This invention relates to high protein concentration formulations containing mannitol
Field of the Invention
The present mvention relates to methods for storing and preparing protein formulations containing mannitol
Background of the Invention
Mannitol has been generally used in protein formulations for maintaining stability and isotonicity of the formulation In the past, liquid nitrogen has been used to quickly freeze protein formulations for storage However, nearly all approaches to large-scale uncontrolled freezing of liquid formulations suffer from negative effects of uncontrolled solidification and meltmg Inadequate control of phase change has been shown to result in product losses due to aggregation, precipitation, oxidation and denaturation Recent technologies have been introduced to control the freeze and thaw process of protem formulations These technologies typically freeze and thaw at a much slower rate As a result, in mannitol-containing protein formulations, the slow freeze-thaw process allows crystallization of mannitol which, in turn, induces protem aggregation In order to avoid mannitol-induced protein aggregation during slow freeze-thaw processes, existmg methods require removmg mannitol from protem formulations and addmg it back during post-thaw operation
Summary of the Invention
The present mvention provides an improved method for storing and preparing protein formulations contaimng mannitol Specifically, the method of the present invention permits frozen storage of protein formulations containing mannitol without first removmg mannitol Therefore, the present mvention reduces costs and processmg steps and time for storing and preparing protein formulations contaimng mannitol
In one aspect, the present invention provides a method for storing a liquid formulation mcludmg gradually cooling the liquid formulation to a temperature lower than about -10 °C The liquid formulation contains mannitol and a protem, the protem being in a concentration greater than 50 mg/ml such that the greater concentration suppresses protein aggregation during cooling
In one embodiment, the method of the present invention includes gradually cooling the liquid formulation to a temperature lower than about -20 °C In another embodiment, the method of the present invention includes gradually cooling the liquid formulation to a temperature at approximately -40 °C or lower In yet another embodiment, the method of the present invention includes gradually cooling the liquid formulation to a temperature at approximately -50 °C or lower
In some embodiments, the present invention can be used for storing the liquid formulations containing mannitol in an amount ranging approximately 0-15% In particular, the liquid formulation may contain mannitol in an amount of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% Percentages are weight/weight when referring to solids and weight/volume when referring to liquids
In some embodiments, the method of the present invention includes gradually coolmg the liquid formulation at a rate of approximately 0 6, 0 5, 0 4, 0 3, 0 2, or 0 1 °C/minute
In some embodiments, the liquid formulation contains a protein in a concentration greater than about 75 mg/ml, 100 mg/ml, 125 mg/ml, 150 mg/ml, or 200 mg/ml Preferably, the liquid formulation contains a protein m a concentration between 50 mg/ml and 200 mg/ml
In some embodiments, the liquid formulation contains a protein that is an antibody In particular, the antibody is a monoclonal antibody In other embodiments the liquid formulation contams a protem that is a pharmaceutical drug substance
In some embodiments, the method for storing a liquid formulation of the present invention is a process intermediate
In another aspect, the present invention provides a method for preparing a liquid formulation mcluding gradually warmmg the liquid formulation from a frozen state to a temperature higher than about 0 °C The liquid formulation contams mannitol and a protein m a concentration greater than 50 mg/ml such that the greater concentration suppresses protein aggregation during warmmg
In some embodiments, the method for preparing a liquid formulation includes gradually warming the liquid formulation from a frozen state to a temperature at approximately 10 °C, 20 °C, 25 °C, 30 °C or higher
In some embodiments, the present invention can be used for preparing the liquid formulations containing mannitol in an amount ranging approximately 0-15% In particular, the liquid formulation contains mannitol m an amount of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% Percentages are weight/weight when referring to solids and weight/volume when referring to liquids
In some embodiments, the method for preparing a liquid formulation includes gradually warming the liquid formulation at a rate of approximately 0 6, 0 5, 0 4, 0 3, 0 2, or 0 1°C/mmute
In some embodiments, the liquid formulation contains a protein in a concentration greater than about 75 mg/ml, 100 mg/ml, 125 mg/ml, 150 mg/ml, or 200 mg/ml Preferably, the liquid formulation contams a protein in a concentration between 50 mg/ml and 200 mg/ml
In some embodiments, the liquid formulation contams a protem that is an antibody In particular, the antibody is a monoclonal antibody In other embodiments, the liquid formulation contains a protem that is a pharmaceutical drug substance
In some embodiments, the method for preparing a liquid formulation of the present invention is a process intermediate
The liquid formulation of the present invention is normally an aqeous formulation
The present invention further provides a composition containing a biologically effective amount of the protein in the liquid formulation prepared by the method of the invention as described m vanous embodiments above
In yet another aspect, the present mvention provides a method for inhibiting manmtol-mduced aggregation of a protein in a liquid formulation by mcreasmg the protein concentration to an amount greater than 50 mg/ml In some embodiments, the method of the present invention inhibits manmtol-mduced aggregation of a protem in a
liquid formulation by increasing the protein concentration to an amount greater than about 75 mg/ml, 100 mg/ml, 125 mg/ml, 150 mg/ml, or 200 mg/ml Typical protem concentration is between 50 mg/ml and 200 mg/ml
In this application, the use of "or" means "and/or" unless stated otherwise As used in this application, the term "comprise" and variations of the term, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers or steps As used in this application, the terms "about" and "approximately" are used as equivalents Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art
Other features, objects, and advantages of the present invention are apparent m the detailed descnption that follows It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation Vanous changes and modifications within the scope of the invention will become apparent to those skilled m the art from the detailed descnption
Brief Description of the Drawings
The drawmgs are for illustration purposes only, not for limitation
Figure 1 illustrates a sample product temperature trace at each exemplary process scale with a CryoPilot (CP) system
Figure 2 shows X-ray diffraction (XRD) patterns of frozen antibody solutions when cooled to -40 °C then wanned to 20 °C both at 0 5 °C/minute
Figure 3 shows a sample thermogram of modulated differential scanning calonmetry (mDSC) when cooling a monoclonal antibody at concentration of 30 mg/ml down to -42 °C
Figure 4 depicts total enthalpy plotted against protein concentration dunng a slow freeze and thaw process
Figure 5 depicts size-exclusive chromatography HPLC (SEC-HPLC) chromatograms of representative antibodies
Figure 6 depicts change m the percentage of high molecular weight (HMW) species plotted against the protem concentration
Figure 7 depicts that same freeze/thaw profile with same mixing speed resulted in 2 sets of traces based on production loads
Figure 8 depicts that the rates for faster freeze and thaw of the lab system were faster than the rates at the minimum production load
Figure 9 depicts that the rates for slow freeze and thaw of the lab system were slower than the rates at maximum production scale
Figure 10 depicts a typical supercoolmg phenomenon observed during lab scale cycle development for slow freeze and thaw
Figure 11 depicts that the revised profile was performed on 5 buffer tnals, followed by 5 MabM tnals with or without manmtol (15 total) and no supercoolmg was observed m any of the 10 thermocouple traces (0% occurrence)
Figure 12 depicts that the product temperature traces of Mab and MabM overlayed and no supercoolmg was observed for any of the 10 thermocouple traces up to 5 Mab runs with or without manmtol
Figure 13 illustrates that the percentage of HMW species mcreased more significantly after multiple freeze and thaw cycles at slow rates as compared to fast rates
Figure 14 illustrates that no increase m HMW species was observed with Mab formulation at concentration of 100mg/mL containing manmtol
Detailed Description of the Invention
The present mvention provides an improved method for storing and preparing protein formulations contaimng manmtol Specifically, the present mvention provides a method for suppressing or eliminating mannitol-induced protem aggregation m a liquid formulation during slow freeze and/or thaw process by mcreasing protem concentration
Various aspects of the invention are described in further detail m the following subsections The use of subsections is not meant to limit the mvention Each subsection may apply to any aspect of the mvention
Pi otein formulations containing manmtol
Proteins are relatively unstable in the aqueous state and undergo chemical and physical degradation resulting in a loss of biological activity during processing and storage Freeze-thaw and lyophihsation are well-established methods for preserving proteins for storage In order to preserve protein conformation, activity and stability, the protein formulations usually contain agents facilitating this, so-called lyoprotectants and cryoprotectants Cryoprotectants are agents which provide stability to the protein from freezmg-induced stresses, however, the term also mcludes agents that provide stability, eg, to bulk drug formulations during storage from non-freezing-induced stresses Lyoprotectants are agents that provide stability to the protein dunng water removal from the system dunng the drying process, presumably by maintaining the proper conformation of the protem through hydrogen bonding Cryoprotectants can also have lyoprotectant effects Examples of frequently used bulking agents include manmtol, glycine, sucrose, lactose, etc The agents also contribute to the tonicity of the formulations
As used herem, "proteins" include any recombmant or purified polypeptides including, but not limited to, antibodies, eg, monoclonal antibodies, single chain antibodies, and other antibody variants, various growth hormones, and any pharmaceutical drug substances Proteins referred to in this application mclude any naturally-occurring, modified or synthesized polypeptides
As used herein, "a protein formulation," "a liquid formulation," or grammatical equivalents mclude any liquid polypeptide-contammg compositions Typically, a liquid formulation of the invention is an aqueous formulation The liquid polypeptide-contammg compositions may further contam "buffering agent" including those agents which maintain the solution pH m an acceptable range and may mclude bulking agents described above and may also mclude histidine, phosphate, citrate, tns, diethanolamine, and the like If the liquid polypeptide-contammg compositions are pharmaceutical compositions, the liquid formulation may further contain "excipients" The term
"excipients" mcludes pharmaceutical acceptable earners as well as lyoprotectants and cryoprotectants that provide proper conformation of the protem during storage so that substantial retention of biological activity and protem stability is maintained
Manmtol induces protein aggregation during slow freeze and thaw
As discussed above, freeze and thaw is a well establish method for long-term storage or as an intermediate step However, nearly all approaches to large-scale freezing of liquid formulations suffer from negative effects of uncontrolled solidification and melting Approaches such as freezing in bags and bottles have been repeatedly shown to result in cryoconcentration and non-uniform temperature profiles within contamers Inadequate control of phase change has been shown to result in product losses due to aggregation, precipitation, oxidation and denaturation By contrast, controlled freeze and thaw (also referred to as slow freeze and thaw) avoids product denaturation typical of uncontrolled methods and eliminates expensive and time-consuming cleaning In addition, overall processes benefit from a well-controlled and predictable operation
Controlled freezing (or slow freezing) typically mcludes gradually cooling a liquid formulation to a temperature suitable for storage at a predetermined rate Typically, a temperature suitable for storage includes, but is not limited to, a temperature at or lower than about -10 °C, -20 °C, -30 °C, -40 °C, -50 °C The gradual step down cooling can be at a rate of approximately 0 6, 0 5, 0 4, 0 3, 0 2, or 0 1 °C/minute
Similarly, controlled thawing (slow thawing) typically mcludes gradually warming a liquid formulation from a frozen state to a desired temperature at a predetermmed rate Typically, a desired temperature for thawing purposes includes, but is not limited to, a temperature at or higher than about 0 °C, 10 °C, 20 °C, or 30 °C The gradual step warming can be at a rate of approximately 0 6, 0 5, 0 4, 0 3, 0 2, or 0 1 °C/minute
Controlled freeze and thaw may be performed in a container, such as a tube, a bag, a bottle, or any other suitable containers The contamers may be disposable Controlled freeze and thaw may also be performed in a large scale or small scale For typical large scale production, a liquid formulation may be frozen in batches of about 1 L
through 300 L, for example, 3L For typical small scale system, a liquid formulation may be frozen m batches of about 1 ml to 500 ml, for example, 30 ml
However, in mannitol-containmg liquid formulations, the slow freezmg and/or thawing allows crystallization of manmtol, which m turn, induces protein aggregation As used herein, "protem aggregation" is meant formation of high molecular weight (HMW) species mcludmg both insoluble species detectable by turbidity measurement and soluble species detectable by size-exclusion chromatography HPLC (SEC-HPLC), cation exchange-HPLC (CEX-HPLC), X-ray diffraction (XRD), modulated differential scanning calonmetry (mDSC) and other means known to one of skill in the art
It is observed that there is a substantial mcrease m the percentage of HMW species in mannitol-contaimng formulations upon multiple freeze and thaw cycles (see the Examples section) Increased amount of manmtol in the formulation also results m higher percentage of HMW species formation Reduced processing volume appears to maintain the percentage of HMW species formed compared to large scale (e g, 125L)
An exothermal event is observed during coolmg in mannitol-contaming formulations The observed enthalpy, which is due to the crystallization of mannital as well as to the unfrozen water, increases as the processing scale mcreases (freeze and thaw rates decreases), or the manmtol level in the formulation mcreases Crystallization event upon thawing in the mannitol-contammg formulation is also observed Without wishing to be bound by theory, the crystallization events in frozen solution suggest that the phase transition due to crystallization may mduce the aggregation of protein upon freeze and thaw Crystallization of manmtol mcreases with the manmtol level, which corresponds to higher %HMW formation There was more manmtol crystallization observed in larger process scale simulation than that of the smaller scale, again correlated to greater rate of HMW formation Decreasing manmtol in the formulation generally favors reducmg HMW species formation in the liquid formulation during freeze and thaw
Increased protein concentration suppresses protein aggregation
The present mvention discovered that mcreasing protem concentration in the liquid formulation suppresses or inhibits protem aggregation during slow freezing and/or thawing process As descnbed in the Examples section, it was found that increasing
protein concentration above 20-30 mg/ml resulted m a decrease in the amount of HMW species formation Without wishing to be bound by theory, it is contemplated that the increased protein aggregation at low protem concentration (eg, <20 mg/ml) may be caused by the increased probability of two molecules coming together during freezing and/or thaw Typically, a protein concentration greater than 50 mg/ml is used to suppress protein aggregation Preferably, a protem concentration greater than about 75 mg/ml, 100 mg/ml, 125 mg/ml, or 150 mg/ml is used to suppress protem aggregation More preferably, a protem concentration between 50 mg/ml to 200 mg/ml is used As used herem, the term "suppresses protein aggregation," or grammatical equivalents, denotes a reduction of the percentage of ITMW species in a liquid formulation as compared to the percentage of HMW species formed m a similar liquid formulation but contammg a protein concentration less than 20 mg/ml The term "suppresses protem aggregation" also includes inhibiting or eliminating formation of HMW species
Thus, by increasing the protem concentration in the liquid formulation contaimng mannitol, the present mvention allows slow freezing and/or thawing of the liquid formulation without mducmg significant protein aggregation The present invention is particularly useful for storing drug product contaimng drug substance For example, the present mvention allows all the excipients including mannitol in a drug product to be present during slow freezing and/or thawing process while keeping the drug substance stable and biologically active Therefore, the present invention eliminates the need for removing mannitol from a drug formulation before storage and adding it back during the drug product filling operation
Thus, the liquid formulations contaimng mannitol and a protein concentration higher than 50 mg/ml may be stored directly in that form for later use, stored in a frozen state as an intermediate step and thawed prior to use, or subsequently prepared m a dried form, such as a lyophilized, air-dned, or spray-dried form, for later reconstitution into a liquid form or other form prior to use In addition, compositions contammg biologically active amount of the protem can be prepared and stored directly m their liquid form m accordance with the present application to take full advantage of the convemence, ease of administration without reconstitution, and ability to supply the formulation m prefilled, ready-to-use synnges or as multidose preparations if the formulation is compatible with bactenostatic agents The present application also provides other forms of compositions
containing biologically active amount of the protein m the liquid formulation stored and prepared as described above
It should be understood that the above-described embodiments and the following examples are given by way of illustration, not limitation The liquid formulation of the present invention is applicable to proteins in general For example, the antibodies used in the liquid formulations described in the Examples section can be any antibodies Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the present description
Examples
Example 1 Slow Freeze and thaw of a monoclonal antibody formulation
A formulation containing a monoclonal antibody (Mab) at various concentrations and lOmM histidme, 10mM methionine, 0-4% mannitol and 0-0 02% polysorbate-80, was frozen and thawed multiple times using a CryoPilot (CP) system (Stedim Biosystems) Each freeze and thaw profile included step-down coolmg to -55°C, and warming to 32°C while the solution was mixed
The CP simulates operation of a CryoVessel (Stedim Biosystems), the full scale production umt The CP set point profiles for various process volumes had been developed prior to this work, to mimic behavior of the Cryo Vessel Figure 1 illustrates a sample of product temperature trace at each process scale with the CP system Freezing (or thawing) rate was defined as the thermocouple reaching -42 °C from 0 °C (or 0 °C from -42 °C) divided by the time
Thawed samples were analyzed primarily by SEC-HPLC and CEX-HPLC to evaluate the level of high molecular weight species (%HMW), and track the levels of acidic and basic species Modulated differential scanning calonmetry (mDSC) and X-Ray Diffraction (XRD) were also used to assess crystallinity and polymorphs of mannitol m frozen solutions
Example 2 Mannitol mduces protem aggregation during slow freeze and thaw
A humanized monoclonal antibody (referred to as MAB-001 m this experiment) was found to aggregate m the presence of mannitol during a slow freeze-thaw process similar to the one described in Example 1 Figure 2 shows XRD patterns of frozen
MAB-001 solutions when cooled to -40 °C then warmed to 20 °C both at 0 5 °C/minute The frozen solution was scanned at -42 °C, -30 °C and -10 °C As shown m Figure 2, the amount of crystallization mcreased with the amount of mannitol in the formulation and higher protein concentration suppressed mannitol crystallization
In addition to XRD, modulated Differential Scanning Calonmetry (mDSC) was also used to examme mannitol crystallization in MAB-001 samples Figure 3 shows a sample thermogram of mDSC when cooling MAB-001 at concentration of 30 mg/ml down to -42 °C The observed enthalpy (brown trace m Figure 3) is due to the crystallization of mannitol as shown in Figure 2 If one assumes total enthalpy equals cooling enthalpy plus warming enthalpy, then the total enthalpy can be plotted against protein concentration as shown m Figure 4 As also shown in Figure 4, increased protein concentration (e g, >30 mg/ml) suppressed the mannitol crystallization
Example 3 Higher protein concentration suppresses mannitol crystallization
Three antibodies referred to as MAB-001, MAB-002 and MAB-003 were dialyzed into 10 mM histidme, 250 mM mannitol, pH 6 0, then subject to five cycles of freeze-thaw, and monitored for HMW species formation The SEC-HPLC chromatograms are shown in Figure 5 As shown in Figure 5, for each of the three proteins MAB-001, MAB-002 and MAB-003, mcreased concentration of the protein m the formulation suppressed formation of HMW species Change in the percentage of HMW species was plotted against the protein concentration in Figure 6 As shown m Figure 6, for each of the three proteins, increasmg the protein concentration above 20-30 mg/ml decreased the amount of HMW formation detected by SEC-HPLC This result was consistent with the mDSC data
Example 4 Lab scale vs production scale
Fast and slow freeze/thaw cycles were developed in a lab system S3 Celsius (Stedrm Biosystems) to match product trace of production scale and to bracket the minimum and maximum production loads FT-100 Celsius (Stedim Biosystems) was used at different production loads For minimum production load, 4 2L (1 bag) were used For maximum production load, 100L (6 bags at 16 6L each) were used Same freeze/thaw set point profile with same mixing speed from FT-100 resulted in 2 sets of product temperature traces based on production loads as shown in Figure 7
Freeze and thaw cycle development was performed using the lab scale S system As shown in Figure 8, the rates for faster freeze and thaw of the lab system were faster than the rates at the minimum production load As shown in Figure 9, the rates for slow freeze and thaw of the lab system were slower than the rates at maximum production scale
Supercoolmg was observed during lab scale cycle development for slow freeze and thaw A typical supercooling phenomenon is shown in Figure 10 The freeze temperature is depressed when supercoolmg happens Super cooling phenomenon was observed m 2 out of 3 tnals of slow freeze/thaw cycles (67% occurrence) Supercoolmg might be a random occurrence with protein and buffer runs or may be caused by bag positions, which were unpredictable Supercooling affected normal freeze trme (NFT) (from 5 C to -5 C) calculation Supercooling may be related to protein concentration level
To avoid supercooling, a monoclonal antibody was used as a model protein (MabM) to develop the lab system slow freeze and thaw cycle MabM was concentrated and dialyzed to Mab formulation buffer solution, then diluted at the following concentrations 50 mg/ml, 100 mg/ml and 150 mg/ml The formulations without mannitol were prepared, followed by frozen and thawed 5 times using the lab system Solid mannitol was then added to the mannitol free formulations These solutions were again frozen and thawed 5 times with the lab system Slow freeze/thaw profile was revised by extending initial deep frozen time and lowering initial freezing temperature to facilitate nucleation As shown in Figure 11, the revised profile was performed on 5 buffer tnals, followed by 5 MabM tnals with or without mannitol (15 total) and no supercoolmg was observed in any of the 10 thermocouple traces (0% occurrence) Manual mix post thaw was required for all protem concentration levels
The fast and slow freeze and thaw profiles developed above were used to assess the feasibility of freeze and thaw of monoclonal antibody (Mab) drug substance The S3 lab scale Celsius was used in this experiment Assay assessments mcluded visual observation of cryoconcentration, SEC HPLC for HMW and low molecular weight (LMW) species, pH, turbidity by A400, concentration and CEX HPLC As shown in Figure 12, the product temperature traces of Mab and MabM overlayed No supercoolmg was observed for any of the 10 thermocouple traces up to 5 Mab runs with or without mannitol Manual mix post thaw was required for all concentration levels
Example 5 Formation of HMW relates to freeze and thaw rates
As shown m Figure 13, the percentage of HMW species mcreased more significantly after multiple freeze and thaw cycles at slow rates as compared to fast rates No changes in the amount of LMW species, pH, turbidity, concentration as well as acidic and basic species were observed As also shown m Figure 13, the increase of HMW species was only seen m a formulation with protein concentration of 50 mg/ml and contammg manmtol As shown m Figure 14, no mcrease m HMW species was observed with Mab formulation at concentration of 100mg/mL contammg manmtol In addition, it was observed that Mab without manmtol remamed stable for up to 5 freeze and thaw cycles in Stedin bags with concentrations of 50 and 150mg/mL
Incorporation by Reference
All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if the contents of each individual publication or patent document were incorporated herein.

WE CLAIM:
1. A method for storing a liquid formulation, the method comprising gradually cooling the liquid formulation to a temperature lower than -10 °C, wherein the liquid formulation comprises mannitol and a protein, the protein being in a concentration greater than 50 mg/ml such that the greater concentration suppresses protein aggregation during cooling.
2. The method of claim 1, wherein the temperature is at approximately -20 °C, -40 °C, or -50 °C.
3. The method of claim 1, wherein the mannitol is in an amount of approximately 0-15%.
4. The method of claim 1, wherein the cooling is at a rate of approximately 0.5
°C/minute,
0.3 °C/minute, or 0.1 °C/minute.
5. The method of claim 1, wherein the protein is in a concentration between 50 mg/ml and 200 mg/ml.
6. The method of claim 1, wherein the protein is in a concentration greater than 75 mg/ml, 100 mg/ml, 125 mg/ml, or 150 mg/ml.
7. The method of claim 1, wherein the protein is an antibody.
8. The method of claim 7, wherein the antibody is a monoclonal antibody.
9. The method of claim 1, wherein the protein is a pharmaceutical drug substance.
10. The method of claim 1, wherein the method is a process intermediate.
11. A method for preparing a liquid formulation, the method comprising gradually
warming the liquid formulation from a frozen state to a temperature higher than 0 °C,
wherein the liquid formulation comprises mannitol and a protein, the protein being in a
concentration greater than 50 mg/ml such that the greater concentration suppresses
protein aggregation during warming.
12. The method of claim 11, wherein the temperature is at approximately 20 °C, or 30
°C.
13. The method of claim 11, wherein the mannitol is in an amount of approximately 0-15%.
14. The method of claim 11, wherein the warming is at a rate of approximately 0.5 °C/minute, 0.3 °C/minute, or 0.1 °C/minute.
15. The method of claim 11, wherein the protein is in a concentration between 50 mg/ml and 200 mg/ml.
16. The method of claim 11, wherein the protein is in a concentration greater than 75 mg/ml, 100 mg/ml, 125 mg/ml, or 150 mg/ml.
17. The method of claim 11, wherein the protein is an antibody.
18. The method of claim 17, wherein the antibody is a monoclonal antibody.
19. The method of claim 11, wherein the protein is a pharmaceutical drug substance.
20. The method of claim 11, wherein the method is a process intermediate.

21. A composition comprising a biologically effective amount of the protein in the liquid formulation prepared by the method of claim 11.
22. A method for inhibiting mannitol-induced aggregation of a protein in a liquid formulation, the method comprising.increasing the protein concentration to an amount greater than 50 mg/ml.
23. The method of claim 22, wherein the protein is in a concentration greater than 75 mg/ml, 100 mg/ml, 125 mg/ml, or 150 mg/ml.
24. A method for storing and preparing protein formulations containing mannitol substantially such as herein described with reference to examples and drawings.