# Patent

## Title
Methods for sterilizing materials containing biologically active agents

## Abstract

Provided are methods for sterilizing a material comprising a biologically-active agent comprising irradiating said material with ionizing radiation at a dose of about 5 kGy to about 25 kGy while maintaining said material in an atmosphere comprising at least 95% by volume inert gas and at a temperature of about 4° C. or lower. Also provided are sterilized materials comprising a biologically-active agent, wherein said materials exhibit substantially the same amount of biological activity as a non-sterilized control.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit to U.S. Provisional Patent Application No. 61/015,350, filed Dec. 20, 2007, the contents of which are incorporated by reference herein, in their entirety.


FIELD OF THE INVENTION
The present application pertains to sterilized biologically-active materials and methods for producing same.
BACKGROUND OF THE INVENTION
The commercial production of therapeutic clinical products containing biological derivatives requires sterilization methods to effectively eliminate bio-burden.
Expanded Human Umbilical Tissue Derived Cells (hUTC) are being developed as a potential cell therapy for the treatment of various degenerative diseases. In addition, cell derivatives (such as trophic factors, proteins, and other molecules) are also being developed as potential therapeutic agents. These derivatives can be used alone or in combination with biomaterials to augment cellular response. Upon deployment directly or indirectly to a target injury site, the derivatives may reduce excessive inflammation, reduce apoptosis and necrosis of endogenous cells of the injury site, induce differentiation of endogenous progenitor cells, increase neovascularization and angiogenesis, and promote new tissue formation. Alone, the derivatives can be deployed as a lyophilized powder, or combined with an aqueous or viscous delivery vehicle. The derivatives can also be combined with and released from biomaterials. For example, the application of the hUTC lysate to biomaterials followed by lyophilization produces a device applicable to tissue engineering and regenerative medicine. Additional information may be found in WO 2005/003334 (filed as PCT/US2004/020931, Jun. 25, 2004), and WO/2006/071794 (filed as PCT/US2005/046851, Dec. 22, 2005), both of which are incorporated herein by reference in their entireties.
However, current methods of sterilization of hUTC and other products containing biological derivatives, while being effective from the sterilization standpoint, reduce the biological activity of the derivatives. Previous approaches have made use of protective agents in combination with the biological sample. See, e.g., U.S. Pat. No. 5,730,933 (disclosing methods that comprise the step of forming a mixture of the biological sample with an extraneous protein, and optionally a free-radical scavenger, prior to irradiation). Such methods have the disadvantage of requiring additional processing steps and materials in addition to the sterilization protocol. Other previous methods perform sterilization in the presence of hydrogen gas. See U.S. 2004/0101958. Improved methods for sterilizing materials that contain active biological derivatives, as well as sterilized yet efficacious clinical products, are needed.
SUMMARY OF THE INVENTION
Provided are methods for sterilizing a material comprising a biologically-active agent comprising irradiating said material with ionizing radiation at a dose of about 5 kGy to about 25 kGy while maintaining said material in an atmosphere comprising at least 95% by volume inert gas and at a temperature of about 4° C. or lower.
Also provided are sterilized materials comprising a biologically-active agent, wherein said materials exhibit substantially the same amount of biological activity as a non-sterilized control.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the measured average quantity of average cells per well of NIH/3T3 mouse fibroblasts co-cultured with gamma-irradiated collagen/ORC samples that are either treated with 150 μg hUTC lysate protein or left untreated.
FIG. 2 depicts the measured average quantity of average cells per well of NIH/3T3 mouse fibroblasts co-cultured with electron beam-irradiated collagen/ORC samples that are either treated with 150 μg hUTC lysate protein or left untreated.
FIG. 3 provides main effects plots for each of the three variable conditions used in the present study.
FIGS. 4A and 4B provide an SDS-PAGE analysis of data generated in accordance with a characterization of hUTC lysate pre-processing.
FIG. 5 details the results of SDS-PAGE analysis in order to determine the effects of sterilization conditions.



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Materials containing biologically-active agents must be subjected to sterilization prior to clinical use. However, this medically-necessary sterilization is traditionally accompanied by the attrition of the biological activity of the therapeutic ingredients, which leads to decreased clinical efficacy and necessitates the use of greater quantities of what are often costly materials.
Provided are methods for sterilizing a material comprising a biologically-active agent comprising irradiating said material with ionizing radiation at a dose of about 5 kGy to about 25 kGy while maintaining said material in an atmosphere comprising at least 95% by volume inert gas and at a temperature of about 4° C. or lower. The instant methods permit the retention of biological activity even while thorough sterilization is accomplished. The biological activity of samples according to the present invention is functionally comparable to that of non-sterilized controls, and the instant methods are therefore highly advantageous over previous methods that effectively represented a trade-off between sterilization and biological efficacy.
In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Where present, all ranges are inclusive and combinable.
The biologically active agent may comprise cells, cell derivatives, growth factors, or any combination thereof. Exemplary cells for use in connection with the present invention include epithelial cells (e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, melanocytes, dermal fibroblasts, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors such as CD34+ or CD34+/CD117+ cells), myoblasts, myocytes, hepatocytes, smooth muscle cells, striated muscle cells, stromal cells, other soft tissue cells or progenitor cells, chondrocytes, osteoblasts, islet cells, nerve cells, placenta-derived cells, human umbilical tissue derived cells, human kidney derived cells, or any combination thereof. Additional information regarding human kidney derived cells may be found in PCT/US2007/021708, filed Oct. 11, 2002, which is herein incorporated by reference in its entirety.
“Cell derivatives” include any material that is acquired from or made by a cell of any type, including, inter alia, organelles, membrane and membrane components, cytoskeletal elements, proteins, hormones, genetic material, and the like. In some embodiments, the biologically active agent is a cell derivative that comprises cell lysate, trophic factors, growth factors, cytokines, conditioned media, or any combination thereof. Exemplary growth factors are readily appreciated by those skilled in the art, e.g., PDGF-BB, bFGF, TGF-beta, HGF, VEGF, or GDF-5. Any biologically active component is considered to fall within the scope of the present invention. For example, expanded human umbilical tissue derived cells (hUTC), as well as cell lysate from hUTCs, are being developed as a potential therapy for the treatment of various degenerative diseases, and sterilization thereof in accordance with the present invention represents a considerable improvement upon such therapeutic protocols.
The material comprising a biologically active agent may further comprise a scaffold material. Thus, biologically active agents such as cells, cell derivatives, growth factors, and other molecules can be used alone or in combination with scaffolds, such as biomaterial scaffolds, to augment cellular response. For example, the application of the hUTC lysate to biomaterial scaffold followed by lyophilization produces a device applicable to tissue engineering and regenerative medicine: hUTC lysate combined with a collagen/oxidized regenerated cellulose (ORC) matrix or polyglactin 910 may be used in these and other contexts. The scaffold material may be naturally or synthetically derived. Exemplary scaffold materials include collagen, cellulose, fibrin, elastin, gelatin, demineralized bone, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polycaprolactone, polyanhydrides, polyhydroxybutyrates, or any combination thereof. Those skilled in the art will readily appreciate other materials that may be used to provide the contemplated structural matrix in accordance with the present invention.
Irradiation of the material comprising a biologically active agent may be performed in accordance with traditional techniques, i.e., using any known sterilization medium or delivery mechanism. For example, the ionizing radiation may comprise, alpha radiation, beta radiation, gamma radiation, X-rays, electron beams, other subatomic particles, or any combination thereof. The dose of ionizing radiation is preferably about 5 kGy to about 25 kGy. In other embodiments, the dose of ionizing radiation is about 6 kGy to about 12 kGy. The dose of ionizing radiation may also be about 6 kGy to about 8 kGy, or may be about 7 kGy.
Pursuant to the present invention, the irradiation of the material comprising a biologically active agent occurs while maintaining the material in an atmosphere that is at least 95% by volume inert gas. The portion of the atmosphere comprising 95% by volume inert gas may comprise a single inert gas, or a combination of two or more inert gases. As used herein, “inert gas” refers to elemental as well as molecular gases that are not reactive under normal circumstances. Exemplary inert gases include noble gases, nitrogen, and other gases and gas mixtures as appreciated by those skilled in the art. The portion of the atmosphere comprising 95% by volume inert gas may comprise solely of nitrogen, and in other embodiments, the atmosphere may comprise about 100% by volume nitrogen.
One skilled in the art will readily appreciate various methods for providing a gaseous atmosphere in accordance with the present invention. The atmosphere in accordance with the present invention may be accomplished by containing the material comprising a biologically active agent in a sealed environment that is continuously flushed with the desired gaseous components, that has been pre-flushed one or more times with the desired gas or gases, or both. These techniques may be used in conjunction with a protocol whereby the material is contained within a vessel prior to irradiation, and wherein the ambient air is purged from the vessel by flushing with the desired atmosphere components prior to irradiating the material.
The temperature at which the irradiation of the material comprising a biologically active agent occurs is preferably about 4° C. or lower. Preferably, the material and atmosphere are equilibrated to the desired temperature prior to irradiation and are maintained at a temperature constantly at or below about 4° C. during the irradiation process. The temperature at which irradiation occurs may be at or less than about 2° C., or may be at or less than about 0° C. If the material and atmosphere are equilibrated to a desired temperature prior to irradiation, the temperature at which equilibration occurs is preferably approximately equal to the temperature at which irradiation is performed.
Also provided are sterilized materials comprising a biologically-active agent, wherein said material exhibits substantially the same amount of biological activity as a non-sterilized control. The identity of the biologically active agent may be as described above with respect to the inventive methods. The amount of biological activity may be assessed according any appropriate assay. For example, the assay may comprise a cell proliferation assay, a transmigration assay, a angiogenesis assay, a cytotoxicity assay, a matrix deposition assay, a lymphocyte activation assay, an apoptosis assay, or any combination of such assays or other assay that is useful in assessing the biological activity in a sample. Those skilled in the art will recognize other assays that may be used to assess the biological activity of the material. “Assessing” the biological activity refers to measuring the degree of biological activity, or to determining the absence or presence of biological activity.
The inventive sterilized materials are preferably those which have been sterilized by irradiating the material with ionizing radiation at a dose of about 5 kGy to about 25 kGy while maintaining the material in an atmosphere comprising at least 95% by volume inert gas and at a temperature of about 4° C. or lower. The conditions of the irradiation, including the type and dose of radiation and the conditions, components, and temperature of the atmosphere are preferably as described above with respect to the inventive methods.
The present invention is further defined in the following examples. It should be understood that these examples, while indicating embodiments of the invention, are given by way of illustration only, and should not be construed as limiting the appended claims. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
EXAMPLE 1
Variation of Sterilization Factors in Sterilization of hUTC Lysate
An evaluation of a full factorial design of experiments was conducted to determine the optimal factor levels for the retention of hUTC lysate biological activity post-sterilization by gamma irradiation or e-beam methods. The sterilization factors tested included 1) sterilization in ambient air vs. nitrogen gas, 2) temperature during sterilization, and 3) irradiation dose. Collagen/oxidized regenerated cellulose (ORC) matrix containing lyophilized hUTC lysate and its respective non-lysate controls were irradiated under test conditions. Biological activity was measured using a mouse NIH/3T3 fibroblasts transwell proliferation assay.
Cell Growth and Harvest. Expanded human umbilical tissue derived cells were seeded at 5,000 cells per cm squared in gelatin-coated flasks with growth medium containing Dulbecco's Modified Eagles Media (DMEM)-low glucose, 15% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), betamercaptoethanol (BME); these cells were expanded for 3 to 4 days (25,000 cells per cm squared target harvest density). Upon 70% confluency, the cells were harvested with trypsin, collected, and centrifuged at 300 rcf for 5 minutes. The trypsin/media was removed by aspiration and cells were washed three times with phosphate buffered saline (PBS).
Cell Wash and Aliquoting. After washing, the cells were re-suspended at 1.0E+07 cell/ml in PBS and delivered as 1 ml aliquots into 1.5 ml sterile siliconized micro-centrifuge tubes. The cells were centrifuged at 300 rcf for 5 minutes and the PBS was removed by aspiration.
Cell Lysis. Tubes containing cell pellets were immersed into liquid nitrogen (LN2) for 60 seconds. The tubes were then removed from LN2 and immediately immersed in a 37° C. water bath for 60 seconds or until thawed (3 min maximum incubation time). This process is repeated three times.
Centrifugation and Lysate Harvest. The freeze-thawed samples were centrifuged for 10 minutes at 13,000 rcf at 4° C. and placed on ice. The supernatant fluid from each tube was removed by pipette and transferred to a single sterile siliconized 1.5 ml tube. This process was repeated until no additional supernatant fluid could be recovered.
Fluid Volume Measurement. To approximate supernatant fluid volume, the tube containing 1.5 ml recovered supernatant fluid was weighed on a balance previously tared with an empty 1.5 ml micro-centrifuge tube (1 mg≈1 μl).
Protein Assay. To determine total protein content, 10 μl of lysate supernatant fluid was diluted into 990 μl PBS and further serially diluted in PBS. The dilution was analyzed by Bradford assay, or other equivalent protein assay (standard range 1.25-25 μg). This value was used to calculate the total protein per cell, the main metric used to ensure the consistency of the process.
Lysate Application and Lyophilization. Collagen/ORC samples (lot#1305263), pre-cut with a 3 mm biopsy punch, were aseptically placed into the wells of 48 well sterile, ultra low cluster cell culture dishes (Cat. No. 3473, Corning Inc., Corning, N.Y.). The supernatant fluid was applied to the material as single 150 μg total protein aliquots. Dishes containing test materials were loaded into the lyophilizer.
Lyophilization. Test materials with applied lysate and control materials without cell lysate were loaded into a FTS Systems Dura-Stop MP Stoppering Tray Dryer and lyophilized using the following ramping program. All steps had a ramping rate of 2.5° C./minute and a 100-mT vacuum (see Table 1, below).









TABLE 1





Step
Shelf Temp (° C.)
Hold Time (min)

















a
−40
180


b
−25
2160


c
−15
180


d
−5
180


e
5
120


f
20
120


g
−20
60









After lyophilization, all samples were aseptically loaded into individual, sterile 1.5 ml screw-cap microfuge tubes (cat. no. 02-681-339, Fisher Scientific, Inc, Hampton, N.H.).

Experimental Design. A full-factorial design of experiments was constructed using MiniTab 14.0 to evaluate the gamma sterilization factors that affect hUTC lysate biological activity (see Table 2, below):







TABLE 2







Gamma Irradiation Design of Experiment










Factor
Levels Tested






hUTC lysate
With, Without



Nitrogen Glove Box
With, Without



Dose (kGy)
7, 12, 25



Temperature (° C.)
0, 4, 25









A second full-factorial design of experiments was constructed using MiniTab 14.0 to evaluate the e-beam sterilization factors that effect hUTC lysate biological activity. All materials were processed in nitrogen glove box and sterilized at 21° C. (see Table 3, below):







TABLE 3







Electron Beam Design of Experiment










Factor
Levels Tested






hUTC lysate
With, Without



Dose (kGy)
7, 12, 25









Table 4, below, provides the experimental run order for hUTC lysate treated collagen/ORC samples sterilized through gamma irradiation.







TABLE 4







Gamma Irradiation













Glove Box




Run Order
Lysate Treatment
Processing
Dose (kGy)
Temp (° C.)














1
Without
With
7
25


2
With
Without
25
25


3
With
With
25
25


4
With
Without
12
4


5
Without
Without
25
0


6
Without
Without
7
25


7
With
With
7
4


8
Without
Without
7
0


9
With
Without
7
0


10
Without
With
25
4


11
Without
Without
7
4


12
Without
With
12
25


13
With
With
7
0


14
Without
Without
12
4


15
Without
With
25
0


16
Without
With
7
0


17
Without
Without
12
0


18
With
Without
12
25


19
With
Without
7
4


20
With
With
25
4


21
With
Without
7
25


22
With
Without
12
0


23
Without
With
12
0


24
With
With
7
25


25
With
With
25
0


26
With
With
12
4


27
With
Without
25
4


28
Without
With
25
25


29
Without
With
12
4


30
With
Without
25
0


31
Without
Without
25
4


32
With
With
12
0


33
With
With
12
25


34
Without
Without
12
25


35
Without
With
7
4


36
Without
Without
25
25









A set of collagen/ORC samples with or without hUTC lysate (n=6 each) was produced and stored at manufacturing facility at −80° C. during sterilization. A second “traveler” set of collagen/ORC samples with or without hUTC lysate (n=6 each) was produced to control for any effects sample shipment may have had on biological activity. This set was shipped with the irradiated samples and stored at gamma sterilization facility at 0° C. during. This set was returned with the irradiated samples for analysis.
Gamma Irradiation. Samples to be sterilized through gamma irradiation were delivered to the gamma sterilization facility on wet ice and stored at 0° C. until sorting and processing.
Samples marked for glovebox processing had the ambient air purged from their respective microfuge containers. The nitrogen purge was accomplished by placing samples into the antechamber of a low-oxygen, nitrogen flushed glovebox (Isolation Technologies, Model: Micro-Inert System 1.5×1, serial no. 5023A, manuf. date 4-94). The cycle designated as “01 ONE CYCLE” was executed prior to bringing samples into the main chamber. This cycle consists of a series of four evacuations, followed by back-flushing with nitrogen to standard atmospheric pressure. The evacuations occur for five minutes, reaching a pressure less than 10 torr. Samples were then transferred into the main chamber to equilibrate. After approximately one hour, their caps, which had been loosened prior to the evacuation cycles, were tightened, and the samples were removed from the glovebox. After the nitrogen purge, these samples were stored at −70° C.
Prior to gamma irradiation, samples were removed from the −70° C. and allowed to equilibrate to room temperature for approximately 30 minutes. During gamma irradiation, the temperature was monitored using an Omega RD-MV 112 Paperless Recorder (S/N S5D803918). The temperature was adjusted to within ±2° C. of the requested set point and controlled manually via a Model 328 Vortex Tube cooler. The radiation cycle was initiated, and the airflow adjusted to maintain and control the temperature. Materials were irradiated with a Gammacell 220 using a cobalt-60 isotope.
E-Beam Irradiation. Samples for e-beam irradiation were delivered to the gamma sterilization facility on wet ice. Samples were purged of ambient air in a nitrogen glovebox, as previously described. After processing, all samples were shipped to the e-beam sterilization facility on dry ice. Six empty microfuge tubes were also sent to calibrate for dose penetration.
E-beam sterilization was performed with a Linac electron beam irradiator. Briefly, the samples were placed in respective treatment boxes containing dry ice. The boxes were passed in front of the scanning horn by conveyor and irradiated by the electron beam. The beam current was held constant, and the speed of the conveyor was adjusted to deliver the requested dose.
Traveler Control. The traveler control set were not subjected to irradiation. These samples were stored at the gamma sterilization facility at −70° C. until the completion of all sterilization cycles. Upon completion of all irradiation cycles, the traveler set was returned to its source with all other samples.
Mouse Fibroblasts. Mouse NIH/3T3 fibroblasts (ATCC CRL-1658) were expanded in growth media (DMEM high glucose with 10% fetal calf serum and 1% penicillin/streptomycin).
Transwell Assay. The mouse NIH/3T3 fibroblasts were plated into the lower portion of a 96 well transwell plate (Cat. No. 3381, Corning Inc., Corning, N.Y.) at 2,500 cells per well and cultured overnight. Media was removed by aspiration and the appropriate media (150 μl per well, 50 μl per transwell) was added, followed by treatments. Positive controls were NIH/3T3 cells in 10% NCS and empty transwell. Negative controls were NIH/3T3 cells in 1% NCS and empty transwell. All test conditions were cultured in 1% NCS.
Cell Harvest and Analysis. On day 4, cells in transwells were harvested by trypsinization (75 μl trypsin followed with 75 μl complete media to neutralize trypsin) and 50 μl of staining solution (48:1:1 media, DMSO, Guava ViaCount Flex reagent (Cat. No. 4500-0110, Guava Technologies, Hayward, Calif.)) was added to each well. Cells were counted using a Guava EasyCyte instrument (Guava Technologies, Hayward, Calif.) with an original volume of 0.2 ml and dilution factor of one.
Results. Table 5, below, provides the results of the measurement of average cells per well for each gamma irradiated condition, as determined by Guava EasyCyte and Guava ViaCount Flex reagent. Condition code refers to samples of collagen/ORC that were subjected to the same conditions, differing only in the treatment with hUTC lysate.







TABLE 5







Gamma Irradiation














Condition
Run
Lysate
Glove Box
Dose

Average



Code
Order
Treatment
Processing
(kGy)
Temp (° C.)
(Cells/Well)
Std. Dev.

















A
13
With
With
7
0
41,053.73
11,450.09



16
Without
With
7
0
16,782.78
4,269.39


B
7
With
With
7
4
22,658.34
8,513.94



35
Without
With
7
4
11,930.00
1,647.71


C
24
With
With
7
25
27,393.44
6,677.86



1
Without
With
7
25
12,479.81
3,724.32


D
32
With
With
12
0
26,715.72
8,810.56



23
Without
With
12
0
18,387.29
5,916.10


E
26
With
With
12
4
35,589.61
9,530.70



29
Without
With
12
4
14,208.16
2,971.11


F
33
With
With
12
25
23,660.07
9,301.86



12
Without
With
12
25
13,680.73
3,682.22


G
25
With
With
25
0
39,387.61
15,636.45



15
Without
With
25
0
18,661.71
4,498.87


H
20
With
With
25
4
23,981.00
7,604.69



10
Without
With
25
4
12,817.93
6,256.35


I
3
With
With
25
25
14,811.75
6,384.01



28
Without
With
25
25
19,408.52
6,223.30


J
9
With
Without
7
0
18,069.07
6,326.40



8
Without
Without
7
0
23,331.11
4,729.10


K
19
With
Without
7
4
32,693.02
15,711.67



11
Without
Without
7
4
11,794.49
978.07


L
21
With
Without
7
25
27,495.82
8,075.72



6
Without
Without
7
25
10,729.76
2,381.61


M
22
With
Without
12
0
27,755.70
12,084.05



17
Without
Without
12
0
19,870.94
2,999.99


N
4
With
Without
12
4
16,613.38
6,319.08



14
Without
Without
12
4
6,040.50
950.71


O
18
With
Without
12
25
9,756.47
6,344.04



34
Without
Without
12
25
11,958.15
1,573.89


P
30
With
Without
25
0
6,306.70
2,485.10



5
Without
Without
25
0
18,179.73
3,669.78


Q
27
With
Without
25
4
13,374.71
6,142.68



31
Without
Without
25
4
13,531.81
1,842.50


R
2
With
Without
25
25
3,891.18
562.15



36
Without
Without
25
25
10,904.23
2,768.38









Table 6, below, provides the results of the measurement of average cells per well for each e-beamed sterilized condition, as determined by Guava EasyCyte and Guava ViaCount Flex reagent. Condition code refers to samples of collagen/ORC that were subjected to the same conditions, differing only in the treatment with hUTC lysate.







TABLE 6







E-Beam













Condition

Lysate
Glove Box

Average



Code
Run Order
Treatment
Processing
Dose (kGy)
(Cells/Well)
Std. Dev.
















A
1
With
With
7
65,452.02
10,316.12



4
Without
With
7
28,025.00
3,077.64


B
2
With
With
12
63,743.84
8,664.52



5
Without
With
12
32,579.22
6,942.06


C
3
With
With
25
55,177.92
10,457.69



6
Without
With
25
32,080.82
13,634.10


Traveler
Treated



49,337.37
12,349.89



Untreated



18,220.36
4,289.74


In-House
Treated



71,417.83
13,972.36



Untreated



37,303.30
7,994.61









FIG. 1 provides the results of the measurement of average cells/well of NIH/3T3 mouse fibroblasts co-cultured with gamma irradiated collagen/ORC samples either treated with 150 μg hUTC lysate protein or untreated and controls. Condition codes are provided in Table 5. FIG. 2 provides the results of the measurement of average cells/well of NIH/3T3 mouse fibroblasts co-cultured with electron beam sterilized collagen/ORC samples either treated with 150 μg hUTC lysate protein or untreated and controls. Condition codes are provided in Table 6. FIG. 3 provides main effects plots for each of the three variable conditions used in the present study.
Biological activity was maintained in approximately 72% of gamma irradiated hUTC lysate containing collagen/ORC samples as compared to collagen/ORC samples alone regardless of test conditions. Samples sterilized at 7 kGy at 0° C. in nitrogen retained the most biological activity and no significant difference was noted between the lysate containing sample and the non-irradiated lysate control. There was a correlation between sterilization in nitrogen, low temperature, and low irradiation dose and lysate biological activity as demonstrated by cell proliferation.
The lysates' biological activity was also maintained in all e-beam irradiated hUTC lysate containing collagen/ORC samples. The lysate containing samples sterilized at 7 kGy retained the most biological activity and no significant difference was noted between the 7 and 25 kGy treated lysate containing samples and the non-irradiated lysate containing traveler control.
EXAMPLE 2
Characterization of Gamma-Ray Sterilized hUTC Cell Lysate by SDS-PAGE
The signature banding-pattern of the major proteins present in hUTC lysate was compared among various sterilization protocols by use of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Lysate was prepared by repeated freeze/thaw cycles of hUTC cell pellets, followed by centrifugation. Samples were then prepared and labeled according to the treatment groups listed in Table 7, below:










TABLE 7





Group
Test condition
Lot/Expiration
Treatment








SURGICEL
UGB325-1,




FIBRILLAR TM
expiration February 2008




Absorbable Hemostat





(SCF)





PROMOGRAN TM Matrix
1305263,




(PGM)
expiration May 2007



A
VICRYL TM (polyglactin
ID: 5248-46-1
Control



910) nonwoven (VNW)
thickness 0.50 mm




without lysate
density 65.2)




(VNW)




B
Lysate lyophilized on VNW
ID: 5248-46-1
Test group




thickness 0.50 mm





density 65.2)



1C
Pre-lyophilized lysate (P)

Control


2C
Post-lyophilized powdered

Control



lysate




3C
Post-lyophilized powdered

Control (not



lysate loaded onto VNW

transported





to SST)









Protein content measurements were conducted, using the Quick Start Bradford Protein Assay (BioRad, Cat. #500-0205). This assay has a reported linear range of 1.25-10 μg/ml in the microassay format and is based on Coomassie Brilliant Blue G-250 dye shifts in absorbance due to protein binding.
For SDS-PAGE, the Invitrogen NOVEX® Pre-cast gel system was utilized. A Tris-Glycine 4% stacking, 4-20% gradient separating gel was used due to its ability to separate a wide molecular weight range of molecules (Invitrogen, Cat. #E660252BOX). For studies conducted at CBAT, the BenchMarkTM 10-220 kDa Protein Ladder (Invitrogen, Cat. #10747-012) was used as a reference. Samples were analyzed in reducing conditions according to the supplied manufacturer's instructions (Invitrogen XCell SureLockTM Mini-Cell, Version H; August 20, Catalog Nos. EI0001, EI0020, and EI0002; NOVEX® Tris-Glycine Gels, Catalog no. IM-6000F).
Running the gel. A 1× Tris-Glycine SDS Running Buffer was prepared by adding 100 ml of 10× NOVEX® Tris-Glycine SDS Running Buffer (Invitrogen, Cat. #LC2675) to 900 ml deionized water. The gel chamber was filled with the prepared 1× Tris-Glycine SDS Running Buffer. The appropriate concentration and volume of the protein sample was loaded onto the gel. The gel was run using the following conditions:















Voltage:
125 V constant



Run Time:
90 minutes



Expected Current:
30-40 mA/gel (start); 8-12 mA/gel (end)









Staining and drying the gel. Gels were stained using the Invitrogen SIMPLYBLUETM SafeStain (Invitrogen, Cat. #LC6060) following the supplied manufacturer's instructions (Basic Protocol). After staining, the gel was dried using the DRYEASE® Mini-Gel Drying System (Invitrogen, Cat. #N12387) according the supplied manufacturer's instructions.
Extraction of lysate proteins from scaffolds. To extract the proteins from scaffolds for analysis, materials were incubated in 1 ml of PBS overnight with shaking at 4° C.
Initial studies were conducted to assess the protein-banding pattern of hUTC lysate, as analyzed by SDS-PAGE to determine the hUTC “protein signature”. Powdered lyophilized lysate (L042205) was dissolved in dH2O to a final concentration of 0.5 μg/μl. Lysate was added to the sample buffer and reducing agent, and heated as described. The lysate+VNW combination was placed in 120 μl of sample buffer with reducing agent and water, then heated and loaded onto the gel as described. Lastly, a sample of basic-FGF was prepared as described as a control.
Following the initial characterization of hUTC lysate, gel electrophoresis was performed to determine the utility of this method to detect changes in protein banding pattern post processing. In these studies, protein-banding pattern was evaluated pre and post lyophilization of the hUTC lysate. In addition, these studies assessed whether hUTC lysate proteins could be assayed after lyophilization onto scaffolds other than VNW (e.g., SCF and PGM).
The scaffold samples were prepared by being placed in a 100 μl mixture of sample buffer, reducing agent and water, then heated as previously described; 17 μl (5 μg) of each sample was loaded onto the gel. The powdered samples and the pre-lyophilized liquid were dissolved in water to a final concentration of 2 μg/μl and prepared for loading as described in the XCell SURELOCKTM Mini-Cell manufacturer's instructions; the final amount of protein loaded onto the gel was 5 μg.
Assay validation studies were conducted. The Invitrogen gel system was used to replicate the system used at CBAT. An extraction step, described in the materials and methods, was added to the previously described protocol to elute lysate from the lyophilized scaffold. VNW scaffolds without lysate were used as a control.
Studies were also conducted to determine stability of hUTC lysate after lyophilization. The powdered and pre-lyophilized samples were dissolved in PBS before being loaded onto the gels, as previously described.
Lysate characterization post-processing (sterilization). Sixteen different sterilization protocols were assessed (see Table 8, below) with four samples tested per condition.







TABLE 8







Matrix Design for Sterilization of Lysate/VNW Samples










Dose (kGy)
Temperature (° C.)
Atmosphere
Sample ID













0
25
Nitrogen
1


0
25
Air
2


0
−70
Nitrogen
3


0
−70
Air
4


7
25
Nitrogen
5


7
25
Air
6


7
−70
Nitrogen
7


7
−70
Air
8


14
25
Nitrogen
9


14
25
Air
10


14
−70
Nitrogen
11


14
−70
Air
12


25
25
Nitrogen
13


25
25
Air
14


25
−70
Nitrogen
15


25
−70
Air
16









Lysate tested in this assay was from the same lot. Samples were sterilized by gamma irradiation. After sterilization, sample Groups A, B and C were analyzed. Additional lots of lysate (L051305 powder and VNW scaffolds, L040405 powder and L042205 powder) were sent for comparison. Samples of VNW containing lysate were extracted using the method previously described. Controls included pre-lyophilized lysate, lysate post-lyophilization, L092605 as a powder (2C) and loaded onto a VNW scaffold (3C). Sample 3C was not irradiated and remained at the inventors' location as a control.

Results. SDS-PAGE data demonstrated that discrete bands were observed in hUTC lysate. When hUTC lysate was compared in samples extracted from VNW scaffolds to the lyophilized lysate powder, the identical core-banding pattern was observed. In addition, when comparing results between lots of hUTC lysate (L042205 and L051305), similar results were observed; FIG. 4A depicts the results of testing two samples of lysate, L042205 as a lyophilized powder and L051305 after lyophilization onto a scaffold (30 μg/scaffold). Lanes were loaded as follows: 1) 7 μl of the BenchMarkTM Protein Ladder, 2) 20 μl (500 ng) of basic-FGF, 3 & 4) L051305 20 μl (5 μg) and 15 μl (3.75 μg) respectively, and lastly 5) μl of L042205 (μg).
Further characterization of post-production processing is shown in FIG. 4B. The gel was loaded in the following order: (1) BENCHMARKTM Protein Ladder, (2) L061305 powder, (3) L061305 on VNW, (4) L061305 on PGM, (5) L062405P, (6) L062405 powder, (7) L062405 on VNW, (8) L062405 on SCF, (9) PGM scaffold without lysate and (10) SCF scaffold without lysate. Multiple lots of lysate were examined (lots L061305 and L062405) before and after lyophilization. Banding patterns of hUTC lysate were consistent before lyophilization (lane 5), and after lyophilization as a powder (lanes 2 and 6). In addition, lysate extracted from VNW scaffolds (lanes 3 and 7) showed consistent results. These data show that a consistent banding pattern was observed for all conditions tested.
Gel results (FIG. 4B) also examined the banding pattern of lysate after lyophilization onto two other types of scaffolds, SURGICEL FIBRILLARTM (SCF) and PROMOGRANTM Matrix (PGM). Lane 8 shows lysate (L062405) after extraction from the SCF scaffold with lane 10 as a comparison (control scaffold without lysate). The results yielded a lysate banding pattern that is consistent with lysate banding pattern observed previously in this report. By contrast, results from PGM scaffolds (lanes 4 and 9), show that collagen component of this collagen/ORC scaffold yields a gel with unresolved bands.
FIG. 5 details the SDS-PAGE analysis of the sterilization study conducted at Lofstrand determining the effects of sterilization conditions outlined in Table 8, supra. FIG. 5 shows the results obtained from the SDS-PAGE analysis. Bradford data (not shown) revealed that little or no proteins were extracted from sample 14B1 and 16B1 (FIG. 5G). At high doses of irradiation, (samples 15B1, 15B2 and 16B2) hUTC lysate retained the characteristic banding-pattern (FIG. 5H). Samples are coded on the gels according to the following scheme: XYZ (e.g., 3A1) where X=Sample ID in Table 8, Y=Treatment codes in Table 7, and Z=sample replicate number. Sample 2C was loaded onto each gel to standardize data.
Bradford data (see Table 9, below) revealed that no proteins were extracted from sample 14B1. Bradford protein content results show inconsistency of protein recovery with the current Bradford method. Despite these differences in protein loading, at high doses of irradiation, (samples 15B1, 15B2 and 16B2) the lysate retained the characteristic banding-pattern.
Table 9, below, depicts Bradford protein concentration assay data for hUTC lysate on 90/10 PGA/PLA scaffolds post-sterilization by gamma irradiation, as performed by Lofstrand Labs Ltd. Samples are coded according to the following scheme: XYZ (e.g., 3A1) where X=Sample ID in Table 8, Y=Treatment codes in Table 7, and Z=sample replicate number. Sample 2C was loaded onto each gel to standardize data. Results from this study show inconsistency of protein recovery with the current Bradford method.











TABLE 9









Initial
Repeat














[Protein]
Recovered
[Protein]
Recovered
Difference


Sample ID
(μg/μl)
(μg)
(μg/μl)
(μg)
(%)















 1C
4.05
238.50
219.8
87.9
22


 2C
5.62
281.50
174
68.8
38


 3C
0.65
22.75
14.77
49.2
35


 1B-1
1.17
58.50


 1B-2
1.35
67.50


 2B-1
1.17
58.50


 2B-2
1.68
58.80


 3B-1
1.00
50.00
0.13
6.50
87


 3B-2
1.37
54.80
1.00
40.00
27


 4B-1
1.22
61.00
0.80
40.00
34


 4B-2
1.32
72.60
0.93
51.15
29.5


 5B-1
1.19
65.45
0.78
42.90
34.0


 5B-2
1.12
50.40
0.81
36.45
27.5


 6B-1
0.66
33.00
0.38
19.00
42.0


 6B-2
1.14
62.70
0.72
39.60
36.0


 7B-1
1.10
60.50
0.71
39.05
35.5


 7B-2
1.24
68.20
0.78
42.90
37.0


 8B-1
1.30
78.00
0.84
50.40
35.0


 8B-2
1.35
78.30
0.93
53.94
31.0


 9B-1
1.10
60.50
0.26
14.30
76.0


 9B-2
1.21
72.60
0.92
55.20
24.0


10B-1
1.03
56.65
0.57
31.35
44.0


10B-2
1.04
60.32
0.74
42.92
28.8


11B-1
1.35
81.00
1.02
61.20
24.0


11B-2
1.07
64.20
0.82
49.20
23.0


12-B-1
1.25
72.50
0.89
51.62
28.8


12-B-2
1.28
70.40
0.98
53.90
23.0


13B-1
1.10
60.50
0.66
36.30
40.0


13B-2
1.17
64.35
0.70
38.50
40.0


14B-1
1.02
0.91
0.00
0.00
100.0


14B-2
1.24
68.20
0.81
44.55
34.6


15B-1
0.64
34.98
0.41
22.55
35.5


15B-2
0.93
53.12
0.73
41.61
21.7


16B-1
0.92
50.38
0.03
1.65
96.7


16B-2
0.79
43.18
0.54
29.70
31.2









This data indicates that hUTC lysate has a consistent banding pattern that can be used as an in vitro method to demonstrate lot-to-lot equivalency of lysate, as well as lysate stability post processing. In addition, this characteristic-banding pattern was not significantly altered by gamma sterilization. hUTC lysate signature banding pattern consisted of five to six bands present at 158.4, 96.3, 87.3, 69.1, 58.3 and 43.3 kd. SDS-PAGE can reliably detect protein-banding pattern of hUTC lysate that has been loaded onto and extracted from nonwoven scaffolds made from VICRYLTM (polyglactin 910) nonwoven fibers.
The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.

## Claims

1. A method for sterilizing a material comprising a biologically-active agent without the addition of a stabilizer or removal of solvent, and without substantial loss in biological activity comprising:
sterilizing said material by irradiating said material with ionizing radiation at a dose of about 5 kGy to about 25 kGy while maintaining said material in an atmosphere comprising at least 95% by volume inert gas and at a temperature of about 4° C. or lower, without adding a stabilizer or removing solvent prior to irradiation, wherein said material retains its biological activity after sterilization.

2. The method according to claim 1 wherein said ionizing radiation comprises X-rays, gamma radiation, or electron beams.

3. The method according to claim 1 wherein said dose of ionizing radiation is about 6 kGy to about 12 kGy.

4. The method according to claim 1 wherein said dose of ionizing radiation is about 7 kGy.

5. The method according to claim 1 wherein said inert gas comprises nitrogen.

6. The method according to claim 1 wherein said atmosphere comprises about 100% by volume nitrogen.

7. The method according to claim 1 wherein said temperature is about 0° C.

8. The method according to claim 1, wherein said material is contained within a vessel, and wherein said method further comprises purging the ambient air from said vessel by flushing with said inert gas prior to irradiating said material.

9. The method according to claim 1 wherein said biologically active agent comprises cells, cell derivatives, growth factors, or any combination thereof.

10. The method according to claim 1 wherein said biologically active agent is selected from the group consisting of epithelial cells, bone marrow cells, adipocytes, stem cells, keratinocytes, melanocytes, dermal fibroblasts, keratinocytes, vascular endothelial cells, myoblasts, myocytes, hepatocytes, smooth muscle cells, striated muscle cells, stromal cells, other soft tissue cells or progenitor cells, chondrocytes, osteoblasts, islet cells, nerve cells, placenta-derived cells, human umbilical tissue derived cells (hUTC), human kidney derived cells, or any combination thereof.

11. The method according to claim 1 wherein said biologically active agent comprises a cell derivative selected from the group consisting of cell lysate, trophic factors, growth factors, cytokines, conditioned media, or any combination thereof.

12. The method according to claim 1 wherein said biologically active agent comprises hUTC lysate.

13. The method according to claim 1 wherein said biologically active agent comprises a growth factor selected from the group consisting of PDGF-BB, bFGF, TGF-beta, HGF, VEGF, GDF-5, or any combination thereof.

14. The method according to claim 1 wherein said material further comprises a scaffold composition.

15. The method according to claim 14 wherein said scaffold composition comprises collagen, cellulose, fibrin, elastin, gelatin, demineralized bone, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polycaprolactone, polyanhydrides, polyhydroxybutyrates, or any combination thereof.

16. The method according to claim 1 wherein said amount of biological activity is assessed according to a cell proliferation assay, transmigration assay, angiogenesis assay, cytotoxicity assay, matrix deposition assay, lymphocyte activation assay, or apoptosis assay.

17. A method for sterilizing a material comprising a biologically-active agent consisting essentially of sterilizing said material by irradiating said material with ionizing radiation at a dose of about 5 kGy to about 25 kGy while maintaining said material in an atmosphere comprising at least 95% by volume inert gas and at a temperature of about 4° C. or lower, wherein said material retains its biological activity after sterilization.

18. The method according to claim 17 wherein said ionizing radiation comprises X-rays, gamma radiation, or electron beams.

19. The method according to claim 17 wherein said dose of ionizing radiation is about 6 kGy to about 12 kGy.

20. The method according to claim 17 wherein said inert gas comprises nitrogen.

21. The method according to claim 17 wherein said atmosphere comprises about 100% by volume nitrogen.

22. The method according to claim 17 wherein said biologically active agent is selected from the group consisting of epithelial cells, bone marrow cells, adipocytes, stem cells, keratinocytes, melanocytes, dermal fibroblasts, keratinocytes, vascular endothelial cells, myoblasts, myocytes, hepatocytes, smooth muscle cells, striated muscle cells, stromal cells, other soft tissue cells or progenitor cells, chondrocytes, osteoblasts, islet cells, nerve cells, placenta-derived cells, human umbilical tissue derived cells (hUTC), human kidney derived cells, or any combination thereof.

23. The method according to claim 17 wherein said biologically active agent comprises a cell derivative selected from the group consisting of cell lysate, trophic factors, growth factors, cytokines, conditioned media, or any combination thereof.

24. The method according to claim 17 wherein said biologically active agent comprises hUTC lysate.

25. The method according to claim 17 wherein said biologically active agent comprises a growth factor selected from the group consisting of PDGF-BB, bFGF, TGF-beta, HGF, VEGF, GDF-5, or any combination thereof.

26. The method according to claim 17 wherein said material further comprises a scaffold composition.

27. The method according to claim 26 wherein said scaffold composition comprises collagen, cellulose, fibrin, elastin, gelatin, demineralized bone, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polycaprolactone, polyanhydrides, polyhydroxybutyrates, or any combination thereof.

28. A method for sterilizing a material comprising a biologically-active agent consisting of sterilizing said material by irradiating said material with ionizing radiation at a dose of about 5 kGy to about 25 kGy while maintaining said material in an atmosphere comprising at least 95% by volume inert gas and at a temperature of about 4° C. or lower, wherein said material retains its biological activity after sterilization.