STERILIZATION AND FILTRATION OF PEPTIDE COMPOSITIONS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. §119(e) of U.S. provisional patent
application serial no. 61/950,536, filed March 10, 2014, which application is hereby incorporated
by reference in its entirety.
SEQUENCE LISTING
[0002] This application makes reference to a sequence listing submitted in electronic form as
an ASCII .txt file named "2004837-0044_Sequences.txt". The .txt file was generated on March
9, 2015 and is 1 kb in size.
BACKGROUND
[0003] 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,
16-amino acid polypeptide with a repeating sequence of arginine, alanine, and aspartic acid (i.e.,
RADARADARADARADA [SEQ ID NO:l], also known as "RADA16"), is commercially
available under the trade names PuraStat®, PuraMatrix®, and PuraMatrix GMP® from 3-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.
SUMMARY
[0004] The present invention provides, among other things, methods for handling peptide
compositions and technologies relating thereto. Teachings provided herein may be particularly
applicable to high-viscosity peptide compositions, and/or compositions of self-assembling
peptides.
[0005] 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).
[0006] 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.
[0007] Thus, the present disclosure provides a variety of technologies relevant to processing
of peptide compositions, and particularly to sterilization.
[0008] In some embodiments, 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).
[0009] The present disclosure provides, among other things, systems for sterilizing peptide
compositions, andor systems for determining appropriate such systems for application to
particular peptide compositions.
[0010] In some embodiments, 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).
[0011] Among other things, the present disclosure provides certain peptide compositions that
may be sterilized by autoclave treatment.
[0012] In some embodiments, the present disclosure provides 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 embodiments, viscosity of
peptide compositions to be filtered may be reduced prior to filtration. In some embodiments,
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 embodiments, such a reduction is temporary.
[0013] In some embodiments, provided technologies 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 2.5% in accordance.
[0014] In some particular embodiments, the present disclosure provides 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 embodiments, a
method does not involve sterilizing filtration.
[0015] In some embodiments, the present disclosure provides 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 embodiments, the heat
treatment performs at about 121 °C for about 25 min.
[0016] In some embodiments, the present disclosure provides a method for sterilizing a
liquid peptide composition having an initial storage modulus within the range of about 300 to
about 5,000 Pa at 1 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 0.01% to 80%> of the initial storage modulus, and
subjecting the composition to filtration while its viscosity is at the reduced level.
[0017] In some embodiments, the step of subjecting the composition to high shear stress
utilizes at least one shear-thinning unit.
[0018] In some embodiments, at least one shear-thinning unit is or comprises at least one
needle. In some embodiments, at least one needle is at least 10 mm long. In some embodiments,
at least one needle has a gauge within the range of about 25 to about 35.
[0019] In some embodiments, at least one shear-thinning unit is or comprises at least one
screen with micro- or nano-sized holes. In some embodiments, micro- or nano-sized holes have
a largest dimension within a range of about 0.5 mih to about 200 mih. In some embodiments, a
pinch between holes is about 5 mih to about 10 mm. In some embodiments, 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 embodiments, thickness of the screen
is about 10 mih to about 10 mm.
[0020] In some embodiments, at least one shear-thinning unit is or comprises at least one
membrane with micro- or nano-sized pores. In some embodiments, the pores gave a size with a
range of about 0.45 mih to about 120 mih.
[0021] In some embodiments, high shear stress for sterilization is with a range of about 30 to
about 200 Pa.
[0022] In some embodiments, a liquid peptide composition comprises RADA16, IEIK13, or
KLD12.
[0023] In some embodiments, a liquid peptide composition is pressurized prior to filtration.
In some embodiments, a peptide liquid composition is further stored the under vacuum.
BRIEF DESCRIPTION OF THE DRAWING
[0024] Figures 1A and IB show exemplary mass spectrometry analysis of RADA16 before
and after autoclave treatment, to assess heat sensitivity. Figure 1A depicts mass spectrometry
before autoclave treatment. Figure IB depicts mass spectrometry after autoclave treatment.
Figure 1C illustrates exemplary RADA16 molecular structure; in the particular peptide
composition that was analyzed, the protein was composed of RADARADARADARADA where
the N-terminus and C-terminus are protected by acetyl and amino groups.
[0025] Figures 2A and 2B show exemplary mass spectrometry analysis of IEIK13 before and
after autoclave treatment, to assess. Figure 2A depicts mass spectrometry before autoclave
treatment. Figure 2B depicts mass spectrometry after autoclave treatment. Figure 2C illustrates
exemplary IEIK13 molecular structure; in the particular peptide composition that was analyzed,
the protein was composed of IEIKIEIKIEIKI, where the N-terminus and C-terminus are
protected by acetyl and amino groups.
[0026] Figure 3A and 3B show exemplary mass spectrometry of KLD12 before and after
autoclave treatment, to assess heat sensitivity. Figure 3A depicts mass spectrometry before
autoclave treatment. Figure 3B depicts mass spectrometry after autoclave treatment. Figure 3C
illustrates exemplary KLD12 molecular structure; in the particular peptide composition that was
analyzed, the protein was composed of KLDLKLDKKLDL, where the N-terminus and Cterminus
are protected by acetyl and amino groups.
[0027] Figure 4 shows exemplary time sweep tests of RADA16 and IEIK13 before and after
autoclave treatment.
[0028] Figure 5 provides a picture of peptides and devices needed for filtering viscous
peptide solutions, for example, RADA16, KLD12, and IEIK13. Shear stress was applied
through 30-gauge needle to peptide solutions (middle), so that the peptide solutions were filtered
(right).
[0029] Figure 6 shows exemplary time sweep tests of 1% and 2.5% RADA16 performed at 1
Pa and 10 rad/s. RADA16 was injected through 30-gauge needle so that applied shear stress
reduced stiffness of the peptides. The measurements were started 1 minute after the injection.
[0030] Figure 7 shows exemplary time sweep tests 1% and 2.5% KLD12 performed at 1 Pa
and 10 rad/s. KLD12 was injected through 30-gauge needle so that the applied shear stress
reduced stiffness of the peptides. The measurements were started 1 minute after the injection.
[0031] Figure 8 shows exemplary time sweep tests 2.5% IEIK13 performed at 1 Pa and 10
rad/s. IEIK13 was injected through 30-gauge needle so that the applied shear stress reduced
stiffness of the peptides. The measurements were started 1 minute after the injection.
[0032] Figure 9 shows an exemplary flow viscosity test performed from 0.003 to 1000 1/sec
of shear rate on 2.5% RADA16 solution.
[0033] Figure 10 shows an exemplary flow viscosity test performed from 0.003 to 1000 1/sec
of shear rate on 1.5% IEIK13 solution.
[0034] Figure 11 shows exemplary viscosity measurements of 2.5% RADA16 as a function
of time to demonstrate viscosity recovery. At time = 0, shear stress was applied to the peptides,
so that the viscosity was reduced. The horizontal line indicates the original viscosity of 2.5%
RADA16.
[0035] Figure 12 shows exemplary viscosity measurements of 1.5% IEIK13 as a function of
time to demonstrate viscosity recovery. At time = 0, shear stress was applied to the peptides, so
that the viscosity was reduced. The horizontal line indicates the original viscosity of 1.5%
IEIK13.
[0036] Figure 13 shows exemplary devices for filtering viscous peptide solutions, for
example, RADA16, KLD12, and IEIK13. Shear stress was applied through a shear-thinning unit
with multiple pores. Peptide solution was dispensed with a syringe on the top (i). Peptide
solution passed through the first shear-thinning chamber (25 mm filter holder, Millipore (ii))
where a shear-thinning unit with pores or holes was inserted to reduce the viscosity of peptide
solution temporarily. Peptide solution then passed into second filtering chamber (25 mm filter
holder, Millipore (iii)) where a filtering membrane was inserted to sterilize peptide solutions or
remove particulates from peptide solutions. Filtered solution was received in a bottle (iv) for
output. A high pressure dispenser was connected to the dispensing syringe (v). High pressure
nitrogen gas was connected to the high pressure dispenser (vii).
[0037] Figure 14 shows visual observation of viscosity after applying shear stress to 2.5%
KLD12 solution and 1.5% IEIK13 solution. The top row includes pictures of 2.5% KLD. The
bottom row includes pictures of 1.5% IEIK13. The solutions in the most left column stay on the
top of the vials. The solutions in the most right column (after applying shear stress) have low
viscosity, so that most materials are located at the bottom of vials.
[0038] Figures 15A, 15B, 15C, and 15D show materials and devices (a micro- or nano-hole
screen) for filtering viscous peptide solutions such as RADA16, KLD12, and IEIK13. Figures
15A, 15B and 15C show features of an exemplary shear thinning unit, a micro-hole screen,
which may be used in the device shown in Figure 13. Holes were generated by laser-drilling
technology. Such a screen may be inserted in the first chamber to reduce viscosity of peptide
solutions before actual filtration through the membrane in the second chamber. Figure 15D
shows visual observation of viscosity after applying shear stress to 2.5% KLD12 using a microor
nano-hole screen
DEFINITIONS
[0039] 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 embodiments, an agent is or comprises a natural
product in that it is found in and/or is obtained from nature. In some embodiments, 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
embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent
may be utilized in crude form. In some embodiments, potential agents are provided as
collections or libraries, for example that may be screened to identify or characterize active agents
within them. Some particular embodiments of agents that may be utilized in accordance with the
present invention 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 embodiments, an agent is or comprises a polymer. In
some embodiments, an agent is not a polymer and/or is substantially free of any polymer. In
some embodiments, an agent contains at least one polymeric moiety. In some embodiments, an
agent lacks or is substantially free of any polymeric moiety.
[0040] 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 embodiments, an amino acid has the general structure H2NC(
H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In
some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino
acid is a D-amino acid; in some embodiments, 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 embodiments, 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 embodiments, an amino acid may be modified by methylation,
amidation, acetylation, and/or substitution as compared with the general structure. In some
embodiments, 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 embodiments, 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
embodiments, the term "amino acid" is used to refer to a free amino acid; in some embodiments
it is used to refer to an amino acid residue of a polypeptide.
[0041] 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
embodiments, the term "approximately" or "about" refers to a range of values that fall within
25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%,
3% , 2%>, 1% , 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 100% of a possible value).
[0042] 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 embodiments, 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 embodiments, two or more entities that
are physically associated with one another are covalently linked to one another; in some
embodiments, 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.
[0043] 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 embodiments, 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.)
[0044] 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
embodiments, determining involves manipulation of a physical sample. In some embodiments,
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
embodiments, determining involves receiving relevant information and/or materials from a
source. In some embodiments, determining involves comparing one or more features of a
sample or entity to a comparable reference.
[0045] The term "gel" as used herein refers to viscoelastic materials whose rheological
properties distinguish them from solutions, solids, etc. In some embodiments, a composition is
considered to be a gel if its storage modulus (G') is larger than its modulus (G"). In some
embodiments, a composition is considered to be a gel if there are chemical or physical crosslinked
networks in solution, which is distinguished from entangled molecules in viscous solution.
[0046] 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.
[0047] 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).
[0048] The term "peptide" as used herein refers to a polypeptide that is typically relatively
short, for example having a length of less than about 100 amino acids, less than about 50 amino
acids, less than 20 amino acids, or less than 10 amino acids.
[0049] The term "polypeptide" as used herein refers to any polymeric chain of amino acids.
In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some
embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some
embodiments, 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 embodiments, a polypeptide may
comprise or consist of natural amino acids, non-natural amino acids, or both. In some
embodiments, a polypeptide may comprise or consist of only natural amino acids or only nonnatural
amino acids. In some embodiments, a polypeptide may comprise D-amino acids, Lamino
acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids.
In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments,
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 embodiments, such pendant
groups or modifications may be selected from the group consisting of acetylation, amidation,
lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments,
a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a
polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a
polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled
polypeptide. In some embodiments, 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 provides 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 embodiments,
such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In
some embodiments, 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 embodiments at a comparable level or
within a designated range) with a reference polypeptide of the class; in some embodiments with
all polypeptides within the class). For example, in some embodiments, a member polypeptide
shows an overall degree of sequence homology or identity with a reference polypeptide that is at
least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a
conserved region that may in some embodiments be or comprise a characteristic sequence
element) that shows very high sequence identity, often greater than 90%> or even 95%, 96%,
97%, 98%o, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20
or more amino acids; in some embodiments, a conserved region encompasses at least one stretch
of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some
embodiments, a useful polypeptide may comprise or consist of a fragment of a parent
polypeptide. In some embodiments, 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.
[0050] The term "reference" as used herein describes a standard or control relative to which
a comparison is performed. For example, in some embodiments, 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 embodiments, a reference or
control is tested and/or determined substantially simultaneously with the testing or determination
of interest. In some embodiments, 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.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0051] The present invention provides technologies for sterilization of peptide compositions.
In some embodiments, disclosed methods are particularly applicable to peptide solutions with
high viscosity and/or stiffness. In some embodiments, the present disclosure defines particular
peptide solutions that may be sterilized by autoclave treatment. In some embodiments, 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 embodiments, the
present disclosure provides technologies that may temporarily reduce peptide solution viscosity
and/or stiffness sufficiently to permit filtration. In some embodiments, 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.
Peptides and Peptide Compositions
[0052] In accordance with one or more embodiments, peptide compositions to which
teachings of the present disclosure may be compositions of amphiphilic peptides having about 6
to about 200 amino acid residues. In certain embodiments, a relevant peptide may have a length
of at least about 7 amino acids. In certain embodiments, a peptide may have a length of between
about 7 to about 17 amino acids. In certain embodiments, a peptide may have a length of at least
8 amino acids, at least about 12 amino acids, or at least about 16 amino acids.
[0053] In some embodiments, as is understood in the art, an amphiphilic polypeptide is one
whose sequence includes both hydrophilic amino acids and hydrophobic amino acids. In some
embodiments, 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 embodiments, such a peptide has an amino acid sequence
that is or comprises repeats of Arg-Ala-Asp-Ala (RADA); in some embodiments, such a peptide
has an amino acid sequence that is or comprises repeats of Lys-Leu-Asp (KLD); in some
embodiments, such a peptide has an amino acid sequence that is or comprises repeats of Ile-Glu-
Ile-Lys (IEIK).
[0054] In some embodiments, 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.
[0055] In some embodiments, 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 [RADAJ4; SEQ ID NO:l). In some embodiments, 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 [KLDLJ3, aka KLD12; SEQ ID NO:2). 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:3).
[0056] In some embodiments, peptide compositions to which the present disclosure may be
relevant are those characterized by certain rheological properties. In some embodiments,
relevant rheological properties may be or include loss modulus, stiffness, rheological recovery
time, storage modulus, viscosity, yield stress, etc. In some embodiments, rheological properties
are assessed via measurement; in some embodiments, one or more rheological properties may be
assessed via visual observation.
[0057] In certain embodiments, storage modulus and stiffness have a positive correlation; in
general, those of ordinary skill appreciate that higher storage modulus is related to higher
stiffness.
[0058] In some embodiments, a high viscosity peptide composition is characterized by a
storage modulus within the range of about 300 to about 5,000 Pa at 1 rad/sec of frequency and 1
Pa of oscillation stress.
[0059] In some embodiments, a peptide composition for use in accordance with the present
invention has a peptide concentration within the range of about 0.01% to about 10%.
[0060] In some embodiments, a peptide composition to which one or more of the
methodologies described herein is applied is of a commercial-scale volume.
[0061] In some embodiments, 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 embodiments, a peptide composition has been stored in a pressure vessel.
[0062] In some embodiments, 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.
Improving Properties
[0063] 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.
[0064] The present disclosure further appreciates that filtration challenges can complicate or
preclude sterilization of such peptide compositions. The present disclosure provides
technologies that permit filtration of certain peptide compositions and/or otherwise permit
sterilization.
Autoclave treatment
[0065] Autoclave treatment is a conventional sterilization method that involves subjecting
materials to high pressure saturated steam at 121 °C. It is generally understood in the art that
application of high heat, such as is involved in autoclave treatment, can degrade peptides.
[0066] 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 embodiments, such compositions may be sterilized by heat
treatment at about 121°C for about 25 minutes.
[0067] In some embodiments, peptide compositions that may be subjected to heat treatment,
and/or to autoclave treatment are IEIK13 compositions. In some such embodiments, IEIK13
compositions have a concentration within the range of about 0.01% to about 10%
[0068] In some embodiments, peptide compositions that may be subjected to heat treatment
and/or to autoclave treatment are KLD12 compositions. In some embodiments, however,
KLD12 compositions are not subjected to autoclave treatment in accordance with the present
invention.
[0069] In some embodiments, RADA16 compositions are not subjected to autoclave
treatment in accordacce with the present invention.
[0070] 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.
[0071] In some embodiments, peptide compositions that can appropriately be subjected to
heat treatment such as autoclave treatment in accordance with the present invention 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 2).
Rheological Property Alteration
[0072] 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).
[0073] In some particular embodiments, rheological property alteration is achieved by
exposure to shear stress.
[0074] 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 selfassembled
structures. The present disclosure further proposes that recovery time may represent
that required for such structures to re-form.
[0075] In some embodiments, shear stress applied to peptide solutions may be at least about
20 Pa. In some embodiments, shear stress applied to peptide solutions may be at least about 30
Pa. In some embodiments, shear stress applied to peptide solutions may be at least about 40 Pa.
In some embodiments, shear stress applied to peptide solutions may be at least about 50 Pa. In
some embodiments, shear stress applied to peptide solutions may be at least about 60 Pa. In
some embodiments, shear stress applied to peptide solutions may be at least about 60 Pa. In
some embodiments, shear stress applied to peptide solutions may be at least about 80 Pa. In
some embodiments, shear stress applied to peptide solutions may be at least about 90 Pa. In
some embodiments, shear stress applied to peptide solutions may be at least about 100 Pa. In
some embodiments, the amount of shear stress may be at least about 30-100 Pa, for example, in
view of the yield stress of RADA16 2.5%, IEIK13 1.5% and 2.5% and KLD12 2.5% noted
above.
[0076] In some embodiments, viscosity of peptide solutions may drop significantly with
shear stress. In some embodiments, viscosity of peptides solutions may drop at least 10%> with
shear stress. In some embodiments, viscosity of peptides solutions may drop at least 30%> with
shear stress. In some embodiments, viscosity of peptides solutions may drop at least 50%> with
shear stress. In some embodiments, viscosity of peptides solutions may drop at least 70%> with
shear stress. In some embodiments, viscosity of peptides solutions may drop at least 90%> with
shear stress.
[0077] In some embodiments, the rheological property alteration is temporary. In some
embodiments, the peptide composition is characterized by rheological recovery characteristics.
For example, in some embodiments, such compositions are characterized in that one or more of
their rheological properties are restored within a time period within a range of about 1 min to
about 48 hours.
[0078] In some embodiments, rheological restoration is considered to be achieved when one
or more rheological properties returns to a level at least 20 % of its initial value.
[0079] In some embodiments, 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
30% reversed.
[0080] In some embodiments, peptide compositions may recover their storage modulus after
application of shear stress. In some embodiments, peptide solutions may recover about 0.1 to
100% of their original storage modulus in 1 min. In some embodiments, peptide solutions may
recover about 0.1 to 10% of their original storage modulus in 1 min. In some embodiments,
peptide solutions may recover about 20 to 100% of their original storage modulus in 20 min. In
some embodiments, peptide solutions may recover about 20 to 60%> of their original storage
modulus in 20 min.
[0081] In some embodiments, peptide solutions may recover their viscosity over time after
filtration. In some embodiments, peptide solutions may recover about 0.1 to 30% of their
original viscosity in 1 min. In some embodiments, peptide solutions may recover about 0.1 to
100% of their original viscosity in 1 min. In some embodiments, peptide solutions may recover
about 20 to 100% of their original viscosity in 20 min. In some embodiments, peptide solutions
may recover about 20 to 60% of their original viscosity in 20 min.
[0082] 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 4). The results
presented in this Example show a logarithmic increase of storage modulus from 1 minute after
injection, as shown in Figure 10 for RADA16, Figure 11 for KLD12, and Figure 12 for IEIK13.
[0083] Among other things, the present disclosure provides 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
embodiments 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).
[0084] 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 embodiments, a shear-thinning unit is or comprises a needle, a membrane, and/or a
screen. In some embodiments, a plurality of individual shear-thinning units is utilized, for
example so that high-throughput filtration can be achieved.
[0085] In some embodiments, the present invention provides devices and methodologies that
can achieve filtration of peptide compositions on a commercial scale.
Needle as a shear-thinning unit
[0086] In some non-limiting embodiments, shear stress may be applied by injection through
one or more needles. Thus, in some embodiments, one or more needles may be used as a shearthinning
unit.
[0087] In some embodiments, a needle may be at least about 1mm long. In some
embodiments, a needle may be at least about 2 mm long. In some embodiments, a needle may
be at least about 5 mm long. In some embodiments, a needle may be at least about a 10 mm
long. In some embodiments, a needle may be at least about 15 mm long. In some embodiments,
a needle may be at least about 20 mm long. In some embodiments, a needle may be at least
about 30 mm long. In some embodiments, a needle may be at least about 40 mm long. In some
embodiments, a needle may be at least about 50 mm long.
[0088] In some embodiments, a needle may have a gauge within a range of about 20 to about
34. In some embodiments, a needle may have a gauge within a range of about 25 to about 34. In
some embodiments, a needle may have a gauge of about 27 to about 34.
[0089] Figure 5 discloses one non-limiting embodiment of a sterilization device in
accordance with one or more non-limiting embodiments. 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.
Membrane as a shear-thinning unit
[0090] In some embodiments, 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.
Figure 13 depicts one non-limiting embodiment of a sterilization device in accordance with one
or more non-limiting embodiments of the present invention. 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.
[0091] In some embodiments, pore size of a shear-thinning unit may be about 0.45 mih to 120
mih. In some embodiments, pore size of a shear-thinning unit may be about 1 mih to 100 mih. In
some embodiments, pore size of a shear-thinning unit may be about 3 mih to 80 mih. In some
embodiments, pore size of a shear-thinning unit may be about 4 mih to 50 mih.
Screen as a shear-thinning unit
[0092] In some embodiments, a shear-thinning unit may have micro- or nano-holes. In some
embodiments, holes may be patterned or drilled on a plate whose thickness may be about 10 mih
to 10 mm in some embodiments. Figure 15 depicts one non-limiting embodiment of a
sterilization device in accordance with one or more non-limiting embodiments of the present
disclosure. A shear-thinning unit may be inserted into the first filtering chamber shown Figure
13.
[0093] In some embodiments, holes in an embodiment of a shear-thinnung unit described
herein may have a largest dimension within the range of about may be about 0.5 mih to 200 mih.
In some embodiments, such dimension may be within the range of about 0.5 mih to 100 mih. In
some embodiments, such dimension may be within the range of about 0.5 mih to 80 mih. In some
embodiments, In some embodiments, such dimension may be within the range of about 0.5 mih
to 50 mih.
[0094] In some embodiments, a shear-thinning unit of this embodiment may have a pitch
between holes within the range of about 5 mih to about 10 mm.
[0095] In some embodiments, 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.
Applications
[0096] In some embodiments, 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).
[0097] 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 embodiments, 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).
[0098] 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).
EXEMPLIFICATION
Example 1: Filtration of high viscous peptide solutions
[0099] 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 1. 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
example2% KLD12, 2.5% KLD12, and 1.5% IEIK13 solutions have 2, 3.4, and 3.2 times higher
maximum viscosities than 2.5% RADA16, respectively.
Table 1 Rheological properties of peptide solutions at selected concentrations
*: at 1 Pa of oscillation stress
# : Maximum viscosity data was adapted in viscosity plots at the range of measured stress.
[00100] Each of the peptide solutions listed in Table 1was subjected to filtration through a 0.2
mih Nalgene syringe filter with 25 mm cellulose acetate membranes. The 1% and 1.5% 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 2% and 2.5% KLD12
solutions and 1.5% 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.
Example 2 : Autoclave treatment of peptide solutions
[00101] 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 121°C for 25 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.
[00102] Figures 1-3 present results of autoclave treatment for certain compositions of
RADA16, IEKI13, and KLD12, respectively.
[00103] The measured molar mass of RADA16, prior to autoclave treatment, was 1712, 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.
[00104] The measured molar mass of IEIK13, prior to autoclave treatment, was 1622, 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.
[00105] The measured molar mass of KLD12, prior to autoclave treatment, is 1467, 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.
[00106] Rheological properties of certain peptide compositions were determined before and
after autoclaving. The data are shown in Figure 4. 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.
[00107] Autoclave treatment may be used for sterilization of IEIK13 compositions as
described herein, but should be avoided for RADA16 compositions.
Example 3 : Rheological properties of peptide compositions with application of shear stress
[00108] 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.
Shearflow test
[00109] Shear flow tests were performed on peptide solutions using a rheometer (DHR-1, TA
Instruments) with 20 mm plates. Results are shown in Figure 9 for 2.5% RADA16 solutions and
Figure 10 for 1.5% IEIK13 solutions. As can be seen, both 2.5% RADA16 and IEIK13 1.5%
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 40 Pa for 2.5% RADA16 solution and about 60 Pa for 1.5% IEIK13 solution.
Viscosity recovery
[00110] The viscosity recovery times of RADA16 and IEIK13 solutions were evaluated after
application of high shear stress. Using a DHR-1 rheomether (TA Instruments), viscosity changes
of 2 .5% RADA16 and 1.5% IEIK13 solutions were measured with flow tests at 0.005 1/sec of
shear rate after applying 1000 1/sec of shear rate to samples for 1 min. 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 re-assembly of peptide molecules
into structures (e.g., nano-fibers) in the solutions. Complete reassembling times of 2.5%
RADA16 and 1.5% IEIK13 solution were about 12 to 48 hours. The results are shown in Figure
11 for 2.5% RADA16 solution and Figure 12 for 1.5% IEIK13 solution.
Storage modulus recovery
[00111] The percentages of recovery back to the original storage modulus at 1 min and 20 min
after injection peptide compositios through a 30 gauge needle are listed in Table 2. The recovery
rate of IEIK13 (specifically, of a 2.5% IEIK13 solution) was the fastest among the peptide
solutions, showing 100% recovery to the original storage modulus in 20 min. KLD12 was the
slowest among those tested to recover; it showed only 23% recovery to the original storage
modulus in 20 min (for 2.5%). In some non-limiting embodiments, it may take about 12 to 48
hours for full recovery to an original modulus after passage through a needle (e.g., injection).
Table 2 Recovery to the original storage modulus at 1 min and 20 min after injection
throu h 30- au e needle
[00112] Rheological measurements were performed for RADA16 and IEIK13 solutions after
injecting them through 30 gauge needles. The results showed a logarithmic increase of storage
modulus from 1minute after injection. The results are shown in Figure 6 for RADA16, Figure 7
for KLD12, and Figure 8 for IEIK13.
Example 4 :A needle as a shear-thinning unit
[00113] 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).
[00114] Figure 5 depicts one non-limiting embodiment 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 30 gauge needle (0.3 mm x 25 mm, Endo irrigation needle with double side vent,
Transcodent, Germany) (middle) and a second syringe with a membrane filter (right). A
viscouse 2.5% 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,
2.5% KLD12 solutions were successfully filtered.
Example 5: High throughput shear-thinning unit
[00115] 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 embodiments, multiple needles or
equivalents may be used as a shear-thinning unit.
Membrane filter
[00116] This Examples demonstrates use of a with membrane filter (pore size > 0.45 mih) as a
shear-thinning unit. Viscous 2.5% KLD12 or 1.5% 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 0.45 mih to 120 m h), and then filtered through a filtering
membrane (for example, pore size: 0.2 mih) in the second chamber.
[00117] To examine the effect of pore size in the shear-thinning unit on viscosity change of
viscous peptide solutions, 2.5% KLD12 and 1.5% IEIK solutions were passed through selected
pore sizes, and their apparent viscosity changes were evaluated. 2.5% KLD solutions passed
through the shear-thinning unit with the pore sizes of 4 1 mih, 20 mih, and 5 mih. Viscosity of the
solutions was decreased enough to flow down when a vessel containing it was flipped over.
Though 2.5% KLD solutions that had passed through the shear-thinning unit with the pore size
of 120 mhi were slightly less viscous than pre-passage 2.5% KLD compositions, they remained
too viscous to flow down in the container-inversion test. The viscosity of 1.5% IEIK13 solutions
was reduced significantly when passed through a membrane with a pore size of 5 mih. Results
are shown in Figure 14.
[00118] 1.0% RADA16 solutions were studied for viscosity reduction with a shear-thinning
unit (shown in Figure 13). 1.0% RADA16 solution, which shows shear thinning and thixotropic
behavior, was passed through a shear-thinning unit at 50 psi of injection pressure. The solution
showed 1.4 ~ 1.7 mL/min of output. The solution could not be passed through a filter (0.2 mih
pore size) at 50 psi 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 3.
Table 3 Filtering abilities of the system shown in Figure 13 for RADA16 1% solution and
water.
*: Diameters of shear-thinning units and filter are 25 mm.
[00119] As demonstrated above, 2.5% KLD12 and 1.5% IEK13 solutions were not able to be
filtered through a 0.2 mih Nalgene syringe filter with 25 mm cellulose acetate membranes. 2.5%
RADA16 is not usually amenable to filtration through 0.2 mih membrane. 2.5% RADA16, 1.5%
IEIK13, and 2.5% KLD12 solutions were able to be filtered after being exposed to a shearthinning
unit at 100 psi of injection pressure showing 3.8, 12.5, and 11.4 mL/min of output,
respectively. The solutions were not able to be filtered without the shear-thinning unit. A shearthinning
unit shown in Figure 16 may be successfully utilized for sterilization and filtration of
viscous peptide solutions which are not easily filtered. The results are shown in Table 4.
Table 4 Filtering abilities filtering system shown in Figure 13 for 2.5% RADA16, 1.5%
IEIK13, 2.5% KLD12 solutions.
*: Diameters of membranes are 25 mm.
Screen
[00120] This Examples are demonstrates successful use of a screen with micro- and/or nanoholes
as a shear-thinning unit. Viscous 2.5% KLD12 or 1.5% IEIK13 solutions may be
transferred to a dispensing syringe (or chamber), injected to a first chamber that includes a shearthinning
unit with micro- and/or nano- holes, and then filtered through the membrane filter (pore
size: 0.2 mih) 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.
Table 5 Filtering abilities of the micro-hole screen system shown in Figure 13 and Figure
15 for 2.5% RADA16, 1.5% IEIK13, 2.5% KLD12 solutions.
Output through filter (0.2 m Output through shear-thinning unit
pore size) at 100 psi.* (screen with micro holes*) + filter
(0.2 mih pore size) at 100 psi.*
RADA16 2.5% 0 mL/min 4.2 mL/min
IEIK13 1.5% 0 mL/min 5.0 mL/min
KLD12 2.5% 0 mL/min 11.5 ml/min
*: Diameters of membranes are 25 mm.
# : hole size is 50 mih in diameter, pitch of holes is 450 mih, and depth of holes is 500 mih.

CLAIMS
What is claimed is:
1. 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.
2. The method of claim 1, wherein the method does not involve sterilizing filtration.
3. 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
4. The method of claim 3, the heat treatment performs at about 121 °C for about 25 min.
5. A method for sterilizing a liquid peptide composition having an initial storage modulus
within the range of about 300 to about 5,000 Pa at 1 rad/sec of frequency and 1 Pa of
oscillation stress,
the method comprising steps of:
subjecting the liquid peptide composition to high shear stress so that storage
modulus of the composition is temporarily reduced to a level within a range of
about 0.01% to 80% of the initial storage modulus; and
subjecting the composition to filtration while its viscosity is at the reduced level.
6. The method of claim 5, wherein the step of subjecting the composition to high shear
stress utilizes at least one shear-thinning unit.
7. The method of claim 6, wherein the at least one shear-thinning unit is or comprises at
least one needle.
8. The method of claim 7, wherein the at least one needle is at least 1mm long.
9. The method of claim 7, wherein the at least one needle has a gauge within the range of
about 25 to about 35.
10. The method of claim 6, wherein the at least one shear-thinning unit is or comprises at
least one screen with micro- or nano-sized holes.
11. The method of claim 10, wherein the micro- or nano-sized holes have a largest dimension
within a range of about 0.5 mih to about 200 mih.
12. The method of claim 10, wherein a pinch between holes is about 5 mih to about 10 mm.
13. The method of claim 10, wherein the 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.
14. The method of claim 10, wherein thickness of the screen is about 10 mih to about 10 mm.
15. The method of claim 6, wherein the at least one shear-thinning unit is or comprises at
least one membrane with micro- or nano-sized pores.
16. The method of claim 15, wherein the pores gave a size with a range of about 0.45 mih to
about 120 mih.
17. The method of claim 5, wherein the high shear stress is with a range of about 30 to about
200 Pa.
18. The method of claim 5, wherein the liquid peptide composition comprises RADA16,
IEIK13, or KLD12.
19. The method of claim 5, wherein the liquid peptide composition is pressurized prior to
filtration.
The method of claim 5, further comprising storing the liquid peptide composition under
vacuum.