectively stores the pharmaceutical composition of hemoglobin at 40 °C temperature, by maintaining the % oxygen in headspace of the packaging apparatus for a period of at least 1 month to 9 months.

[0222] In an embodiment, the present invention discloses a packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a primary' packaging apparatus;b. a secondary packaging apparatus;34IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein the primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and suitable excipients;wherein, the secondary packaging apparatus envelops the primary packaging apparatus thereby forming the packaging apparatus;wherein, the pharmaceutical composition of hemoglobin stored in primary packaging apparatus remains physiologically active.

[0223] In an embodiment, the % oxygen in the headspace is maintained in the secondary packaging apparatus such that the % oxygen in the secondary packaging apparatus is maintained below 5%.

[0224] In an embodiment, the present invention provides a packaging apparatus, wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%, below 4%, below 3%, below 2%, and below 1% analysed by oximetry.

[0225] In an embodiment, the present invention provides a packaging apparatus, wherein the stored pharmaceutical composition of hemoglobin has a purity of more than 90%, more than 95% and about 99%.

[0226] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin, wherein the stored pharmaceutical composition has stability for at least 3 months to about 54 months at refrigerated temperatures.

[0227] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin, wherein the stored pharmaceutical composition has stability for at least 3 months to about 24 months at room temperatures.

[0228] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin, wherein the stored pharmaceutical composition has stability for at least 1 month to about 9 months at 40°C.

[0229] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and other excipients;b. a secondary packaging apparatus enveloping the primary packaging apparatus to form the packaging apparatus;wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5% analyzed by oximetry;35IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein, the pharmaceutical composition of hemoglobin stored in primary packaging apparatus remains physiologically active;wherein, the stored pharmaceutical composition has stability for at least 3 months to about 54 months at about 2°C to about 8°C.

[0230] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and other excipients;b. a secondary packaging apparatus enveloping the primary packaging apparatus to form the packaging apparatus;wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5% analyzed by oximetry;wherein, the pharmaceutical composition of hemoglobin stored in primary packaging apparatus remains physiologically active;wherein, the stored pharmaceutical composition has stability for at least three months to about 24 months at 25°C±2°C.

[0231] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and other excipients;b. a secondary packaging apparatus enveloping the primary packaging apparatus to form the packaging apparatus;wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5% analyzed by oximetry;wherein, the pharmaceutical composition of hemoglobin stored in primary packaging apparatus remains physiologically active;wherein, the stored pharmaceutical composition has stability for about 1 month to about 9 months at 40°C.

[0232] In an embodiment, the present invention provides an packaging apparatus suitable to store physiologically active hemoglobin comprising:a. a pri mary packaging apparatus;b. a secondary packaging apparatus;wherein the primary packaging apparatus comprising a pharmaceutical composition of hemoglobin;36IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein the secondary packaging apparatus enveloped the primary packaging apparatus;wherein the secondary packaging apparatus is opaque metallized plastic barrier that is gas and moisture impermeable;wherein the secondary packaging apparatus maintains less than about 5% Oxygen in headspace;wherein the packaging apparatus stores the hemoglobin for at least 3 months to about 54 months at 2°C to 8°C and the hemoglobin remains physiologically active;wherein the pharmaceutical composition of hemoglobin maintains metHb below 5%.

[0233] In another embodiment, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0234] In another embodiment, the secondary packaging apparatus maintains less than about 4% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0235] In another embodiment, the secondary packaging apparatus maintains less than about 3% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity7more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0236] In another embodiment, the secondary packaging apparatus maintains less than about 2% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is 37IPTS / 200205494.1Attorney Docket No.: PLG-028WOmaintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0237] In another embodiment, the secondary packaging apparatus maintains less than about 1% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0238] In another embodiment, the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity7more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0239] In another embodiment, the secondary packaging apparatus maintains less than about 0% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0240] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary7packaging comprising physiologically active hemoglobin wherein the metHb is below 4%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0241] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less 38IPTS / 200205494.1Attorney Docket No.: PLG-028WOthan about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 3%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0242] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary' packaging comprising physiologically active hemoglobin wherein the metHb is below 2%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0243] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 1 %; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0244] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3% below 2% below 1%; wherein the physiologically active hemoglobin has a purity' more than 95%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0245] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging 39IPTS / 200205494.1Attorney Docket No.: PLG-028WOcomprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3% below 2% below 1%; wherein the physiologically active hemoglobin has a purity about 99%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at about 2°C to about 8°C.

[0246] In another embodiment, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0247] In another embodiment, the secondary packaging apparatus maintains less than about 4% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0248] In another embodiment, the secondary packaging apparatus maintains less than about 3% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0249] In another embodiment, the secondary packaging apparatus maintains less than about 2% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.40IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0250] In another embodiment, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 1%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0251] In another embodiment, the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0252] In another embodiment, the secondary packaging apparatus maintains 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0253] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 4%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0254] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 3%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the 41IPTS / 200205494.1Attorney Docket No.: PLG-028WOhemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0255] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 2%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0256] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 1%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0257] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3%, below 2%, below 1%; wherein the physiologically active hemoglobin has a purity more than 95%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0258] In another embodiment, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5% in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3%, below 2%, below 1%; wherein the physiologically active hemoglobin has a purity about 99%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 1642IPTS / 200205494.1Attorney Docket No.: PLG-028WOmmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 25°C±2°C.

[0259] In another embodiment, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0260] In another embodiment, the secondary packaging apparatus maintains less than about 4% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0261] In another embodiment, the secondary packaging apparatus maintains less than about 3% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0262] In another embodiment, the secondary packaging apparatus maintains less than about 2% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity7more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0263] In another embodiment, the secondary packaging apparatus maintains less than about 1% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is 43IPTS / 200205494.1Attorney Docket No.: PLG-028WOmaintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0264] In another embodiment, the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0265] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 % , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0266] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 % , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 4%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0267] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 3%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.44IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0268] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 2%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0269] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 1%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0270] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3% below 2% below 1% ; wherein the physiologically active hemoglobin has a purity more than 95%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.

[0271] In another embodiment, the secondary packaging apparatus maintains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% , less than about 0.5%, 0% or negligent oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%, below 4%, below 3% below 2% below 1% ; wherein the physiologically active hemoglobin has a about 99%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for suitable time selected from at least 3 months, at least 6 months, at least 12 months, at least 18 months, and at least 24 months at 40°C.45IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0272] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains 0% or negligent oxygen amount in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity7of more than about 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for at least about 24 months at 2-8°C or the composition is stable for at 12 months at 25°C±2°C or the composition is stable for at least 9 months at 40°C.

[0273] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 2% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 3%; wherein the physiologically active hemoglobin has a purity7of 91%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for at least about 18 months at 2-8°C.

[0274] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 1.6%; wherein the physiologically active hemoglobin has a purity of about 97%; wherein the p50 value of the hemoglobin is maintained is about 14 mmHg; wherein composition is stable for about 6 months at 2-8°C.

[0275] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 2.0%; wherein the physiologically active hemoglobin has a purity7of purity7of about 97%; wherein the p50 value of the hemoglobin is maintained is about 12 mmHg; wherein composition is stable for about 12 months at 2-8°C.

[0276] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the 46IPTS / 200205494.1Attorney Docket No.: PLG-028WOmetHb is about 2.3%; wherein the physiologically active hemoglobin has a purity of about 96%; wherein the p50 value of the hemoglobin is maintained is about 12 mmHg; wherein composition is stable for about 24 months at 2-8°C.

[0277] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 2.5%; wherein the physiologically active hemoglobin has a purity of more than about 90%; wherein the p50 value of the hemoglobin is maintained is about 12 mmHg; wherein composition is stable for about 54 months at 2-8°C.

[0278] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 2.0%; wherein the physiologically active hemoglobin has a purity of about 98%; ; wherein the p50 value of the hemoglobin is maintained is about 12 mmHg; wherein composition is stable for about 3 months at 25°C±2°C.

[0279] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 1.7% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 2.0%; wherein the physiologically active hemoglobin has a purity of about 96%; wherein the p50 value of the hemoglobin is maintained is about 13 mmHg; wherein composition is stable for about 6 months at 25°C±2°C.

[0280] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 2.3%; wherein the physiologically active hemoglobin has a purity of about 96%; wherein the p50 value of the hemoglobin is maintained is about 12 mmHg; wherein composition is stable for about 12 months at 25°C±2°C.

[0281] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 0.5% oxygen in the headspace;47IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 2.3%; wherein the physiologically active hemoglobin has a purity of about 96%; wherein the p50 value of the hemoglobin is maintained is about 12 mmHg; wherein composition is stable for about 24 months at 25°C±2°C.

[0282] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains about 1.8% oxygen amount in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 2.0%; wherein the physiologically active hemoglobin has a purity7of more than about 90%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for at least about 6 months at 40°C.

[0283] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains 0% oxygen amount in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 1.7%; wherein the physiologically active hemoglobin has a purity of more than about 95%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for at least about 7 days at 40°C.

[0284] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains about 5.3% oxygen amount in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is about 5.1%; wherein the physiologically active hemoglobin has a purity of more than about 85%; wherein the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein composition is stable for at least about 7 days at 40°C.

[0285] In an embodiment, the present invention provides a packaging apparatus, wherein the hemoglobin in the pharmaceutical composition is conjugated with polyethylene glycol (PEG).

[0286] In an embodiment, hemoglobin in the pharmaceutical composition is PEGylated hemoglobin or PEG-Hb-CO.

[0287] In an embodiment, the present invention provides a primary packaging apparatus comprising the pharmaceutical composition, wherein the composition is ready to use.48IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0288] In an embodiment, the present invention provides a primary packaging apparatus comprising the pharmaceutical composition, wherein the pharmaceutical composition is free of oxygen scavenger.

[0289] In an embodiment, the present invention discloses a packaging apparatus, wherein the primary packaging apparatus is an intravenous bag (IV Bag) as shown in FIG. 7 A.

[0290] In an embodiment, the present invention discloses a packaging apparatus, wherein the primary packaging apparatus is transparent, or opaque or translucent.

[0291] In an embodiment, the present invention discloses a packaging apparatus, wherein the primary packaging apparatus is rectangular in shape having dimensions of about 10”x6.5” inches.

[0292] In an embodiment, the present invention discloses a packaging apparatus, comprising a primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and other excipients; and a secondary packaging apparatus enveloping the primary packaging apparatus to form the packaging apparatus as shown in FIG. 7C.

[0293] In an embodiment, the present invention discloses a packaging apparatus, comprising a primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and other excipients; and a secondary packaging apparatus enveloping the primary packaging apparatus to form the packaging apparatus, wherein the packaging apparatus provides mechanical and environmental protection to pharmaceutical composition.

[0294] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin.

[0295] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin, wherein the hemoglobin is biologically active and isolated from animal; wherein the hemoglobin is PEGylated; wherein the hemoglobin is carboxylated; wherein the hemoglobin is reoxygenated; wherein the hemoglobin is free of viruses and other pathogens; wherein the hemoglobin is de-oxygenated and heated at high temperature more than 60°C for about more than 5 hours; thereafter the deoxygenated hemoglobin is re-oxygenated; wherein the hemoglobin is non-natural.

[0296] In an embodiment, the present invention discloses a packaging apparatus, wherein the primary packaging apparatus is enveloped within the secondary packaging apparatus to form a packaging apparatus as shown in FIG. 7C.49IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0297] In an embodiment, the present invention provides a primary packaging apparatus, wherein the primary apparatus comprises two or more ports as shown in FIG. 7A.

[0298] In an embodiment, the present invention provides a primary packaging apparatus, wherein the primary apparatus comprises a filling port, a sampling port and a spike port.

[0299] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the primary packaging apparatus comprises a moisture vapour transmission rate of about 0.05g / 100 in2 / day to about 0.15 g / 100 in2 / day.

[0300] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the primary packaging apparatus comprises a moisture vapour transmission rate of about 0.05g / 100 in2 / day, about 0.06 g / 100 in2 / day, about 0.07g / 100 in2 / day, about 0.08 g / 100 in2 / day, about 0.09 g / 100 in2 / day, about 0.10 g / 100 in2 / day, about 0.11 g / 100 in2 / day, about 0.12 g / 100 in2 / day, about 0.13 g / 100 in2 / day, about 0.14 g / 100 in2 / day and about 0.15 g / 100 in2 / day.

[0301] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the primary packaging apparatus comprises a moisture vapour transmission rate of about 0.11g / 100 in2 / day.

[0302] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the pri mary packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the primary packaging apparatus comprises an oxygen permeability of about 0.15 cm3 / 100 in2 / day to about 0.3 cm3 / 100 in2 / day.

[0303] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary' packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the primary packaging apparatus comprises an oxygen permeability of about 0.15 cm3 / 100 in2 / day, 0.16 cm3 / 100 in2 / day, 0.17 cm3 / 100 in2 / day, 0.18 cm3 / 100 in2 / day, 0.19 cm3 / 100 in2 / day, 0.2 cm3 / 100 in2 / day, 0.21 cm3 / 100 in2 / day, 0.22 cm3 / 100 in2 / day, 0.23 cm3 / 10050IPTS / 200205494.1Attorney Docket No.: PLG-028WOin2 / day, 0.24 cm3 / 100 in2 / day, 0.25 cm3 / 100 in2 / day, 0.26 cm3 / 100 in2 / day, 0.27 cm3 / 100 in2 / day, 0.28 cm3 / 100 in2 / day, 0.29 cm3 / 100 in2 / day, and about 0.30 cm3 / 100 in2 / day.

[0304] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the primary packaging apparatus comprises an oxygen permeability of about 0.28 cm3 / 100 in2 / day.

[0305] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the primary packaging apparatus comprises a moisture vapour transmission rate of about of about 0.05g / 100 in2 / day to about 0.15 g / 100 in2 / day; wherein, the primary packaging apparatus comprises an oxygen permeability of about 0.15 cm3 / 100 in2 / day to about 0.3 cm3 / 100 in2 / day.

[0306] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the primary packaging apparatus comprises a moisture vapour transmission rate of about 0.1 lg / 100 in2 / day; wherein, the primary packaging apparatus is having an oxygen permeability of about 0.28 cm3 / 100 in2 / day.

[0307] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus has an inward layer and an outward layer.

[0308] In an embodiment, the inward layer of the primary packaging apparatus holds the pharmaceutical composition as shown in 7 (c).

[0309] In an embodiment, the outward layer of the primary packaging apparatus is in contact with the secondary packaging apparatus as shown in 7 (c).

[0310] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is a multi-layered metallized polymer bag as shown in FIG. 7B.

[0311] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is Mylar pouches.51IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0312] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is gas impermeable.

[0313] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus has an inward layer and an outward layer.

[0314] In an embodiment, the inward layer of the secondary packaging apparatus envelops the primary packaging apparatus as shown in 7 (c).

[0315] In an embodiment, the outward layer of the secondary packaging apparatus is in contact with the outer environment as shown in 7 (c).

[0316] In an embodiment, the outward layer of the secondary packaging apparatus prevents the contact of the primary packaging apparatus with the external environmental conditions as shown in 7 (c).

[0317] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is oxygen impermeable.

[0318] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus. In yet another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is configured to shield the primary packaging from gaseous exchange, humidity, and light, preventing oxygenation and degradation of the hemoglobin composition, thereby preserving the integrity and stability of the pharmaceutical composition.

[0319] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus comprises inert gas; wherein the headspace of secondary packaging is substantially free of oxygen.

[0320] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus bears the weight of primary packaging apparatus.

[0321] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus holds primary packaging apparatus.52IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0322] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% less than about 1%, less than about 0.5% oxygen in the headspace.

[0323] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% less than about 1%, less than about 0.5% oxygen in the headspace. In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 5% oxygen in the headspace.

[0324] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains the oxygen in the headspace through inert gas sparging.

[0325] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein sparged inert gas in the secondary packaging apparatus is selected from nitrogen, carbon dioxide, helium, neon, argon, krypton, xenon, radon, and oganesson.

[0326] In yet another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein sparged inert gas in the secondary packaging apparatus is argon.

[0327] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains gaseous mixture of argon and less than about 5% oxygen in the headspace.

[0328] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains gaseous mixture of an inert gas and oxygen in the headspace, wherein the secondary packaging apparatus is substantially free of oxygen.53IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0329] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains gaseous mixture of an inert gas and oxygen in the headspace, wherein the secondary packaging apparatus is free of oxygen; comprising gases other than oxygen.

[0330] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein, the secondary packaging comprises a moisture vapour transmission rate of less than about 0.0010 g / 100 in2 / day.

[0331] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein, the secondary packaging comprises a moisture vapour transmission rate of less than about 0.0010 g / 100 in2 / day, less than about 0.0009 g / 100 in2 / day, less than about 0.0008 g / 100 in2 / day, less than about 0.0007 g / 100 in2 / day, less than about 0.0006 g / 100 in2 / day, less than about 0.0005 g / 100 in2 / day, and less than about 0.0004 g / 100 in2 / day.

[0332] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein, the secondary packaging comprises a moisture vapour transmission rate of about 0.0006 g / 100 in2 / day.

[0333] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein, the secondary packaging comprises an oxygen permeability of less than about 0.0010 cm3 / 100 in2 / day.

[0334] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein, the secondary packaging comprises an oxygen permeability of less than about 0.0010 cm3 / 100 in2 / day, less than about 0.0009 cm3 / 100 in2 / day, less than about 0.0008 cm3 / 100 in2 / day, less than about 0.0007 cm3 / 100 in2 / day, less than about 0.0006 cm3 / 100 in2 / day, less than about 0.0005 cm3 / 100 in2 / day and less than about 0.0004 cm3 / 100 in2 / day.

[0335] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein, the secondary packaging comprises an oxygen permeability of about 0.0005 cm3 / 100 in2 / day.

[0336] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the 54IPTS / 200205494.1Attorney Docket No.: PLG-028WOprimary packaging apparatus holds the pharmaceutical composition of hemoglobin; wherein, the secondary packaging apparatus envelops the primary packaging apparatus; wherein the secondary packaging apparatus maintains less than 5% oxygen in the headspace; wherein, the secondary packaging apparatus is gas impermeable; wherein, the secondary packaging comprises a moisture vapour transmission rate of less than about 0.0010 g / 100 in2 / day; wherein, the secondary packaging comprises an oxygen permeability of less than about 0.0010 cm3 / 100 in2 / day.

[0337] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus holds the pharmaceutical composition of hemoglobin; w herein, the secondary packaging apparatus envelops the primary packaging apparatus; wherein the secondary packaging apparatus maintains less than 5% oxygen in the headspace; wherein, the secondary packaging apparatus is gas impermeable; wherein, the secondary packaging comprises a moisture vapour transmission rate of about 0.0006 g / 100 in2 / day; wherein, the secondary packaging comprises an oxygen permeability of about 0.0005 cm3 / 100 in2 / day.

[0338] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains less than about 5% oxygen and less than 5% metHb in the headspace.

[0339] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is configured to shield the primary packaging apparatus containing the pharmaceutical composition from one or more condition selected from fluctuation in oxygen, fluctuation in humidity, effect of light, thereby preserving the integrity and stability of the pharmaceutical composition.

[0340] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus envelops the primary packaging apparatus to form a single unit packaging apparatus.

[0341] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the packaging apparatus holds the pharmaceutical composition.

[0342] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the 55IPTS / 200205494.1Attorney Docket No.: PLG-028WOprimary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition maintains p50 value in the range of about 5mmHg to about 18 mmHg.

[0343] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition maintains p50 value in the range of about 7mmHg to about 16 mmHg.

[0344] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition maintains p50 value about 5 mmHg, about 6 mmHg, about 7 mmHg, about 8 mmHg, about 9 mmHg, about 10 mmHg, about 11 mmHg, about 12 mmHg, about 13 mmHg, about 14 mmHg, about 15 mmHg, about 16 mmHg, about 17 mmHg, and about 18 mmHg as analysed by Hemox analyzer.

[0345] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition maintains p50 value 7 mmHg, about 8 mmHg, about 9 mmHg, about 10 mmHg, about 11 mmHg, about 12 mmHg, about 13 mmHg, about 14 mmHg, about 15 mmHg, and about 16 mmHg, as analysed by Hemox analyzer.

[0346] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a primary’ packaging apparatus;b. a secondary packaging apparatus;wherein the primary packaging apparatus comprises a pharmaceutical composition of hemoglobin;wherein, the hemoglobin in the pharmaceutical composition is conjugated with polyethylene glycol (PEG);wherein, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace;wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%;wherein, the pharmaceutical composition has a purity of more than 90%;56IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein, the stored pharmaceutical composition has stability for at least 3 months to about 54 months at about 2°C to about 8°C.

[0347] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a primary' packaging apparatus;b. a secondary packaging apparatus;wherein the primary packaging apparatus comprises a pharmaceutical composition of hemoglobin;wherein, the hemoglobin in the pharmaceutical composition is conjugated with polyethylene glycol (PEG);wherein, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace;wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%;wherein, the pharmaceutical composition has a purity of more than 90%; wherein, the stored pharmaceutical composition has stability for at least three months to about 24 months at 25°C±2°C.

[0348] In an embodiment, the present invention provides a packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a primary’ packaging apparatus;b. a secondary packaging apparatus;wherein the primary packaging apparatus comprises a pharmaceutical composition of hemoglobin;wherein, the hemoglobin in the pharmaceutical composition is conjugated with polyethylene glycol (PEG);wherein, the secondary packaging apparatus maintains less than about 5% oxygen in the headspace;wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%;wherein, the pharmaceutical composition has a purity of more than 90%; wherein, the stored pharmaceutical composition has stability for about 1 month to about 9 months at 40°C.wherein, the stored physiologically active composition of maintains the p50 value in the range of 7mmHg to about 16 mmHg as analysed by Hemox analyser.57IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0349] In an embodiment, the packaging apparatus stores the hemoglobin at least for at least 3 months at room temperature and the hemoglobin remains physiologically active.

[0350] In an embodiment, the packaging apparatus stores the hemoglobin for at least 3 months at 25°C±2°C and the hemoglobin remains physiologically active.

[0351] In an embodiment, the packaging apparatus stores the hemoglobin for at least 6 months at 25°C±2°C and the hemoglobin remains physiologically active.

[0352] In an embodiment, the packaging apparatus stores the hemoglobin for at least 9 months at 25°C±2°C and the hemoglobin remains physiologically active.

[0353] In an embodiment, the packaging apparatus stores the hemoglobin for at least 12 months at 25°C±2°C and the hemoglobin remains physiologically active.

[0354] In an embodiment, the packaging apparatus stores the hemoglobin for at least 15 months at 25°C±2°C and the hemoglobin remains physiologically active.

[0355] In an embodiment, the packaging apparatus stores the hemoglobin for at least 18 months at 25°C±2°C and the hemoglobin remains physiologically active.

[0356] In an embodiment, the packaging apparatus stores the hemoglobin for at least 21 months at 25°C±2°C and the hemoglobin remains physiologically active.

[0357] In an embodiment, the packaging apparatus stores the hemoglobin for at least 24 months at 25°C±2°C and the hemoglobin remains physiologically active

[0358] In an embodiment, the packaging apparatus stores the hemoglobin with metHb less than 5% for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least for 15 months, at least 18 months, at least 21 months, and at least 24 months, at 25°C±2°C and the hemoglobin remains physiologically active.

[0359] In an embodiment, the packaging apparatus stores the hemoglobin for at least 1 month at 40°C and the hemoglobin remains physiologically active.

[0360] In an embodiment, the packaging apparatus stores the hemoglobin for at least 2 months at 40°C and the hemoglobin remains physiologically active.

[0361] In an embodiment, the packaging apparatus stores the hemoglobin for at least 3 months at 40°C and the hemoglobin remains physiologically active.

[0362] In an embodiment, the packaging apparatus stores the hemoglobin for at least 4 months at 40°C and the hemoglobin remains physiologically active.

[0363] In an embodiment, the packaging apparatus stores the hemoglobin for at least 5 months at 40 °C and the hemoglobin remains physiologically active.

[0364] In an embodiment, the packaging apparatus stores the hemoglobin for at least 6 months at 40°C and the hemoglobin remains physiologically active.58IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0365] In an embodiment, the packaging apparatus stores the hemoglobin for at least 7 months at 40°C and the hemoglobin remains physiologically active.

[0366] In an embodiment, the packaging apparatus stores the hemoglobin for at least 8 months at 40°C and the hemoglobin remains physiologically active.

[0367] In an embodiment, the packaging apparatus stores the hemoglobin for at least 9 months at 40°C and the hemoglobin remains physiologically active.

[0368] In an embodiment, the packaging apparatus stores the hemoglobin with metHb less than 5% for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months at 40°C and the hemoglobin remains physiologically active.In an embodiment, the packaging apparatus stores the hemoglobin at least for 18 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0369] In an embodiment, the packaging apparatus stores the hemoglobin at least for 21 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0370] In an embodiment, the packaging apparatus stores the hemoglobin at least for 24 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0371] In an embodiment, the packaging apparatus stores the hemoglobin at least for 27 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0372] In an embodiment, the packaging apparatus stores the hemoglobin at least for 30 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0373] In an embodiment, the packaging apparatus stores the hemoglobin at least for 33 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0374] In an embodiment, the packaging apparatus stores the hemoglobin at least for 36 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0375] In an embodiment, the packaging apparatus stores the hemoglobin at least for 39 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0376] In an embodiment, the packaging apparatus stores the hemoglobin at least for 42 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0377] In an embodiment, the packaging apparatus stores the hemoglobin at least for 45 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0378] In an embodiment, the packaging apparatus stores the hemoglobin at least for 48 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0379] In an embodiment, the packaging apparatus stores the hemoglobin at least for 51 months at 2°C to 8°C and the hemoglobin remains physiologically active.59IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0380] In an embodiment, the packaging apparatus stores the hemoglobin at least for 54 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0381] In an embodiment, the packaging apparatus stores the hemoglobin with metHb less than 5% for at least 24 months, at least 27 months, at least 30 months, at least 33 months, at least 36 months, at least 39 months, at least 42 months, at least 45 months, at least 48 months, at least 51 months, and at least 54 months, at 2-8°C and the hemoglobin remains physiologically active.

[0382] In an embodiment, the packaging apparatus fits in the standard equipment in an operating or emergency room such as, for example, a pressure infuser and / or warmer. Either manual "pressure cuffs" or automated infusers.

[0383] In an embodiment, the present invention discloses a packaging apparatus that is cost effective.

[0384] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition is stable for at least about 1 month to about 9 months at 40°C, and for even longer periods, which is very difficult as the level of metHb in hemoglobin gradually increases, even when the composition is not in contact of oxygen.

[0385] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition is stable for at least about 3 months to at least about 54 months at 2-8°C, and for even longer periods, which is very difficult as the level of metHb in hemoglobin gradually increases, even when the composition is not in contact of oxygen.

[0386] In another embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin and other excipients, wherein the composition is stable for at least about 1 month to at least about 24 months at 25°C±2°C, and for even longer periods, which is very difficult as the level of metHb in hemoglobin gradually increases, even when the composition is not in contact of oxygen.

[0387] In an embodiment, the present invention discloses a packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the primary packaging apparatus alone is not sufficient because the primary packaging permits oxygen ingress, leading to hemoglobin oxygenation and degradation during storage. Therefore,60IPTS / 200205494.1Attorney Docket No.: PLG-028WOa secondary, gas-impermeable packaging apparatus is required to prevent oxygen exposure, ensuring the stability and efficacy of the hemoglobin composition.

[0388] In an embodiment, the present invention provides an improved packaging apparatus suitable to store physiologically active hemoglobin comprising:a. a primary' packaging apparatus;b. a secondary packaging apparatus;wherein, the primary packaging apparatus comprises pharmaceutical composition of PEGylated hemoglobin;wherein the primary packaging apparatus is enveloped in the secondary packaging apparatus;wherein, the secondary packaging apparatus is sparged with an inert gas to displace oxygen;wherein, the stored hemoglobin composition purity is more than about 90%; wherein, the secondary packaging apparatus maintains oxygen less than about 5% in the headspace.

[0389] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin, in a suitable packaging apparatus, the method comprising:a. filling the pharmaceutical composition in a primary packaging apparatus:b. enveloping the primary packaging apparatus with a secondary packaging apparatus to form the packaging apparatusc. sealing the secondary packaging;d. storing the packaging apparatus containing the pharmaceutical composition; wherein the primary packaging apparatus comprises a pharmaceutical composition of hemoglobin and other excipients;wherein, pharmaceutical composition stored in the packaging apparatus remains physiologically active.

[0390] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of PEGylated hemoglobin, in a packaging apparatus, the method comprising:a. filling the pharmaceutical composition in a primary' packaging apparatus;b. enveloping the primary packaging apparatus with a secondary packaging apparatus to form the packaging apparatusc. sealing the secondary packaging;61IPTS / 200205494.1Attorney Docket No.: PLG-028WOd. storing the packaging apparatus containing the pharmaceutical composition; wherein the primary packaging apparatus comprises a pharmaceutical composition of PEGylated hemoglobin and other excipients;wherein, the secondary packaging apparatus maintains oxygen less than about 5% in the headspace;wherein the stored pharmaceutical composition maintains metHb below 5% analyzed by oximetry;wherein, the stored hemoglobin composition purity is more than about 90%; wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C.

[0391] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is sealed at seal dwell of about 1 second, about 1.1 second, about 1.2 second, about 1.3 second, about 1.4 second, about 1.5 second, about 1.6 second, about 1.7 second, about 1.8 second, about 1.9 second, about 2.0 second, about 2.1 second, about 2.2 second, about 2.3 second, about 2.4 second, about 2.5 second, about 2.6 second, about 2.7 second, about 2.8 second, about 2.9 second, and about 3.0 second.

[0392] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is sealed at seal dwell of about 1.3 second, about 1.4 second, about 1.5 second, about 1.6 second, about 1.7 second, about 1.8 second, about 1.9 second, and about 2.0 second.

[0393] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is sealed at seal dwell of about 1.5 second.

[0394] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is sealed at takt time selected from about 1.5 second, about 1.6 second, about 1.7 second, about 1.8 second, about 1.9 second, about 2.0 second, about 2.1 second, about 2.2 second, about 2.3 second, about 2.4 second, about 2.562IPTS / 200205494.1Attorney Docket No.: PLG-028WOsecond, about 2.6 second, about 2.7 second, about 2.8 second, about 2.9 second, about 3.0 second, about 3.1 second, about 3.2 second, about 3.3 second, about 3.4 second, and about 3.5 second.

[0395] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is sealed at takt time selected from about 1.8 second, about 1.9 second, about 2.0 second, about 2.1 second, about 2.2 second, about 2.3 second, about 2.4 second, and about 2.5 second.

[0396] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is sealed at takt time of about 2.0 second.

[0397] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus sealed at seal pressure selected from about 40 psi, about 41 psi, about 42 psi, about 43 psi, about 44 psi, about 45 psi, about 46 psi, about 47 psi, about 48 psi, about 49 psi, about 50 psi, about 51 psi, about 52 psi, about 53 psi, about 54 psi, about 55 psi, about 56 psi, about 57 psi, about 58 psi, about 59 psi, about 60 psi, about 61 psi, about 62 psi, about 63, psi, about 64 psi, about 65 psi, about 66psi, about 67 psi, about 68 psi, about 69 psi, about 70 psi, about 71 psi, about 72 psi, about 73 psi about 74 psi and about 75psi.

[0398] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus sealed at seal pressure selected from about 45 psi, about 46 psi, about 47 psi, about 48 psi, about 49 psi, about 50 psi, about 51 psi, about 52 psi, about 53 psi, about 54 psi, about 55 psi, about 56 psi, about 57 psi, about 58 psi, about 59 psi, and about 60 psi.

[0399] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus sealed at seal pressure of about 50 psi.63IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0400] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus sealed at temperature selected from about 330°F, about 340°F, about 350°F, about 360°F, about 370°F, about 380°F, about 390°F, and about 400°F.

[0401] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus sealed at temperature of about 370°F.

[0402] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of PEGylated hemoglobin, in a packaging apparatus, comprising:a. filling the pharmaceutical composition in a primary packaging apparatus;b. enveloping the primary packaging apparatus with a secondary packaging apparatus to form the packaging apparatusc. sealing the secondary packaging;d. storing the packaging apparatus containing the pharmaceutical composition; wherein the primary packaging apparatus comprises a pharmaceutical composition of PEGylated hemoglobin and other excipients;wherein, the secondary packaging apparatus maintains oxygen less than about 5% in the headspace;wherein, the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein, the stored pharmaceutical composition maintains metHb below 5% analyzed by oximetry;wherein, the secondary packaging apparatus is sealed at seal dwell of about 1.5 second; wherein, the secondary' packaging apparatus is sealed at takt time of about 2.0 second; wherein, the secondary packaging apparatus is sealed at seal pressure of about 50 psi; wherein, the secondary packaging apparatus is sealed at a temperature of about 370°F.

[0403] In an embodiment, the present invention discloses a method for storing of pharmaceutical composition of hemoglobin in a packaging apparatus, wherein the packaging apparatus comprising a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus is sealed at seal dwell of about 1.5 Sec, Takt Time of about 2.0 Sec, Seal pressure at about 50 psi and temperature at about 370 °F.64IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0404] In an embodiment, the present invention provides a pharmaceutical composition packaging system, comprising:a. a primary packaging apparatus comprising PEGylated hemoglobin composition; b. a secondary packaging apparatus enveloping the primary packaging apparatus; wherein the packaging apparatus stores the hemoglobin for at least 3 months; wherein, the secondary packaging apparatus is sealed at seal dwell of about 1 second to about 3.0 second;wherein, the secondary packaging apparatus is sealed at takt time of about 1.5 second to about 3.5 seconds;wherein, the secondary packaging apparatus is sealed at temperature of about 330°F to about 400°F;wherein, the secondary packaging apparatus is sealed at seal pressure of about 25 psi to about 75 psi.

[0405] In another embodiment, the present invention discloses a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, comprising, storing the pharmaceutical composition of hemoglobin in a suitable packaging apparatus consisting of:a. a primary' packaging apparatus;b. a secondary' packaging apparatus;wherein the primary packaging apparatus comprises a pharmaceutical composition of hemoglobin and other excipients;wherein, the secondary packaging apparatus envelops the primary packaging apparatus; wherein the secondary packaging apparatus maintains less than about 5% Oxygen in the headspace through inert gas sparging;wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein the hemoglobin composition remains physiologically active;wherein, the stored hemoglobin composition purity is more than about 90%; wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%.

[0406] In an embodiment, the present invention discloses a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, wherein the hemoglobin composition has a purity of more than about 90%, more than about 91 %. More65IPTS / 200205494.1Attorney Docket No.: PLG-028WOthan about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, and more than about 99%.

[0407] In an embodiment, the present invention discloses a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, wherein the hemoglobin composition has a purity of more than about 95%, more than about 96%, more than about 97%, more than about 98%, and about 99%.

[0408] In an embodiment, the present invention discloses a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, wherein the hemoglobin composition has a purity of more than about 90%.

[0409] In an embodiment, the present invention discloses a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%.

[0410] In an embodiment, the present invention discloses a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%, below 4%, below 3%, below 2%, and below 1%.

[0411] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, comprising hemoglobin molecules covalently conjugated with PEG polymers, and other suitable excipients selected from buffer, stabilizers, tonicity modifier, and rehydrating salts;wherein, the packaging apparatus comprising, a primary packaging apparatus and a secondary packaging apparatus;wherein, the secondary packaging apparatus envelops the primary packaging apparatus; wherein, the pharmaceutical composition of hemoglobin is contained in the primary packaging apparatus;wherein, the pharmaceutical composition has stability for at least three months at room temperature;wherein, the pharmaceutical composition has stability for at least 3 months at refrigerated conditions;wherein, the pharmaceutical composition has stability for at least 1 month at 40°C.

[0412] In another embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein pharmaceutical composition has stability for at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, and at least 54 months at 2°C to 8°C; wherein the pharmaceutical 66IPTS / 200205494.1Attorney Docket No.: PLG-028WOcomposition maintains metHb below 5%; wherein, the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein, the pharmaceutical composition maintains hemoglobin purity more than 90%.

[0413] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein the pharmaceutical composition has stability for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, and at least 24 months, at 25°C±2°C; wherein the pharmaceutical composition maintains metHb below 5%; wherein, the p50 value of the hemoglobin is maintained in the range of 7 mmHg to about 16 mmHg; wherein the pharmaceutical composition maintains hemoglobin purity more than 90%.

[0414] In another embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein, the packaging apparatus comprising, a primary packaging apparatus and a secondary packaging apparatus, wherein the secondary packaging apparatus maintains oxygen below 5%, enabling consistency in the pharmaceutical composition product quality across batches.

[0415] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein the pharmaceutical composition has stability for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months and at least 9 months at 40°C, wherein the pharmaceutical composition maintains metHb below 5%, wherein the pharmaceutical composition maintains hemoglobin purity more than 90%.

[0416] In an embodiment, the pharmaceutical composition, the primary packaging apparatus comprises PEGylated hemoglobin molecules and other excipients selected from buffer, stabilizers, tonicity modifier, and rehydrating salts.

[0417] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises buffer selected from citrate, Tris, MOPS, HEPES, phosphate buffer, and sodium acetate and ammonia buffers in an amount of about 1 mM to about 5 mM.

[0418] In an embodiment, the buffer is phosphate buffer in an amount of about 1 mM to about 5 mM.

[0419] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises stabilizer selected from amine stabilizers, sodium metabisulfite, polyethylene glycol, EDTA and polysorbates.67IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0420] In an embodiment, the stabilizer is an amine stabilizer preferably cysteine. In an embodiment, the cysteine is maintained in an amount of more than 5 mM.

[0421] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises tonicity modifier selected from potassium chloride, glycerin, lactose, sodium chloride, dextrose, and mannitol in an amount of about 100 mM to about 200 mM.

[0422] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises tonicity modifier is sodium chloride in an amount of about 150 mM.

[0423] In an embodiment, the present invention discloses a pharmaceutical composition of hemoglobin, wherein the composition comprises rehydrating salts are selected from sodium chloride, sodium bicarbonate, magnesium sulfate hetpahydrate, calcium chloride dihydrate, and potassium chloride in an amount of about 0.5 mM to about 5 mM.

[0424] In another embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein pharmaceutical composition has stability for at least 24 months at refrigerated conditions.

[0425] In another embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein pharmaceutical composition has stability for at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, and at least 54 months at 2°C to 8°C.

[0426] In another embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein pharmaceutical composition has stability for at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, and at least 54 months at 2°C to 8°C, wherein the pharmaceutical composition maintains metHb below 5%.

[0427] In another embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein pharmaceutical composition has stability for at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, and at least 54 months at 2°C to 8°C, wherein the pharmaceutical composition maintains metHb below 5%, wherein the pharmaceutical composition maintains hemoglobin purity more than 90%.

[0428] In another embodiment, the present invention discloses a pharmaceutical composition of hemoglobin stored in a packaging apparatus, wherein pharmaceutical composition has stability for at least 24 months at room temperature, wherein the 68IPTS / 200205494.1Attorney Docket No.: PLG-028WOpharmaceutical composition maintains metHb below 5%, wherein the pharmaceutical composition maintains hemoglobin purity more than 90%.

[0429] In certain embodiment, the present invention discloses a method of treating a disease condition in a subject in need thereof, by administering to the subject a therapeutically effective amount of pharmaceutical composition, wherein the pharmaceutical composition comprising hemoglobin and suitable excipients; wherein, the hemoglobin composition is stored in a suitable packaging apparatus, the packaging apparatus consisting of:a. a primary packaging apparatus;b. a secondary packaging apparatus;wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein the hemoglobin composition remains physiologically active;wherein the stored pharmaceutical composition of hemoglobin maintains metHb below 5%.

[0430] In an embodiment, the present invention discloses a method of treating a disease condition, wherein, the disease condition is selected from cancer, haemorrhagic shock, intracranial haemorrhage, hypoxia, necrosis, myocardial injury, acute myocardial infarction, hypertension, pulmonary hypertension.

[0431] In another embodiment, the present invention discloses a method of administering physiologically active hemoglobin composition stored in a suitable packaging apparatus to a subject in need thereof, wherein the packaging apparatus consists of a primary packaging apparatus and a secondary packaging apparatus, the method comprising:a. opening the secondary packaging apparatus to access the primary packaging apparatus comprising pharmaceutical composition;b. administering to the subject a therapeutically effective amount of pharmaceutical composition stored in the primary packaging;wherein the pharmaceutical composition comprising PEGylated hemoglobin and suitable excipients;wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein the hemoglobin composition in the packaging apparatus remains physiologically active;69IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C.

[0432] In an embodiment, the present invention discloses a method of administering physiologically active hemoglobin composition stored in a suitable packaging apparatus to a subject in need thereof, wherein the subject in need is administered with a therapeutically effective amount of pharmaceutical composition; wherein the therapeutically effective amount of PEGylated hemoglobin is selected from about 300 mg / kg to about 1500 mg / kg. In an embodiment, the therapeutic effective amount of PEG-HB-CO is selected from about 500 mg / kg to about 1200 mg / kg. In an embodiment, the therapeutic effective amount of PEG-HB-CO is selected from about 800 mg / kg to about 1000 mg / kg.

[0433] In an embodiment, the present invention discloses a method of administering physiologically active hemoglobin composition stored in a suitable packaging apparatus to a subject in need thereof, wherein the subject in need is administered with a therapeutically effective amount of pharmaceutical composition; wherein the therapeutically effective amount of PEGylated hemoglobin 1000 mg / kg.

[0434] In an embodiment, the therapeutic effective amount of PEGylated hemoglobin is administered in at least two cycles in a subject in need thereof.

[0435] In an embodiment, the therapeutic effective amount of PEGylated hemoglobin is administered in at least two cycles in a subject in need thereof, wherein the first infusion cycle has higher flow rate than the second cycle.

[0436] In an embodiment, the first infusion cycle delivers a lower amount of therapeutic effective amount of PEGylated hemoglobin than the second cycle.

[0437] In an embodiment, the first infusion cycle maintains a flow rate of about 3 ml / min, about 3.5 ml / min, about 4 ml / min, about 4.5 ml / min, about 5 ml / min, about 5.5 ml / min, about 6ml / min, about 6.5ml / min, about 7ml / min, about 7.5 ml / min, 8 ml / min based on the weight of subject and effective amount of pegylated hemoglobin needs to be administrated.

[0438] In an embodiment, the first infusion cycle delivers PEG-Hb-CO in the range of about 100 ml, about 105 ml, about 110 ml, about 115 ml, about 120 ml, about 125 ml, about 130 ml, about 135 ml, about 140 ml, about 145 ml, about 150 ml, about 155 ml, about 160 ml, about 165 ml about 170 ml, about 175 ml, about 180 ml, about 185 ml, about 190 ml, and about 195 ml, about 200 ml wherein the total dose volume is at least 500ml.

[0439] In an embodiment, the first infusion cycle maintains higher flow rate than second or subsequent cycle at least by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 70IPTS / 200205494.1Attorney Docket No.: PLG-028WO65%, about 70%, about 75%, about 80%, about 90%, about 95% and about 100% than the second cycle.

[0440] In an embodiment, the first infusion cycle maintains a flow rate of about 6.7 ml / min and delivers about 200ml.

[0441] In an embodiment, the second cycle maintains flow rate of about 1.5ml / min, about 1.6ml / min, about 1.3 ml / min, about 1.4 ml / min, about 1.5 ml / min, about 1.6 ml / min, about 1.7 ml / min, about 1.8ml / min, about 1.9 ml / min, about 2.0 ml / min, about 2.1 ml / min, about 2.2 ml / min, about 2.3 ml / min, about 2.4 ml / min, about 2.5 ml / min, about 2.6 ml / min, about 2.7 ml / min, about 2.8ml / min, about 2.9 ml / min, about 3.0 ml / min, about 3.1 ml / min, about 3.2 ml / min, about 3.3 ml / min, about 3.4 ml / min, about 3.5 ml / min, about 3.6 ml / min, about 3.7 ml / min, about 3.8ml / min, about 3.9 ml / min, about 4.0 ml / min, about 4.1 ml / min, about 4.2 ml / min, about 4.3 ml / min, about 4.4 ml / min, and about 4.5 ml / min.

[0442] In an embodiment, the second cycle delivers PEG-HB-CO in the range of about 275 ml, about 300 ml, about 350 ml, about 355 ml, about 360 ml, about 365 ml, about 370 ml, about 375 ml, about 380 ml, about 385 ml, about 390 ml, about 395 ml, about 400 ml, about 405 ml, about 410 ml, about 415 ml, about 420 ml, about 425 ml, about 430 ml, about 435 ml, about 440 ml, about 445 ml, about 450 ml, about 455 ml, about 460 ml, about 465 ml, about 470 ml, about 475 ml, about 480 ml, and about 485 ml, about 490 ml, about 495 ml, and about 500 ml.

[0443] In certain embodiment, the second cycle last for least about 10 min, about 20 min, about 30 min, about 40 min, about 50 min, about 60 min, about 70 min, about 80 min and about 90 min.

[0444] In an embodiment, the second infusion cycle maintains a flow rate of about 3.3 ml / min and delivers about 300ml.

[0445] In an embodiment, the present invention discloses a process for storing a physiologically active hemoglobin composition stored in a suitable packaging apparatus; wherein the packaging apparatus consists of a primary packaging apparatus and a secondary packaging apparatus, the process comprising:a. washing fresh whole blood collected from animal sources to produce washed RBCs; b. extracting hemoglobin from the RBC’s;c. performing filtration of the extracted hemoglobin;d. performing ultrafiltration and concentration of hemoglobin;e. performing deoxygenation of ultrafiltered and concentrated hemoglobin;71IPTS / 200205494.1Attorney Docket No.: PLG-028WOf. performing heat inactivation of deoxygenated hemoglobin for reduction of virus and / or prion by heat treatment at suitable temperature;g. performing reoxygenation of heat-treated deoxygenated hemoglobin to produce oxygenated hemoglobin composition;h. optionally performing PEGylation of the oxygenated hemoglobin composition; i. optionally performing fdtration of the pegylated hemoglobin;j. optionally performing carboxylation process to produce carboxy lated PEGylated hemoglobin composition;k. filling the pharmaceutical composition of carboxylated PEGylated hemoglobin obtained from (i) in the primary packaging apparatus;l. enveloping the primary packaging apparatus with a secondary packaging apparatus to form a packaging apparatus;m. sealing the packaging apparatus; and,n. storing the packaging apparatus containing the pharmaceutical composition; wherein the viral inactivation of deoxygenated hemoglobin is performed by heat treatment and maintains L-cysteine concentration in deoxygenated hemoglobin during step f) for more than 5 mM;wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein the hemoglobin composition stored in the packaging apparatus remains physiologically active;wherein the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C.

[0446] In an embodiment, the present invention discloses a process for storing a physiologically active hemoglobin stored in a suitable packaging apparatus, wherein the hemoglobin composition comprises impurities selected from charge variants, Low molecular weight impurities, high molecular weight impurities, HCPs and HC DNA.

[0447] In an embodiment, the charge variants are below 25%.

[0448] In an embodiment, the charge variants are selected from acidic variants and basic variants.

[0449] In another embodiment, the acidic variants are less than 15%, less than 14%, less than 13%, less than 12%, less than 11%. less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% or less.

[0450] In another embodiment, the acidic variants are less than 15%.72IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0451] In another embodiment, the basic variants are less than 15%, less than 14%, less than 13%, less than 12%, less than 11%. less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5% or less.

[0452] In another embodiment, the basic variants are less than 15%.

[0453] In an embodiment, the present invention discloses a process for storing a physiologically active hemoglobin stored in a suitable packaging apparatus, wherein the hemoglobin obtained from (f) is re-oxygenated.

[0454] In an embodiment, the present invention discloses a process for storing a physiologically active hemoglobin stored in a suitable packaging apparatus, wherein the L-cysteine concentration in deoxygenated hemoglobin is maintained more than 5 mM.Pharmaceutical Composition

[0455] In an embodiment, the present disclosure provides a pharmaceutical composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability for at least three months at 25°C±2°C.

[0456] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 6 months at 25°C±2°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0457] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 12 months at 25°C±2°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0458] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 24 months at 25°C±2°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0459] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 30 months at 2°C to 8°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.73IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0460] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 36 months at 2°C to 8°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0461] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 42 months at 2°C to 8°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0462] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 54 months at 2°C to 8°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace.

[0463] In an embodiment, the present disclosure also provides a stable pharmaceutical composition of hemoglobin comprising with hemoglobin and metHb less than about 5% for at least 12 months at 25°C ± 2°C; wherein the composition is stored in suitable packaging and maintains less than about 5% oxygen in the headspace; wherein the composition improved the stability of hemoglobin for at least 12 months at 2°C to 8°C compared to the composition stored in suitable packaging and maintains more than 5% oxygen.

[0464] In an embodiment, the present disclosure provides a pharmaceutical composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability for at least three months at 25°C±2°C and at least for two years at 2°C to 8°C.

[0465] In an embodiment, the present disclosure provides a pharmacal composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability7for at least three months at room temperature and at least for two years at 2°C to 8°C; wherein the pharmaceutical composition comprises less than 5% metHb formation.

[0466] In an embodiment, the primary container is an intravenous bag (IV Bag) and the secondary container is a multi-layered metallized polymer bag that is gas impermeable and opaque (e.g. Mylar pouches).

[0467] In an embodiment, the present disclosure provides a pharmaceutical composition contained in a primary container comprising PEG polymers covalently conjugated 74IPTS / 200205494.1Attorney Docket No.: PLG-028WOwith a hemoglobin molecule; wherein the primary container is enclosed in a secondary container; wherein the secondary container is gas impermeable; wherein the headspace of the secondary container is controlled to contain less than about 5% Oxygen; wherein the pharmaceutical composition has stability for at least three months at room temperature and at least for two years at 2°C to 8°C; wherein the pharmaceutical composition comprises less than 5% metHb formation.

[0468] In an embodiment, the present disclosure provides a pharmaceutical composition contained in a primary container comprising PEG polymers covalently conjugated with a hemoglobin molecule; wherein the primary7container is enclosed in a secondary container; wherein the secondary7container is a multi-layered metallized polymer bag that is gas impermeable and opaque (e.g. Mylar pouches); wherein the headspace of the secondary container is controlled to contain less than about 5% oxygen by flushing an inert gas and followed by heat sealing the secondary7container; wherein the pharmaceutical composition has stability7for at least three months at room temperature and at least for two years at 2°C to 8°C; wherein the pharmaceutical composition comprises less than 5% metHb formation.

[0469] In an embodiment, the present disclosure provides an improved packaging apparatus suitable to store physiologically active hemoglobin comprising:a) Primary7packaging apparatus;b) Secondary7packaging apparatus;wherein the primary packaging apparatus comprising a pharmaceutical composition of hemoglobin;wherein the secondary packaging apparatus enveloped the primary packaging apparatus;wherein the secondary packaging apparatus is opaque metallized plastic barrier that is gas and moisture impermeable;wherein the secondary packaging apparatus maintains less than about 5% Oxygen in headspace;wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2°C to 8°C and the hemoglobin remains physiologically active;wherein the pharmaceutical composition of hemoglobin maintains metHb below 5%; wherein the pharmaceutical composition of hemoglobin maintains p50 value in between 7 to 16 mmHg analysed by Hemox analyser; wherein the hemoglobin is conjugated with polyethylene glycol (PEG).75IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0470] In an embodiment, the packaging apparatus stores the hemoglobin at least for 18 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0471] In an embodiment, the packaging apparatus stores the hemoglobin at least for 24 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0472] In an embodiment, the packaging apparatus stores the hemoglobin at least for 30 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0473] In an embodiment, the packaging apparatus stores the hemoglobin at least for 36 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0474] In an embodiment, the packaging apparatus stores the hemoglobin at least for 40 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0475] In an embodiment, the present disclosure provides an improved packaging apparatus suitable to store physiologically active hemoglobin comprising:a) Primary packaging apparatus;b) Secondary' packaging apparatus;wherein the primary packaging apparatus comprising hemoglobin, buffer, stabilizer; wherein the secondary packaging apparatus enveloped the primary packaging apparatus; wherein the secondary packaging apparatus is gas impermeable;wherein the secondary packaging apparatus maintains gaseous mixture comprising predominantly higher argon and less than about 5% Oxygen;wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0476] In an embodiment, the present disclosure provides a method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, comprising, storing the pharmaceutical composition of hemoglobin in suitable packaging consisting of primary packaging and secondary packaging wherein the primary packaging apparatus comprising pharmaceutical composition of hemoglobin; wherein the secondary packaging apparatus enveloped the primary' packaging apparatus; wherein the secondary' packaging apparatus is sparged with argon; wherein the secondary packaging apparatus maintains less than about 5% Oxygen in the headspace; wherein the packaging apparatus stores the hemoglobin at least for 12 months at 2 to 8°C and the hemoglobin remains physiologically active.

[0477] In an embodiment, the present disclosure provides a packaging and composition system comprising a primary packaging with a hemoglobin composition and a secondary gas-76IPTS / 200205494.1Attorney Docket No.: PLG-028WOimpermeable packaging, wherein the packaging system is configured to maintain less than 5% oxygen in the headspace and preserves hemoglobin stability for at least 24 months.

[0478] In an embodiment, the present disclosure provides a process for preparing a pharmaceutical composition for storage, comprising:a) Primary' packaging apparatus;b) Secondary- packaging apparatus;wherein the pharmaceutical composition is enclosed in a primary packaging apparatus; wherein the primary packaging apparatus is further enclosed in the secondary packaging apparatus;wherein, sparging the secondary packaging apparatus with an inert gas to displace oxygen; and sealing the secondary packaging apparatus with controlled oxygen content in the headspace;wherein secondary container that maintains less than about 5% oxygen in its headspace; wherein the pharmaceutical composition is Pegylated hemoglobin.

[0479] In an embodiment, the present disclosure provides a method for enhancing the stability of a hemoglobin pharmaceutical composition, comprising controlling the oxygen level in the headspace to below 5% through argon sparging; wherein the hemoglobin pharmaceutical composition maintains metHb below 5%; wherein the stability' of the hemoglobin pharmaceutical composition at least for 18 months at 2°C to 8°C and the hemoglobin remains physiologically active.

[0480] In an embodiment, the headspace in the secondary packaging apparatus is maintained below 5%, below 4%, below 3%, below 2%.

[0481] In an embodiment, the present disclosure provides a pharmaceutical composition comprising PEG polymers covalently conjugated with a hemoglobin molecule contained in a primary container; wherein the primary container is further enclosed in an additional outer packaging; wherein the pharmaceutical composition has stability for at least three months at room temperature and at least for two years at 2°C to 8°C; wherein the pharmaceutical composition comprises less than 5% metHb formation.

[0482] The present disclosure describes a heat-sealing recipe that produces a desired secondary packaging seal, free of visual imperfections as well as maintains the seal integrity and subsequently the headspace oxygen content for at least 5 years at refrigerated condition and 2 years at room temperature storage.

[0483] In an embodiment, the secondary bag is sealed at seal dwell of about 1.5 Sec, Takt Time of about 2.0 Sec, Seal pressure at about 50 psi and temperature at about 370 °F.77IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0484] The present disclosure describes a heat-sealing recipe that produces a desired secondary packaging seal, free of visual imperfections as well as maintains the seal integrity and subsequently the headspace oxygen content for at least 5 years at refrigerated condition and 2 years at room temperature storage.

[0485] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at a temperature selected from about 330 F to about 400 F.

[0486] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at a temperature selected from about 330 F, about 340 F, about 350 F, about 360 F, about 370 F, about 380 F, about 390 F, about 400 F.

[0487] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at seal dwell selected from about 1.0 second to about 3.0 second.

[0488] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at seal dwell at about 1.0 second, about 1.1 second, about 1.2 second, about 1.3 second, about 1.4 second, about 1.5 second, about 1.6 second, about 1.7 second, about 1.8 second, about 1.9 second, about 2.0 second.

[0489] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at seal dwell of about 1.5 second.

[0490] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at takt time selected from about 1.5 second to about 3.5 Second.

[0491] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at takt time selected from about 1.5 second, about 1.6 second, about 1.7 second, about 1.8 second, about 1.9 second, about 2.0 second, about 2.1 second, about 2.2 second, about 2.3 second, about 2.4 second, about 2.5 second, about 2.6 second, about 2.7 second, about 2.8 second, about 2.9 second, about 3.0 second, about 3.1 second, about 3.2 second, about 3.3 second, about 3.4 second, about 3.5 Second.

[0492] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at takt time about 2.0 Second.

[0493] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at seal pressure selected from about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29 psi, about 30 psi, about 31 psi, about 32 psi, about 33 psi, about 34 psi, about 35 psi, about 36 psi, about 37 psi, about 38 psi, about 39 psi, about 40 psi, about 41 psi, about 42 psi, about 43 psi, about 44 psi, about 45 psi, about 46 psi, about 47 psi, about 48 psi, about 49 psi, about 50 psi, about 51 psi, about 52 psi, about 53 psi, about 54 psi, about 55 psi, about 56 psi, about 57 psi, about 58 psi, about 59 psi, about 60 psi, about 61 psi, about 62 psi, about 63 psi,78IPTS / 200205494.1Attorney Docket No.: PLG-028WOabout 64 psi, about 65 psi, about 66 psi, about 67 psi, about 68 psi, about 69 psi, about 70 psi, about 71 psi, about 72 psi, about 73 psi, about 74 psi, about 75 psi.

[0494] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at seal pressure selected from about 40 psi, about 41 psi, about 42 psi, about 43 psi, about 44 psi, about 45 psi, about 46 psi, about 47 psi, about 48 psi, about 49 psi, about 50 psi, about 51 psi, about 52 psi, about 53 psi, about 54 psi, about 55 psi, about 56 psi, about 57 psi, about 58 psi, about 59 psi, about 60 psi.

[0495] In an embodiment, the secondary bag and / or secondary packaging apparatus is sealed at seal pressure about 50 psi.

[0496] In an embodiment, the primary container is a flexible IV bag, and the secondary container is a polyester film made from stretched polyethylene terephthalate (PET).

[0497] In an embodiment, the hemoglobin molecule in the present disclosure is PEGylated carboxyhemoglobin (PEG-Hb-CO) molecule.

[0498] In some embodiments, the PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) or hemoglobin composition in the present disclosure maintains metHb below 5%, preferably below 4% during storage at 2-8°C for more than 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, 36 months, 39 months, 42 months, 45 months, 48 months, 51 months, 54 months, 57 months, 61 months, 64 months, 67 months and 71 months. In some embodiments, the p50 value of hemoglobin is 7-16 mmHg analyzed by Hemox Analyzer.

[0499] In some embodiments, the PEGylated hemoglobin or carboxylated hemoglobin (PEG-Hb-CO) or hemoglobin composition in the present disclosure maintains metHb below 5%, preferably below 4% during storage at room temperature (25°C±2°C) for more than 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 27 months, 30 months, 33 months, and 36 months.EXAMPLESExample 1:

[0500] Pharmaceutical composition of PEG-Hb-CO-filled primary packaging apparatus (IV bags) is placedinside a 7.0 mil thickness 8”xl6” secondary packaging (metalized Mylar bags; Packdryl500, Impack). The secondary packaging is placed on a P-series pouch sealer from Ceratek and aligned with the nozzle. The secondary packaging bag is flushed with vacuum to remove all headspace air, followed by flush with Argon such that the headspace79IPTS / 200205494.1Attorney Docket No.: PLG-028WOoxygen content is NMT 5%. The secondary bag is sealed to achieve a strong, visual defect free seal (Seal dwell = 1.5 Sec, Takt Time = 2.0 Sec, Seal Pressure = 50psi, temperature 370 °F). The content of headspace oxygen is verified by a portable gas headspace analyzer to be NMT 5%. The packaging apparatus comprising pharmaceutical is stored at 2-8°C.

[0501] Results: The following table 1 compares two lots tested at 18M refrigerated condition with different headspace oxygen content with their TO attributes. Secondary packaging with high headspace oxygen resulted in significantly higher degradation across all parameters.Table 1: 18 Months refrigerated condition with <2% headspace oxygen content vs 16.1% headspace oxygen content with their TO attributes.<Table 2: 54 Months stability data stored at 2-8°C.<>Example 2:

[0502] Pharmaceutical composition of PEG-Hb-CO -filled primary packaging apparatus (IV bags) is placed inside a 7.0 mil thickness 8”xl6” secondary packaging (metalized Mylar bags; Packdry 1500, Impack). The secondary packaging is placed on a P-series pouch sealer from Ceratek and aligned with the nozzle. The secondary packaging bag is flushed with 80IPTS / 200205494.1Attorney Docket No.: PLG-028WOvacuum to remove all headspace air, followed by flush with Argon such that the headspace oxygen content is NMT 5%. The secondary bag is sealed to achieve a strong, visual defect free seal (Seal dwell = 1.5 Sec, Takt Time = 2.0 Sec, Seal Pressure = 50psi, temperature 370 °F). The content of headspace oxygen is verified by a portable gas headspace analyzer to be NMT 5%. The packaging apparatus comprising pharmaceutical is stored at Room Temperature.

[0503] Results: The following Table 3, FIG. 4 and FIG. 5 shows stability of pharmaceutical composition when stored at 25°C ± 2°C for 24 months. It is evident that when the pharmaceutical composition is stored in the packaging apparatus at 25°C±2°C with Headspace <2% O2, the pharmaceutical composition remains physiologically active about 24 months.Table 3: 24 Months stability data stored at Room Temperature (25°C ± 2°C / 60% RH ± 5%RH)<>Example 3:

[0504] Pharmaceutical composition of PEG-Hb-CO -filled primary packaging apparatus (IV bags) placed inside secondary packaging (metalized Mylar bags) and heat-sealed are studied to evaluate the relationship between headspace oxygen (%), MetHb (%) and HbCO (%) levels of the product. The secondary packaging is flushed with an inert gas preferably argon gas prior to heat sealing, with the goal of achieving less than 5% headspace oxygen. Herein, the headspace oxygen concentration is adjusted to specific levels to assess the impact81IPTS / 200205494.1Attorney Docket No.: PLG-028WOof oxygen fluctuations on the MetHb (%) of the pharmaceutical composition within the primary packaging apparatus. Headspace oxygen content is measured using a headspace analyzer. On day 7, HbCO (%) levels are recorded, and a trend analysis is performed. The packaging apparatus comprising pharmaceutical is stored at 40°C to achieve faster degradation profile and simulate long-term stability.

[0505] Results: The following Table 4 and FIG. 6 illustrates the change in MetHb (%) levels as headspace oxygen (%) increases under 40°C conditions, observed over a 7-day period for 6 different packaging bags. It is evident from the result that the Secondary packaging with higher headspace oxygen results in significantly higher degradation across all parameters. The Initial Headspace oxygen % relates to % oxygen level in the headspace on day 0; effect on MetHb levels and HbCO levels is observed on day 7. It is evident that an increase in headspace oxygen (%) within the secondary packaging corresponded to an increase in MetHb (%) and a decrease in HbCO (%) of the product in the IV bag.Table 4: Results for 7-day study, demonstrating the impact of headspace oxygen% in secondary packaging on MetHb and HbCO levels in product inside IV bag (primary packaging).Example 4:

[0506] A Pharmaceutical composition of PEG-Hb-CO is filled in a primary packaging apparatus (IV bags) is placed inside a 7.0 mil thickness 8”xl6” secondary packaging (metalized Mylar bags; Packdry 1500, Impack). The secondary packaging is placed on a P-series pouch sealer from Ceratek and aligned with the nozzle. The secondary packaging bag is flushed with vacuum to remove all headspace air, followed by flush with Argon such that the headspace oxygen content is NMT 5%. The secondary bag is sealed to achieve a strong, visual defect free seal (Seal dwell = 1.5 Sec, Takt Time = 2.0 Sec, Seal Pressure = 50psi, temperature 370 °F). The content of headspace oxygen is verified by a portable gas headspace analyzer to be NMT82IPTS / 200205494.1Attorney Docket No.: PLG-028WO5%. The packaging apparatus comprising pharmaceutical is stored at 2-8°C and another packaging bag is stored at room temperature. The effect of different headspace maintained less than 5% on the MetHb levels and HbCO levels in packaging bags stored at 2-8°C at room temperature and at 40°C is observed. The acidic and basic charge variants in the stored composition are analyzed by cIEF (capillary Iso Electric Focusing) method generally known in the art.Table 5 : Data corresponding to a packaging bag B 1 with headspace oxygen % in secondary packaging observed to be between 1% - 5% under 2-8°C, 25±2°C and at 40°C temperature conditions.• Initial MetHb% (TO, 2-8°C): 1.6%• After 60 months at 2-8°C: MetHb% rises only slightly to 1.7%, showing minimal increase over a long storage period.• After 6 months at RT (25±2°C): MetHb% is 2.0%, only a slight increase from the initial value.• After 6 months at 40°C: MetHb% is 2.0%, only a slight increase from the initial value.

[0507] Result: The following table 5 illustrates that with headspace oxygen maintained below 2%, the MetHb content remains stable and does not show drastic increases, even after extended storage at 2-8°C (over 54 months), at 40°C for about 6 months, at room temperature 25±2°C for 6 months or more. In addition, when analysed the composition had less than 15% acidic variants and less than 15% basic variants at varied temperatures.

[0508] This supports our observation that headspace oxygen less than 5% helps preserve product quality over time.83IPTS / 200205494.1Attorney Docket No.: PLG-028WOExample 5: Pouch Sealing Testing (Secondary packaging)

[0509] Product pouches made from Impak Corp. Packdry 1500 (P / N# P15C0816FID; Size: 8.0in x 16.0in. I.D.) were sealed using Sencorp White Ceratek equipment to determine optimal sealing parameters for secondary packaging. The machine was set up with a seal dwell of 1.5 seconds, takt time of 2.0 seconds, and seal pressure of 50 psi.

[0510] A series of tests were conducted at varying temperatures to evaluate seal integrity:• At temperatures from 250°F to 300°F, no seal was formed.• At 310°F and 320°F, only very marginal seals were observed.• At 330°F, a marginal seal was achieved.• From 340°F to 400°F, strong seals were consistently formed, with 340°F yielding a seal that could be pulled apart, while 350°F and above produced robust seals.

[0511] Further testing at 400°F with increased dwell and takt times revealed progressive blistering and distortion: Dwell times from 2 to 5 seconds and takt times from 2.5 to 5.5 seconds resulted in slight to moderate blistering, with poly sealant squeezed out at the sides and significant distortion at the highest dwell / takt settings.Table 6: Data corresponding to series of tests conducted at varying temperatures to evaluate seal integrity.84IPTS / 200205494.1Attorney Docket No.: PLG-028WO

[0512] Result: The example demonstrates that effective and reliable seals for secondary packaging are achieved at 370°F with the specified dwell time, takt time, and pressure. Lower temperatures do not produce adequate seals, while higher temperatures and longer dwell times can lead to blistering and distortion of the seal, potentially compromising package integrity.85IPTS / 200205494.1

Claims

Attorney Docket No.: PLG-028WOCLAIMSWhat is claimed:

1. A packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a primary’ packaging apparatus;b. a secondary packaging apparatus;wherein, the primary packaging apparatus comprising a pharmaceutical composition of hemoglobin and suitable excipients;wherein, the secondary packaging apparatus envelops the primary packaging apparatus thereby forming the packaging apparatus;wherein, the pharmaceutical composition of hemoglobin stored in primary packaging apparatus remains physiologically active.

2. A packaging apparatus suitable to store pharmaceutical composition of hemoglobin comprising:a. a pri mary packaging apparatus;b. a secondary packaging apparatus;wherein, the primary packaging apparatus comprises a pharmaceutical composition of hemoglobin and suitable excipients;wherein, the secondary packaging apparatus envelops the primary packaging apparatus thereby forming the packaging apparatus;wherein, the stored pharmaceutical composition of hemoglobin maintains metHb below 5% analyzed by oxymetry;wherein, the stored pharmaceutical composition has stability for at least 24 months at about 2°C to about 8°C.

3. The packaging apparatus as claimed in claim 1 or 2, wherein the stored pharmaceutical composition has stability for at least three months at 25°C±2°C.

4. The packaging apparatus as claimed in claim 1 or 2, wherein the secondary packaging apparatus maintains less than about 5% oxygen in the headspace.

5. The packaging apparatus as claimed in claim 4, wherein, the secondary packaging apparatus maintains oxygen less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% and less than about 0.5% in the headspace.86IPTS / 200205494.1Attorney Docket No.: PLG-028WO6. The packaging apparatus as claimed in claim 1 or 2, wherein, the secondary packaging apparatus maintains 0% or negligent oxygen amount in the headspace due to which the hemoglobin composition remains physiologically active.

7. The packaging apparatus as claimed in claim 1 or 2, wherein the secondary packaging apparatus maintains the oxygen in the headspace through inert gas sparging, wherein, the sparged inert gas is selected from helium, neon, argon, krypton, xenon, radon, and oganesson.

8. The packaging apparatus as claimed in claim 1 or 2, wherein the pharmaceutical composition of hemoglobin maintains p50 value of about 7 mmHg, about 8 mmHg, about 9 mmHg, about 10 mmHg, about 11 mmHg, about 12 mmHg, about 13 mmHg, about 14 mmHg, about 15 mmHg, and about 16 mmHg as analysed by Hemox analyser.

9. The packaging apparatus as claimed in claim 1 or 2, wherein the hemoglobin is PEGylated hemoglobin or PEG-Hb-CO.

10. The packaging apparatus as claimed in claim 1 or 2, wherein the stored hemoglobin composition has a purity of more than about 90%.

11. A method for storing pharmaceutical composition of PEGylated hemoglobin, in a suitable packaging apparatus, comprising:a. filling the pharmaceutical composition in a primary packaging apparatus:b. enveloping the primary packaging apparatus with a secondary packaging apparatus to form the packaging apparatus;c. sealing the packaging apparatus;d. storing the packaging apparatus containing the pharmaceutical composition; wherein, the primary packaging apparatus comprises a pharmaceutical composition of PEGylated hemoglobin and suitable excipients;wherein, the secondary packaging apparatus maintains oxygen less than about 5% in the headspace;wherein, the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein, the stored pharmaceutical composition maintains metHb below 5% analyzed by oxymetry.87IPTS / 200205494.1Attorney Docket No.: PLG-028WO12. The method as claimed in claim 11, wherein the secondary packaging apparatus is sealed at seal dwell of about 1 second, about 1.1 second, about 1.2 second, about 1.3 second, about 1.4 second, about 1.5 second, about 1.6 second, about 1.7 second, about 1.8 second, about 1.9 second, about 2.0 second, about 2.1 second, about 2.2 second, about 2.3 second, about 2.4 second, about 2.5 second, about 2.6 second, about 2.7 second, about 2.8 second, about 2.9 second, and about 3.0 second.

13. The method as claimed in claim 12, wherein the secondary packaging apparatus is sealed at seal dwell of about 1.5 seconds.

14. The method as claimed in claim 11, wherein secondary packaging apparatus is sealed at takt time selected from about 1.5 second, about 1.6 second, about 1.7 second, about 1.8 second, about 1.9 second, about 2.0 second, about 2.1 second, about 2.2 second, about 2.3 second, about 2.4 second, about 2.5 second, about 2.6 second, about 2.7 second, about 2.8 second, about 2.9 second, about 3.0 second, about 3.1 second, about 3.2 second, about 3.3 second, about 3.4 second, and about 3.5 second.

15. The method as claimed in claim 14, wherein the secondary packaging apparatus is sealed at takt time of about 2.0 seconds.

16. The method as claimed in claim 11, wherein the secondary packaging apparatus is sealed at seal pressure selected from about 40 psi, about 41 psi, about 42 psi, about 43 psi, about 44 psi, about 45 psi, about 46 psi, about 47 psi, about 48 psi, about 49 psi, about 50 psi, about 51 psi, about 52 psi, about 53 psi, about 54 psi, about 55 psi, about 56 psi, about 57 psi, about 58 psi, about 59 psi, about 60 psi.

17. The method as claimed in claim 16, wherein the secondary packaging apparatus is sealed at seal pressure of about 50 psi.

18. The method as claimed in claim 11, wherein the secondary packaging apparatus is sealed at a temperature selected from about 330°F, about 340°F, about 350°F, about 360°F, about 370°F, about 380°F, about 390°F, and about 400°F.

19. The method as claimed in claim 18, wherein the secondary packaging apparatus is sealed at a temperature of about 370°F.88IPTS / 200205494.1Attorney Docket No.: PLG-028WO20. A method for prevention of metHb formation during the storage of pharmaceutical composition of hemoglobin, comprising, storing the pharmaceutical composition of hemoglobin in a suitable packaging apparatus consisting of:a. a primary packaging apparatus;b. a secondary packaging apparatus;wherein, the primary packaging apparatus comprises a pharmaceutical composition of hemoglobin and other excipients;wherein, the secondary packaging apparatus envelops the primary packaging apparatus; wherein, the secondary packaging apparatus maintains less than about 5% Oxygen in the headspace through inert gas sparging;wherein, the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein, the hemoglobin composition remains physiologically active;wherein, the stored hemoglobin composition purity is more than about 90%; wherein, the stored pharmaceutical composition of hemoglobin maintains metHb below 5%.

21. The method as claimed in claim 20, wherein the hemoglobin composition has a purity of more than about 90%, more than about 91%. More than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, and more than about 99%.

22. The method as claimed in claim 21, wherein the hemoglobin composition has a purity of more than about 90%.

23. A pharmaceutical composition of hemoglobin stored in a packaging apparatus, comprising hemoglobin molecules covalently conjugated with PEG polymers, and other excipients; wherein, the packaging apparatus comprising, a primary packaging apparatus and a secondary packaging apparatus;wherein, the secondary packaging apparatus envelops the primary packaging apparatus wherein, the pharmaceutical composition of hemoglobin is contained in the primary packaging apparatus:wherein, the hemoglobin molecules in the pharmaceutical composition are conjugated with polyethylene glycol (PEG);89IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein, the pharmaceutical composition has stability for at least three months to about 24 months at room temperature;wherein, the pharmaceutical composition has stability for at least 3 months to about 54 months at refrigerated conditions;wherein the pharmaceutical composition of hemoglobin maintains metHb below 5% analyzed by oximetry;wherein, the pharmaceutical composition remains physiologically active.

24. The pharmaceutical composition as claimed in claim 23, wherein, the pharmaceutical composition maintains metHb below 5%, below 4%, below 3%, below 2% and below 1%.

25. The pharmaceutical composition as claimed in claim 23, wherein, the pharmaceutical composition has stability for at least 3 months, at least 6 months, at least 12 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, and at least 54 months at 2°C to 8 °C.

26. The pharmaceutical composition as claimed in claim 25, wherein, the pharmaceutical composition has stability for at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, and at least 54 months at 2°C to 8°C, wherein the pharmaceutical composition maintains metHb below 5%.

27. The pharmaceutical composition as claimed in claim 25, wherein, the pharmaceutical composition has stability for at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, and at least 54 months at 2°C to 8°C, wherein the pharmaceutical composition maintains metHb below 5%, wherein the pharmaceutical composition maintains hemoglobin purity more than 90%.

28. The pharmaceutical composition as claimed in claim 23, wherein, the pharmaceutical composition has stability for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, and at least 24 months, at 25°C±2°C.

29. The pharmaceutical composition as claimed in claim 25, wherein, the pharmaceutical composition has stability for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, and at least 24 months, at 25°C±2°C, wherein the pharmaceutical composition maintains metHb below 5%.90IPTS / 200205494.1Attorney Docket No.: PLG-028WO30. The pharmaceutical composition as claimed in claim 25, wherein, the pharmaceutical composition has stability for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, and at least 24 months, at 25°C±2°C, wherein the pharmaceutical composition maintains metHb below 5%, wherein the pharmaceutical composition maintains hemoglobin purity more than 90%.

31. The pharmaceutical composition as claimed in claim 23, wherein, the pharmaceutical composition has stability for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months, at 40°C.

32. The pharmaceutical composition as claimed in claim 31, wherein, the pharmaceutical composition has stability for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months, at 40°C, wherein the pharmaceutical composition maintains metHb below 5%.

33. The pharmaceutical composition as claimed in claim 31, wherein, the pharmaceutical composition has stability for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, and at least 9 months, at 40°C, wherein the pharmaceutical composition maintains metHb below 5%, wherein the pharmaceutical composition maintains hemoglobin purity more than 90%.

34. The pharmaceutical composition as claimed in claim 23, wherein, the pharmaceutical composition, comprises PEGylated hemoglobin and other excipients selected from buffer, stabilizers, tonicity modifier, and rehydrating salts.

35. The pharmaceutical composition as claimed in claim 23, wherein, the buffer is selected from citrate, Tris, MOPS, HEPES, phosphate buffer, and sodium acetate and ammonia buffers in an amount of about 1 mM to about 5 mM.

36. The pharmaceutical composition as claimed in claim 35, wherein, the buffer is phosphate buffer in an amount of about 1 mM to about 5 mM.

37. The pharmaceutical composition as claimed in claim 23, wherein, stabilizer is selected from amine stabilizers, sodium metabisulfite, polyethylene glycol, EDTA and polysorbates.91IPTS / 200205494.1Attorney Docket No.: PLG-028WO38. The pharmaceutical composition as claimed in claim 37, wherein, the stabilizer is cysteine maintained in an amount more than 5 mM.

39. The pharmaceutical composition as claimed in claim 23, wherein, the tonicity modifier is selected from potassium chloride, glycerin, lactose, sodium chloride, dextrose, and mannitol in an amount of about 100 mM to about 200 mM.

40. The pharmaceutical composition as claimed in claim 39, wherein, the tonicity modifier is sodium chloride used in an amount of about 150 mM.

41. The pharmaceutical composition as claimed in claim 23, wherein, the rehydrating salts are selected from sodium chloride, sodium bicarbonate, magnesium sulfate hetpahydrate, calcium chloride dihydrate, and potassium chloride in an amount of about 0.5 mM to about 5 mM.

42. The packaging apparatus as claimed in any of the preceding claims, wherein the primary packaging apparatus is made of an IV bag.

43. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus is a multilayered metallized polymer bag.

44. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus is gas impermeable.

45. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus is oxygen impermeable.

46. The packaging apparatus as claimed in any of the preceding claims holds primary packaging apparatus.

47. The packaging apparatus, as claimed in any of the preceding claims, bears the weight of primary packaging apparatus.

48. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus is free of visual imperfections.

49. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus maintains a gaseous mixture of an inert gas and oxygen in the headspace, wherein the packaging apparatus is substantially free of oxygen.92IPTS / 200205494.1Attorney Docket No.: PLG-028WO50. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus maintains a gaseous mixture of argon and less than about 5% oxygen in the headspace.

51. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus maintains less than about 5% oxygen and less than 5% metHb in the headspace.

52. The packaging apparatus as claimed in any of the preceding claims, wherein the packaging apparatus provides mechanical and environmental protection to the pharmaceutical composition.

53. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus is configured to shield the primary packaging apparatus containing the pharmaceutical composition from one or more condition selected from fluctuation in oxygen, fluctuation in humidity, effect of light thereby preserving the integrity and stability of the pharmaceutical composition.

54. The packaging apparatus as claimed in any of the preceding claims, wherein the pharmaceutical composition is free of oxygen scavenger.

55. A method of treating a disease condition in a subject in need thereof, by administering to the subject a therapeutically effective amount of pharmaceutical composition, wherein the pharmaceutical composition comprising hemoglobin and other excipients; wherein, the hemoglobin composition is stored in a suitable packaging apparatus, the packaging apparatus consisting of:a. a primary’ packaging apparatus;b. a secondary packaging apparatus;wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein, the secondary packaging apparatus maintains less than about 5% Oxygen in the headspace;wherein, the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C;wherein, the hemoglobin composition remains physiologically active;93IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein, the stored pharmaceutical composition of hemoglobin maintains metHb below 5%.

56. The method of treating a disease condition as claimed in claim 55, wherein, the diease condition is selected from cancer, haemorrhagic shock, intracranial haemorrhage, hypoxia, necrosis, myocardial injury, acute myocardial infarction, hypertension, pulmonary hypertension.

57. The method of treating a disease condition as claimed in claim 55, wherein the therapeutic effective amount of PEGylated hemoglobin is selected from about 300 mg / kg to about 1500 mg / kg.

58. The method of treating a disease condition as claimed in claim 56, wherein the therapeutic effective amount of PEGylated hemoglobin is administered in at least two cycles in a subject in need thereof.

59. The method of treating a disease condition as claimed in claim 56, wherein the first infusion cycle has higher flow rate than the second cycle.

60. The method of treating a disease condition as claimed in claim 56, wherein the first infusion cycle delivers a lower amount of therapeutic effective amount of PEGylated hemoglobin than the second cycle.

61. The method of treating a disease condition as claimed in claim 56, wherein the first infusion cycle delivers about 200 ml of therapeutic effective amount of PEGylated hemoglobin maintaining a flow rate of about 6.7 ml / min.

62. The method of treating a disease condition as claimed in claim 56, wherein the second infusion cycle delivers about 300 ml of therapeutic effective amount of PEGylated hemoglobin maintaining a flow rate of about 3.3 ml / min.

63. A method of administering physiologically active hemoglobin composition stored in a suitable packaging apparatus to a subject in need thereof, wherein the packaging apparatus consists of a primary packaging apparatus and a secondary packaging apparatus, the method comprising:a. opening the secondary packaging apparatus to access the primary packaging apparatus comprising pharmaceutical composition;94IPTS / 200205494.1Attorney Docket No.: PLG-028WOb. administering to the subject a therapeutically effective amount of pharmaceutical composition stored in the primary packaging;wherein, the pharmaceutical composition comprising PEGylated hemoglobin and suitable excipients;wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein, the hemoglobin composition in the packaging apparatus remains physiologically active;wherein, the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C.

64. A process for storing a physiologically active hemoglobin composition stored in a suitable packaging apparatus; wherein the packaging apparatus consists of a primary packaging apparatus and a secondary packaging apparatus, the process comprising:a. washing fresh whole blood collected from animal sources to produce washed RBCs; b. extracting hemoglobin from the RBC’s;c. performing filtration of the extracted hemoglobin;d. performing ultrafiltration and concentration of hemoglobin;e. performing deoxygenation of ultrafiltered and concentrated hemoglobin;f. performing heat inactivation of deoxygenated hemoglobin for reduction of virus and / or prion by heat treatment at suitable temperature;g. performing reoxygenation of heat-treated deoxygenated hemoglobin to produce oxygenated hemoglobin composition;h. optionally performing PEGylation of the oxygenated hemoglobin composition; i. optionally performing filtration of the pegylated hemoglobin;j. optionally performing carboxylation process to produce carboxylated PEGylated hemoglobin composition;k. filling the pharmaceutical composition of carboxylated PEGylated hemoglobin obtained from (i) in the primary packaging apparatus;l. enveloping the primary packaging apparatus with a secondary packaging apparatus to form a packaging apparatus;m. sealing the packaging apparatus; and.n. storing the packaging apparatus containing the pharmaceutical composition;95IPTS / 200205494.1Attorney Docket No.: PLG-028WOwherein, the viral inactivation of deoxygenated hemoglobin is performed by heat treatment and maintains L-cysteine concentration in deoxygenated hemoglobin during step f) for more than 5 mM;wherein, the secondary packaging apparatus envelops the primary packaging apparatus to form the packaging apparatus;wherein, the hemoglobin composition stored in the packaging apparatus remains physiologically active;wherein, the packaging apparatus stores the pharmaceutical composition for at least 24 months at 2-8°C.

65. The process as claimed in claim 64 wherein the hemoglobin obtained from (f) is reoxygenated.

66. The process as claimed in claim 64 wherein the L-cysteine concentration in deoxygenated hemoglobin is maintained at more than 5 mM.

67. The pharmaceutical composition as claimed in any of the preceding claims, wherein the hemoglobin composition comprises impurities selected from charge variants, Low molecular weight impurities, high molecular weight impurities, HCPs and HC DNA.

68. The pharmaceutical composition as claimed in claim 67, wherein the charge variants are below 25%.

69. The pharmaceutical composition as claimed in claim 68, the acidic variants are about 15% and less.

70. The pharmaceutical composition as claimed in claim 68, the basic variants are about 15% and less.

71. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a urity more than 90%; wherein composition is stable for suitable time selected from at least 3 months, to about 24 months at about 2°C to about 8°C.96IPTS / 200205494.1Attorney Docket No.: PLG-028WO72. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein composition is stable for suitable time selected from at least 3 months, to about 24 months at 25°C±2°C.

73. The packaging apparatus as claimed in any of the preceding claims, wherein the secondary packaging apparatus maintains less than about 5% oxygen in the headspace; wherein the primary packaging comprising physiologically active hemoglobin wherein the metHb is below 5%; wherein the physiologically active hemoglobin has a purity more than 90%; wherein composition is stable for suitable time selected from at least 1 month, to about 3 months at 40°C.97IPTS / 200205494.1

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