Patent Description:
Peptide agents with the ability to self-assemble into gel structures have a wide variety of uses in therapeutic and research contexts. One such peptide agent, for example, a synthetic, <NUM>-amino acid polypeptide with a repeating sequence of arginine, alanine, and aspartic acid (i.e., RADARADARADARADA [SEQ ID NO: <NUM>], also known as "RADA16"), is commercially available under the trade names PuraStat®, PuraMatrix®, and PuraMatrix GMP® from <NUM>-D Matrix Medical Technology, and has demonstrated utility in a wide range of laboratory and clinical applications, including cell culture, drug delivery, accelerated cartilage and bone growth, and regeneration of CNS, soft tissue, and cardiac muscle, and furthermore as a matrix, scaffold, or tether that can be associated with one or more detectable agents, biologically active agents, cells, and/or cellular components. <CIT> discloses the removal of antiplatelet agents and antocoagulants previously added to a platelet preparation to improve the shelf life of the platelet preparation prior to administration to a patient. <CIT> discloses commercial scale sterilization and packaging of <NUM>% RADA16. <CIT> discloses "shear-thinning" of a self-assembled hydrogel through a syringe needle.

The present invention provides methods for sterilizing a liquid peptide composition as set out in the claims appended hereto.

Among other things, the present disclosure demonstrates that certain peptide compositions (e.g., compositions of particular peptides, at particular concentrations, and/or having particular rheological properties) have certain characteristics and/or may not be amenable to certain handling and/or processing steps such as, for example, filtration (e.g., sterilizing filtration).

The present disclosure also demonstrates that certain particular peptide compositions are surprisingly stable to one or more treatments (e.g., heat treatment, as is applied in autoclave procedures) that damage many peptide compositions.

Thus, the present disclosure describes a variety of technologies relevant to processing of peptide compositions, and particularly to sterilization.

The present disclosure demonstrates that particular peptide compositions may have one or more useful and/or surprising characteristics (e.g., resistance to damage from heat treatment, rheological responsiveness to and/or recovery from application of shear stress, etc).

The present disclosure describes, among other things, systems for sterilizing peptide compositions, andor systems for determining appropriate such systems for application to particular peptide compositions.

Particular peptide compositions may be defined, for example, by one or more features selected from the group consisting of: peptide sequence, peptide concentration, viscosity, stiffness, sensitivity to heat treatment, rheological responsiveness to application of shear stress, rheological recovery from application of shear stress, etc).

Among other things, the present disclosure describes certain peptide compositions that may be sterilized by autoclave treatment.

The present disclosure describes certain technologies for achieving filtration of certain peptide compositions, and particularly for altering rheological properties of peptide compositions (as defined by identity and sequence of the peptide) so that they are rendered amenable to filtration. For example, in some cases, viscosity of peptide compositions to be filtered may be reduced prior to filtration. In some cases, shear stress may be applied to peptide compositions, so that rheological properties may be altered. For example, viscosity and/or stiffness of a peptide composition may be reduced prior to filtration; in some cases, such a reduction is temporary.

The technologies described enable filtration of peptide compositions at higher concentrations than is feasible with conventional filtration techniques. For example, technologies described herein permit RADA16 to be filtered, and particularly to be sterilized by filtration, at concentrations higher than <NUM>% in accordance.

The present disclosure describes a method for sterilizing a liquid peptide composition whose sequence comprises a series of repeating units of IEIK comprising subjecting the composition to autoclave treatment. In some cases, a method does not involve sterilizing filtration.

The present disclosure describes a method for sterilizing a liquid peptide composition whose sequence comprises a series of repeating units of IEIK comprising subjecting the composition to heat treatment. In some cases, the heat treatment performs at about <NUM> for about <NUM>.

The present disclosure describes a method for sterilizing a liquid peptide composition having an initial storage modulus within the range of about <NUM> to about <NUM>,<NUM> Pa at <NUM> Pa of oscillation stress, the method comprising steps of subjecting the composition to high shear stress so that storage modulus of the composition is temporarily reduced to a level within a range of about <NUM>% to <NUM>% of the initial storage modulus, and subjecting the composition to filtration while its viscosity is at the reduced level.

In some cases, the step of subjecting the composition to high shear stress utilizes at least one shear-thinning unit.

In some cases, at least one shear-thinning unit is or comprises at least one needle. In some cases, at least one needle is at least <NUM> long. In some cases, at least one needle has a gauge within the range of about <NUM> to about <NUM>.

In some cases, at least one shear-thinning unit is or comprises at least one screen with micro- or nano-sized holes. In some cases, micro- or nano-sized holes have a largest dimension within a range of about <NUM> to about <NUM>. In some cases, a pinch between holes is about <NUM> to about <NUM>. In some cases, a screen is made at least in part of a material selected from the group consisting of stainless-steel, tungsten, titanium, silicon, ceramic, plastic, and combination thereof. In some cases, thickness of the screen is about <NUM> to about <NUM>.

In some cases, at least one shear-thinning unit is or comprises at least one membrane with micro- or nano-sized pores. In some cases, the pores gave a size with a range of about <NUM> to about <NUM>.

In some cases, high shear stress for sterilization is with a range of about <NUM> to about <NUM> Pa.

In some cases, a liquid peptide composition comprises RADA16, IEIK13, or KLD12.

In some cases, a liquid peptide composition is pressurized prior to filtration. In some cases, a peptide liquid composition is further stored the under vacuum.

The term "agent" as used herein may refer to a compound or entity of any chemical class including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. In some cases, an agent is or comprises a natural product in that it is found in and/or is obtained from nature. In some cases, an agent is or comprises one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some cases, an agent may be utilized in isolated or pure form; in some cases, an agent may be utilized in crude form. In some cases, potential agents are provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. Some particular agents that may be utilized in accordance with the present teaching include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNAs, shRNAs, DNA/RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptide mimetics, etc. In some cases, an agent is or comprises a polymer. In some cases, an agent is not a polymer and/or is substantially free of any polymer. In some cases, an agent contains at least one polymeric moiety. In some cases, an agent lacks or is substantially free of any polymeric moiety.

As used herein, the term "amino acid," in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some cases, an amino acid has the general structure H2N-C(H)(R)-COOH. In some cases, an amino acid is a naturally-occurring amino acid. In some cases, an amino acid is a synthetic amino acid; in some cases, an amino acid is a D-amino acid; in some cases, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some cases, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some cases, an amino acid may be modified by methylation, amidation, acetylation, and/or substitution as compared with the general structure. In some cases, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some cases, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some cases, the term "amino acid" is used to refer to a free amino acid; in some cases it is used to refer to an amino acid residue of a polypeptide.

As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain cases, the term "approximately" or "about" refers to a range of values that fall within <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed <NUM>% of a possible value).

Two events or entities are "associated" with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some cases, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some cases, two or more entities that are physically associated with one another are covalently linked to one another; in some cases, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

The term "comparable" is used herein to describe two (or more) sets of conditions, circumstances, individuals, or populations that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed. In some cases, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied. Those skilled in the art will appreciate that relative language used herein (e.g., enhanced, activated, reduced, inhibited, etc) will typically refer to comparisons made under comparable conditions.

Certain methodologies described herein include a step of "determining". Those of ordinary skill in the art, reading the present specification, will appreciate that such "determining" can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some cases, determining involves manipulation of a physical sample. In some cases, determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some cases, determining involves receiving relevant information and/or materials from a source. In some cases, determining involves comparing one or more features of a sample or entity to a comparable reference.

The term "gel" as used herein refers to viscoelastic materials whose rheological properties distinguish them from solutions, solids, etc. In some cases, a composition is considered to be a gel if its storage modulus (G') is larger than its modulus (G"). In some cases, a composition is considered to be a gel if there are chemical or physical cross-linked networks in solution, which is distinguished from entangled molecules in viscous solution.

The term "in vitro" as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

The term "in vivo" as used herein refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

The term "peptide" as used herein refers to a polypeptide that is typically relatively short, for example having a length of less than about <NUM> amino acids, less than about <NUM> amino acids, less than <NUM> amino acids, or less than <NUM> amino acids.

The term "polypeptide" as used herein refers to any polymeric chain of amino acids. In some cases, a polypeptide has an amino acid sequence that occurs in nature. In some cases, a polypeptide has an amino acid sequence that does not occur in nature. In some cases, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some cases, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some cases, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some cases, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some cases, a polypeptide may comprise only D-amino acids. In some cases, a polypeptide may comprise only L-amino acids. In some cases, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some cases, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some cases, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some cases, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some cases, a polypeptide is linear. In some cases, a polypeptide may be or comprise a stapled polypeptide. In some cases, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification describes and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some cases, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some cases, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some cases at a comparable level or within a designated range) with a reference polypeptide of the class; in some cases with all polypeptides within the class). For example, in some cases, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about <NUM>-<NUM>%, and is often greater than about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or more and/or includes at least one region (e.g., a conserved region that may in some cases be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than <NUM>% or even <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%. Such a conserved region usually encompasses at least <NUM>-<NUM> and often up to <NUM> or more amino acids; in some cases, a conserved region encompasses at least one stretch of at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more contiguous amino acids. In some cases, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide. In some cases, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

The term "reference" as used herein describes a standard or control relative to which a comparison is performed. For example, in some cases, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some cases, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some cases, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

The reference technical disclosure set out below may in some respects go beyond the disclosure of the invention per se, and may also provide technical background for related technical developments. It will therefore be appreciated that this reference technical disclosure is not intended to define the invention as such, but rather to place it in a broader technical context.

The present disclosure describes technologies for sterilization of peptide compositions. In some cases, disclosed methods are particularly applicable to peptide solutions with high viscosity and/or stiffness. In some cases, the present disclosure defines particular peptide solutions that may be sterilized by autoclave treatment. In some cases, the present disclosure defines particular peptide solutions that may not be amenable to filtration unless and until treated so as to alter their rheological properties. In some cases, the present disclosure describes technologies that may temporarily reduce peptide solution viscosity and/or stiffness sufficiently to permit filtration. In some cases, the present disclosure teaches technologies for facilitating handling, processing, and/or filtration of certain peptide solutions, for example by applying high shear stress that modify rheological properties thereof.

Peptide compositions to which teachings of the present disclosure may be relevant include compositions of amphiphilic peptides having about <NUM> to about <NUM> amino acid residues. In certain cases, a relevant peptide may have a length of at least about <NUM> amino acids. In certain cases, a peptide may have a length of between about <NUM> to about <NUM> amino acids. In certain cases, a peptide may have a length of at least <NUM> amino acids, at least about <NUM> amino acids, or at least about <NUM> amino acids.

In some cases, as is understood in the art, an amphiphilic polypeptide is one whose sequence includes both hydrophilic amino acids and hydrophobic amino acids. In some cases, such hydrophilic amino acids and hydrophobic amino acids may be alternately bonded, so that the peptide has an amino acid sequence of alternating hydrophilic and hydrophobic amino acids. In some cases, such a peptide has an amino acid sequence that is or comprises repeats of Arg-Ala-Asp-Ala (RADA); in some cases, such a peptide has an amino acid sequence that is or comprises repeats of Lys-Leu-Asp (KLD); in some cases, such a peptide has an amino acid sequence that is or comprises repeats of Ile-Glu-Ile-Lys (IEIK).

In some cases, a peptide for use in accordance with the present disclosure, may generally be self-assembling, and/or may exhibit a beta-sheet structure in aqueous solution under certain conditions.

In some cases, a peptide for use in accordance with the present disclosure has an amino acid sequence as found in the commercial product known as PuraMatrix®, i.e., has the amino acid sequence Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala (i.e., RADA16, aka [RADA]<NUM>; SEQ ID NO: <NUM>). In some cases, a peptide for use in accordance with the present disclosure has an amino acid sequence: Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu (i.e., KLDL12, aka [KLDL]<NUM>, aka KLD12; SEQ ID NO:<NUM>). a peptide for use in accordance with the present disclosure has an amino acid sequence: Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile (i.e., IEIK13, aka (IEIK)3I; SEQ ID NO:<NUM>).

In some cases, peptide compositions to which the present disclosure may be relevant are those characterized by certain rheological properties. In some cases, relevant rheological properties may be or include loss modulus, stiffness, rheological recovery time, storage modulus, viscosity, yield stress, etc. In some cases, rheological properties are assessed via measurement; in some cases, one or more rheological properties may be assessed via visual observation.

In certain cases, storage modulus and stiffness have a positive correlation; in general, those of ordinary skill appreciate that higher storage modulus is related to higher stiffness.

In some cases, a high viscosity peptide composition is characterized by a storage modulus within the range of about <NUM> to about <NUM>,<NUM> Pa at <NUM> rad/sec of frequency and <NUM> Pa of oscillation stress.

In some cases, a peptide composition has a peptide concentration within the range of about <NUM>% to about <NUM>%.

In some cases, a peptide composition to which one or more of the methodologies described herein is applied is of a commercial-scale volume.

In some cases, a peptide composition to which one or more of the methodologies described herein is applied is one that has been stored for a period of time. In some cases, a peptide composition has been stored in a pressure vessel.

In some cases, a peptide composition to which one or more methodologies described herein is applied is then stored, for example, in a reservoir vessel prior to packaging.

The present disclosure appreciates that preparation and/or handling of certain peptide compositions (e.g., particularly compositions of certain self-assembling peptides and/or of high peptide concentrations) has been complicated by difficulties related, for example, to high viscosity and/or stiffness. The present disclosure particularly demonstrates that certain peptide compositions are not amenable to filtration, and in particular to filtration through sterilizing filters.

The present disclosure further appreciates that filtration challenges can complicate or preclude sterilization of such peptide compositions. The present disclosure describes technologies that permit filtration of certain peptide compositions and/or otherwise permit sterilization.

Autoclave treatment is a conventional sterilization method that involves subjecting materials to high pressure saturated steam at <NUM>. It is generally understood in the art that application of high heat, such as is involved in autoclave treatment, can degrade peptides.

The present disclosure surprisingly demonstrates that certain peptide compositions are stable to heat treatment, and particularly to autoclave treatment. Among other things, the present disclosure demonstrates that such peptide compositions may be sterilized with the autoclave treatment. In some cases, such compositions may be sterilized by heat treatment at about <NUM> for about <NUM> minutes.

In some cases, peptide compositions that may be subjected to heat treatment, and/or to autoclave treatment are IEIK13 compositions. In some such cases, IEIK13 compositions have a concentration within the range of about <NUM>% to about <NUM>%.

In some cases, peptide compositions that may be subjected to heat treatment and/or to autoclave treatment are KLD12 compositions. In some cases, however, KLD12 compositions are not subjected to autoclave treatment in accordance with the present teaching.

In some cases, RADA16 compositions are not subjected to autoclave treatment in accordance with the present teaching.

Without wishing to be bound by any particular theory, the present disclosure proposes that the stability of certain IEIK13 compositions to heat treatment such as autoclave treatment may be attributable, at least in part, to the absence of aspartic acid (Asp, D) in compositions, while RADA16 and KLD12 have aspartic acids.

In some cases, peptide compositions that can appropriately be subjected to heat treatment such as autoclave treatment in accordance with the present teaching are characterized by resistance to degradation when exposed to such treatment and/or by stability of rheological properties (e.g., viscosity and/or stiffness) when subjected to such treatment. In accordance with the present disclosure, peptide compositions of interest may be exposed to heat treatment such as autoclave treatment, and one or more properties of the composition (e.g., peptide degradation and/or one or more rheological properties) can be assessed, for example before and after treatment, so that appropriateness of sterilizing such composition via autoclave treatment may be determined (see, e.g., Example <NUM>).

The present disclosure demonstrates that certain peptide compositions can be rendered amenable to filtration via exposure to treatment that alters one or more rheological properties (e.g., that alters viscosity and/or stiffness).

In some particular cases, rheological property alteration is achieved by exposure to shear stress.

Without wishing to be bound by any particular theory, the present disclosure proposes that subjecting peptide compositions as described herein to high shear stress can disrupt self-assembled structures. The present disclosure further proposes that recovery time may represent that required for such structures to re-form.

In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, shear stress applied to peptide solutions may be at least about <NUM> Pa. In some cases, the amount of shear stress may be at least about <NUM>~<NUM> Pa, for example, in view of the yield stress of RADA16 <NUM>%, IEIK13 <NUM>% and <NUM>% and KLD12 <NUM>% noted above.

In some cases, viscosity of peptide solutions may drop significantly with shear stress. In some cases, viscosity of peptides solutions may drop at least <NUM>% with shear stress. In some cases, viscosity of peptides solutions may drop at least <NUM>% with shear stress. In some cases, viscosity of peptides solutions may drop at least <NUM>% with shear stress. In some cases, viscosity of peptides solutions may drop at least <NUM>% with shear stress. In some cases, viscosity of peptides solutions may drop at least <NUM>% with shear stress.

In some cases, the rheological property alteration is temporary. In some cases, the peptide composition is characterized by rheological recovery characteristics. For example, in some cases, such compositions are characterized in that one or more of their rheological properties are restored within a time period within a range of about <NUM> to about <NUM> hours.

In some cases, rheological restoration is considered to be achieved when one or more rheological properties returns to a level at least <NUM> % of its initial value.

In some cases, rheological restoration is considered to be achieved when the change observed in one or more rheological properties upon application of shear stress is at least <NUM>% reversed.

In some cases, peptide compositions may recover their storage modulus after application of shear stress. In some cases, peptide solutions may recover about <NUM> to <NUM>% of their original storage modulus in <NUM>. In some cases, peptide solutions may recover about <NUM> to <NUM>% of their original storage modulus in <NUM>. In some cases, peptide solutions may recover about <NUM> to <NUM>% of their original storage modulus in <NUM>. In some cases, peptide solutions may recover about <NUM> to <NUM>% of their original storage modulus in <NUM>.

In some cases, peptide solutions may recover their viscosity over time after filtration. In some cases, peptide solutions may recover about <NUM> to <NUM>% of their original viscosity in <NUM>. In some cases, peptide solutions may recover about <NUM> to <NUM>% of their original viscosity in <NUM>. In some cases, peptide solutions may recover about <NUM> to <NUM>% of their original viscosity in <NUM>. In some cases, peptide solutions may recover about <NUM> to <NUM>% of their original viscosity in <NUM>.

The present disclosure specifically exemplifies appropriate adjustment of rheological properties of certain peptide compositions upon application of shear stress (e.g., specifically upon passage through a needle, for example of particular structure) (see Example <NUM>). The results presented in this Example show a logarithmic increase of storage modulus from <NUM> minute after injection, as shown in <FIG> for RADA16, <FIG> for KLD12, and <FIG> for IEIK13.

Among other things, the present disclosure describes methodologies in accordance with which one or more certain peptide compositions are subjected to high shear stress so that one or more of their rheological properties is adjusted (e.g., viscosity is decreased) to an appropriate level so that the composition(s) become amenable to filtration, and in some cases to sterilizing filtration, and the composition(s) are subjected to such filtration, within a time period after the subjecting to shear stress selected so that filtration occurs while the rheological properties remain adjusted (e.g., before significant or complete restoration of such propert(ies) has occurred).

In general, as described herein, shear stress may be applied by application of a peptide composition to (and/or passage of a peptide composition through) a shear-thinning unit. In some cases, a shear-thinning unit is or comprises a needle, a membrane, and/or a screen. In some cases, a plurality of individual shear-thinning units is utilized, for example so that high-throughput filtration can be achieved.

In some cases, the devices and methodologies can achieve filtration of peptide compositions on a commercial scale.

In some non-limiting cases, shear stress may be applied by injection through one or more needles. Thus, in some cases, one or more needles may be used as a shear-thinning unit.

In some cases, a needle may be at least about <NUM> long. In some cases, a needle may be at least about <NUM> long. In some cases, a needle may be at least about <NUM> long. In some cases, a needle may be at least about a <NUM> long. In some cases, a needle may be at least about <NUM> long. In some cases, a needle may be at least about <NUM> long. In some cases, a needle may be at least about <NUM> long. In some cases, a needle may be at least about <NUM> long. In some cases, a needle may be at least about <NUM> long.

In some cases, a needle may have a gauge within a range of about <NUM> to about <NUM>. In some cases, a needle may have a gauge within a range of about <NUM> to about <NUM>. In some cases, a needle may have a gauge of about <NUM> to about <NUM>.

<FIG> discloses one non-limiting case of a sterilization device in accordance with one or more non-limiting cases. As depicted, peptide composition (e.g., viscous solution of a self-assembling peptide) (left) may be transferred to the first syringe with a needle, injected to the second syringe (right), and then filtered.

In some cases, a shear-thinning unit utilized to apply shear stress to a peptide composition as described herein may be a device or entity characterized by micro- or nano-pores. <FIG> depicts one non-limiting case of a sterilization device in accordance with one or more non-limiting cases of the present teaching. As depicted, a peptide solution (e.g., a viscous solution of a self-assembling peptide) may be transferred to a dispensing syringe (or a pressure vessel), delivered to a first chamber with pores for shear stress, and then filtered in the second chamber. As will be understood by those skilled in the art, diameter size of membrane may vary depending on the amount of peptide solution.

In some cases, pore size of a shear-thinning unit may be about <NUM> to <NUM>. In some cases, pore size of a shear-thinning unit may be about <NUM> to <NUM>. In some cases, pore size of a shear-thinning unit may be about <NUM> to <NUM>. In some cases, pore size of a shear-thinning unit may be about <NUM> to <NUM>.

In some cases, a shear-thinning unit may have micro- or nano-holes. In some cases, holes may be patterned or drilled on a plate whose thickness may be about <NUM> to <NUM> in some cases. <FIG> depicts one non-limiting case of a sterilization device in accordance with one or more non-limiting cases of the present disclosure. A shear-thinning unit may be inserted into the first filtering chamber shown <FIG>.

In some cases, holes in an case of a shear-thinnung unit described herein may have a largest dimension within the range of about may be about <NUM> to <NUM>. In some cases, such dimension may be within the range of about <NUM> to <NUM>. In some cases, such dimension may be within the range of about <NUM> to <NUM>. In some cases, In some cases, such dimension may be within the range of about <NUM> to <NUM>.

In some cases, a shear-thinning unit of this case may have a pitch between holes within the range of about <NUM> to about <NUM>.

In some cases, shear-thinning unit may be made, in whole or in part, of a material selected from the group consisting of stainless-steel, tungsten, titanium, similar metal, silicon, ceramic or plastic materials, and combinations thereof.

In some cases, peptide compositions to which technologies described herein are applied are then utilized in one or more applications that involve biological cells, tissues, or organisms (e.g., so that sterilized compositions are of particular utility).

As is known in the art, certain peptide compositions (e.g., certain compositions of self-assembling peptides) have proven to be particularly useful as matrices for cell growth in vivo and/or in vitro, and/or as void fillers, hemostats, barriers to liquid movement, wound healing agents, etc. In some cases, such compositions form peptide hydrogels with one or more desirable characteristics (e.g., pore and/or channel size, strength, deformability, reversibility of gel formation, transparency, etc).

Those skilled in the art, reading the present disclosure, will immediately appreciate its usefulness in a variety of contexts in which such peptide compositions, including gel compositions and especially including reversibly gelling compositions, are employed. Of particular interest are in vivo applications (e.g., surgical applications or other applications, particularly that permit or benefit from delivery via a cannula-type device, such as a needle, through which composition may be administered or applied).

The present Example describes, among other things, rheological properties of various peptide compositions (i.e., specifically of compositions of self-assembling peptides), and demonstrates significant variability of parameters such as viscosity, storage modulus (e.g., stiffness), loss modulus, and yield stress for different peptides and/or for different concentrations of the same peptide. The Example also demonstrates that certain of these solutions are not readily amenable to filtration. In particular, the Example demonstrates that high viscosity solutions of such peptides present challenges for filtration technologies. Rheological properties were determined for a variety of peptide solutions. Specifically, solutions of RADA16, IEIK13, and KLD12 per prepared at concentrations indicated below in Table <NUM>. As can be seen, in general, higher concentration solutions showed higher max viscosity. Furthermore, peptides of different sequence showed different max viscosities in solutions of the same concentration. For example, <NUM>% KLD12, <NUM>% KLD12, and <NUM>% IEIK13 solutions have <NUM>, <NUM>, and <NUM> times higher maximum viscosities than <NUM>% RADA16, respectively.

Each of the peptide solutions listed in Table <NUM> was subjected to filtration through a <NUM> Nalgene syringe filter with <NUM> cellulose acetate membranes. The <NUM>% and <NUM>% KLD12 solutions (which, as can be seen, are characterized by relatively low concentration, viscosity and/or stiffness) passed successfully through the filter. By contrast, the <NUM>% and <NUM>% KLD12 solutions and <NUM>% IEIK13 solutions (which, as can be seen, are characterized by relatively high concentration, viscosity and/or stiffness) could not be passed successfully through the filter; instead, the filter burst.

The present Example demonstrates that some peptide compositions (i.e., specifically compositions of self-assembling peptides as described herein) are surprisingly stable to heat treatment. In particular, this Example demonstrates that certain peptide compositions maintain a stable molar mass even upon application of autoclave treatment at <NUM> for <NUM> minutes. The present Example therefore establishes that such compositions can successfully be sterilized through application of high heat (e.g., autoclave) technologies. The Example simultaneously demonstrates, however, that certain peptide compositions are not stable to such treatment.

<FIG> present results of autoclave treatment for certain compositions of RADA16, IEKI13, and KLD12, respectively.

The measured molar mass of RADA16, prior to autoclave treatment, was <NUM>, which matches its calculated molar mass. However, the mass spec analysis demonstrated that RADA16 was degraded during the autoclave treatment, thereby demonstrating that this technique cannot be used for sterilization of such a RADA16 composition.

The measured molar mass of IEIK13, prior to autoclave treatment, was <NUM>, which also matches its calculated molar mass. Mass spec analysis demonstrated that IEIK13 was not degraded after the autoclave treatment, thereby demonstrating that this technique can usefully be employed for sterilization of such an IEIK13 composition.

The measured molar mass of KLD12, prior to autoclave treatment, is <NUM>, which matches its calculated molar mass. KLD12 was partially degraded during autoclave treatment. As KLD12 was degraded during autoclave treatment, it was determined that autoclave treatment is not a preferred technique for sterilization of such KLD12 compositions; a conventional filtration approach to sterilization was carried out on KLD12 at several concentrations of peptide.

Rheological properties of certain peptide compositions were determined before and after autoclaving. The data are shown in <FIG>. As can be seen, autoclaved IEIK13 surprisingly exhibited almost identical rheological strength as non-autoclaved IEIK13, while RADA16 displayed a dramatic decrease of rheological strength.

Autoclave treatment may be used for sterilization of IEIK13 compositions as described herein, but should be avoided for RADA16 compositions.

The present Example demonstrates that applied shear stress may decrease viscosity and/or stiffness of certain peptide solutions, and furthermore demonstrates that such decrease in viscosity and/or stiffness can render the compositions amenable to various and/or processing technologies (e.g., filtration) to which the compositions are not amenable absent such treatment.

Shear flow tests were performed on peptide solutions using a rheometer (DHR-<NUM>, TA Instruments) with <NUM> plates. Results are shown in <FIG> for <NUM>% RADA16 solutions and <FIG> for <NUM>% IEIK13 solutions. As can be seen, both <NUM>% RADA16 and IEIK13 <NUM>% solutions showed a typical shear thinning properties. That is, as shear rate increased, their viscosities were dramatically dropped. As shear rate increased, shear stress immediately increased, and then slightly decreased when viscosity reached a plateau. The yield stress was about <NUM> Pa for <NUM>% RADA16 solution and about <NUM> Pa for <NUM>% IEIK13 solution.

The viscosity recovery times of RADA16 and IEIK13 solutions were evaluated after application of high shear stress. Using a DHR-<NUM> rheomether (TA Instruments), viscosity changes of <NUM>% RADA16 and <NUM>% IEIK13 solutions were measured with flow tests at <NUM><NUM>/sec of shear rate after applying <NUM><NUM>/sec of shear rate to samples for <NUM>. RADA16 and IEIK13 solutions showed a typical thixotropic behavior, which means their viscosity were slowly recovered. Without wishing to be bound by any particular theory, we propose that rheological property recovery times for these solutions may be based on reassembly of peptide molecules into structures (e.g., nano-fibers) in the solutions. Complete reassembling times of <NUM>% RADA16 and <NUM>% IEIK13 solution were about <NUM> to <NUM> hours. The results are shown in <FIG> for <NUM>% RADA16 solution and <FIG> for <NUM>% IEIK13 solution.

The percentages of recovery back to the original storage modulus at <NUM> and <NUM> after injection peptide compositios through a <NUM> gauge needle are listed in Table <NUM>. The recovery rate of IEIK13 (specifically, of a <NUM>% IEIK13 solution) was the fastest among the peptide solutions, showing <NUM>% recovery to the original storage modulus in <NUM>. KLD12 was the slowest among those tested to recover; it showed only <NUM>% recovery to the original storage modulus in <NUM> (for <NUM>%). In some non-limiting cases, it may take about <NUM> to <NUM> hours for full recovery to an original modulus after passage through a needle (e.g., injection).

Rheological measurements were performed for RADA16 and IEIK13 solutions after injecting them through <NUM> gauge needles. The results showed a logarithmic increase of storage modulus from <NUM> minute after injection. The results are shown in <FIG> for RADA16, <FIG> for KLD12, and <FIG> for IEIK13.

The present Example describes a filtration process for peptide compositions (specifically, of self-assembling peptides as described herein) using a needle as a shear-thinning unit. In particular, the present Example demonstrates that application of appropriate shear stress (e.g., via passage through a shear-thinning unit) can alter rheological properties of the composition (e.g., can reduce viscosity and/or stiffness, etc) so that it can successfully be passed through a filter such as, for example, a sterilizing filter).

<FIG> depicts one non-limiting case of a sterilization device in accordance with the present disclosure. As depicted, the device includes a first syringe that applies sheer stress to the composition sufficient to alter its rheological properties such that it successfully passes through a second syringe that is fitted with a membrane filter of appropriate pore size to achieve sterilization of the composition. Specifically, the depicted device includes a first syringe with a <NUM> gauge needle (<NUM> x <NUM>, Endo irrigation needle with double side vent, Transcodent, Germany) (middle) and a second syringe with a membrane filter (right). A viscouse <NUM>% KLD12 solution (left) was transferred to the first syringe, and was then injected into the second syringe (right) and then filtered through the membrane filter. Using this method, <NUM>% KLD12 solutions were successfully filtered.

The present Example describes certain shear thinning units. The principle of operation is like that for the first needle described above. Specifically, each shear-thinning unit applies shear stress appropriate and sufficient to adjust one or more rheological properties of an applied peptide composition so that the composition becomes amenable to filtration, and specifically to filtration through a sterilizing filter. In some cases, multiple needles or equivalents may be used as a shear-thinning unit.

This Examples demonstrates use of a with membrane filter (pore size > <NUM>) as a shear-thinning unit. Viscous <NUM>% KLD12 or <NUM>% IEIK13 solutions may be transferred to a dispensing syringe (or a pressure vessel), delivered to a first chamber with a shear-thinning unit (for example, pore size ranging from <NUM> to <NUM>), and then filtered through a filtering membrane (for example, pore size: <NUM>) in the second chamber.

To examine the effect of pore size in the shear-thinning unit on viscosity change of viscous peptide solutions, <NUM>% KLD12 and <NUM>% IEIK solutions were passed through selected pore sizes, and their apparent viscosity changes were evaluated. <NUM>% KLD solutions passed through the shear-thinning unit with the pore sizes of <NUM>, <NUM>, and <NUM>. Viscosity of the solutions was decreased enough to flow down when a vessel containing it was flipped over. Though <NUM>% KLD solutions that had passed through the shear-thinning unit with the pore size of <NUM> were slightly less viscous than pre-passage <NUM>% KLD compositions, they remained too viscous to flow down in the container-inversion test. The viscosity of <NUM>% IEIK13 solutions was reduced significantly when passed through a membrane with a pore size of <NUM>. Results are shown in <FIG>.

<NUM>% RADA16 solutions were studied for viscosity reduction with a shear-thinning unit (shown in <FIG>). <NUM>% RADA16 solution, which shows shear thinning and thixotropic behavior, was passed through a shear-thinning unit at <NUM> kPa of injection pressure. The solution showed <NUM> ~ <NUM>/min of output. The solution could not be passed through a filter (<NUM> pore size) at <NUM> kPa of injection pressure (i.e., without prior exposure to a shear-thinning unit). However, water, which is a representative Newtonian fluid, showed that output flow rate was relatively consistent. The results are shown in Table <NUM>.

As demonstrated above, <NUM>% KLD12 and <NUM>% IEK13 solutions were not able to be filtered through a <NUM> Nalgene syringe filter with <NUM> cellulose acetate membranes. <NUM>% RADA16 is not usually amenable to filtration through <NUM> membrane. <NUM>% RADA16, <NUM>% IEIK13, and <NUM>% KLD12 solutions were able to be filtered after being exposed to a shear-thinning unit at <NUM> kPa of injection pressure showing <NUM>, <NUM>, and <NUM>/min of output, respectively. The solutions were not able to be filtered without the shear-thinning unit. A shear-thinning unit shown in Figure <NUM> may be successfully utilized for sterilization and filtration of viscous peptide solutions which are not easily filtered. The results are shown in Table <NUM>.

This Examples are demonstrates successful use of a screen with micro- and/or nano- holes as a shear-thinning unit. Viscous <NUM>% KLD12 or <NUM>% IEIK13 solutions may be transferred to a dispensing syringe (or chamber), injected to a first chamber that includes a shear-thinning unit with micro- and/or nano- holes, and then filtered through the membrane filter (pore size: <NUM>) in the second chamber. Instead of syringe for injection, a high pressure chamber may be used to deliver a peptide composition. Membrane size (e.g., diameter) and/or other characteristics (e.g., pore size, etc) may be selected to accommodate amount of peptide composition to be passed through it.

Claim 1:
A method for sterilizing a liquid peptide composition characterized in that the composition has an initial storage modulus within the range of <NUM> to <NUM>,<NUM> Pa at <NUM> rad/sec of frequency and <NUM> Pa of oscillation stress as measured with <NUM> plates, the method comprising the steps of:
subjecting the liquid peptide composition to high shear stress within a range of <NUM> to <NUM> Pa so that the storage modulus of the composition is temporarily reduced to a level within a range of <NUM>% to <NUM>% of the initial storage modulus; and
subjecting the composition to sterilizing filtration while its viscosity is at the reduced level, wherein the peptide composition is in aqueous solution and comprises a peptide at a concentration range of <NUM>% to <NUM>% having the amino acid sequence selected from: Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala (RADA16); Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile (IEIK13); and Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu (KLD12), wherein the step of subjecting the composition to high shear stress utilizes at least one shear-thinning unit selected from: a needle, a screen with micro- or nano-sized holes and a membrane with micro- or nano-sized pores.