WO2018031954A1

WO2018031954A1 - Identifying components of dry powder mixtures using raman spectroscopy

Info

Publication number: WO2018031954A1
Application number: PCT/US2017/046636
Authority: WO
Inventors: Catherine LYNES; Jason E. DICKENS
Original assignee: Biogen Ma Inc.
Priority date: 2016-08-12
Filing date: 2017-08-11
Publication date: 2018-02-15

Abstract

Provided herein, in some embodiments, are methods for evaluating a dry powder mixture (e.g., dry-powder cell culture medium) using Raman spectroscopy. In other embodiments, the methods include Raman spectroscopy and/or morphological analysis of a plurality of particles of the dry-powder cell culture medium. In some embodiments, the methods provided herein are useful for identifying media suitable for use in a cell culture process.

Description

IDENTIFYING COMPONENTS OF DRY POWDER MIXTURES USING RAMAN

SPECTROSCOPY

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/374,733, filed August 12, 2016, which is hereby incorporated by reference in its entirety.

FIELD

The disclosure relates to the field of spectroscopic analysis of complex dry powder mixtures.

BACKGROUND

Input raw materials quality remains one of the most common sources of variation in biotherapeutic cell culture processes. Raw materials are designed to provide cell nutrients required for growth as well as to fulfill key functions, and include organic and inorganic single components, polymers, and dry powder cell-culture media, such as chemically defined media (CDM) formulations. Composition inconsistencies in raw material can lead to various biotherapeutic production issues and impact drug substance critical quality attributes.

Deficient components within CDM, such as amino acids, are a considerable risk because they impact both cell growth rates and drug substance critical quality attributes. For example, glutamine deficiencies affect cell apoptosis and lower growth rates. Missing vitamins can frequently cause apoptosis and elevation of oxidation processes. Cell culture processes lacking key vitamins, such as cyanocobalamin, result in lower levels of cell proliferation and demonstrate degraded protein production. Without organic sugars such as glucose, fructose, and sucrose, energy-dependent processes may be incomplete. Optimal cell culture-based protein production depends, in large part to the presence of a number of individual cell culture medium components.

Historic characterization of cell culture input raw materials has been based upon pharmacopeial modalities. In certain cases these methods have been inadequate to assess critical material attributes for certain raw materials that impact cell culture processes. With the rapid growth in biopharmaceutical cell culture-based production worldwide, the development of more sensitive and robust analytical methods remains a focused effort within the industry to understand and mitigate raw material variation. Such characterization most often involves analytical techniques such as chromatography, spectroscopy, microscopy, and physical property modalities. Multicomponent raw materials, such as CDM, are assessed by complex traditional analytical techniques including LC-MS, amino acid analysis (AAA), and ICP-MS. AAA demonstrated success in quantifying amino acids, whereas LC-MS affords analysis of amino acids, vitamins, organic components, and organic impurities. There are several clear disadvantages of using these techniques. Both methods require a skilled operator to acquire and interpret data and laborious sample preparation. Moreover, both methods require solvents, thereby impacting the carbon footprint. Such modalities also only assess chemical characteristics of CDM. An alternative technique demonstrated the use of Raman spectroscopy for single particle detection of complex formulations (Li et al., Anal Chem. 2015; 87: 3419-28). However, while Raman spectroscopy offers certain advantages of rapid, noninvasive analysis for some raw materials, Raman spectroscopy has been shown to be an insufficient technique for the identification of complex chemically defined dry powder cell-culture media. Kent K.P., et al., "Tool for Dry Powder Media Identification," Genetic Engineering & Biotechnology News, Nov 15, 2015, Vol. 35, No. 20. Based on the pivotal role of CDMs used in cell culture media, the development of methods for the compositional analysis and physical properties of dry powder cell-culture media is of interest.

SUMMARY

In one aspect, the disclosure provides methods for using Raman spectroscopy to evaluate dry powder mixtures, for example, a dry powder cell-culture medium. In some embodiments, the methods include dispersing a dry powder mixture (e.g., a dry powder cell- culture medium) onto a substrate and analyzing a plurality of individual particles of the dry powder mixture using Raman spectroscopy to confirm the presence or absence of one or more components of the dry powder mixture. The methods provided herein may be useful for confirming the presence of one or more components of a dry powder cell-culture medium prior to using it for a cell culture process.

In another aspect, methods include image analysis of individual particles of a dry powder mixture (e.g., a dry powder cell-culture medium), which may facilitate the identification of one or more individual components of the dry powder mixture, and/or be used to predict performance, for example, dissolution time of the dry powder mixture.

In some embodiments, provided herein are analytical methods developed to assess chemically defined media at the particulate level by Raman spectroscopy. In some embodiments, methods described herein can be conducted by Raman microscopy. For example, in some aspects, the application provides methods that involve obtaining a Raman spectrum from a discrete region of a sample of particles. In some embodiments, Raman microscopy allows individual particles in a dry powder mixture to be located and analyzed via Raman spectroscopy. In some embodiments, methods described herein relate to evaluating chemically defined media at the particulate level by Raman spectroscopy and static imaging, such as Morphologically Directed Raman Spectroscopy. In some

embodiments, qualitative physical -chemical characterization provides insight into raw material lot variations and supplier differences which affect cell growth and media dissolution. This green analytical chemistry method requires negligible sample preparation and eliminates the need for organic solvents. In one example, autonomous acquisition and data analysis of spectra and morphological data afforded identification of approximately 30 components within one hour based on detection of 150 particles. For a 72 hour scan, morphological information was obtained for 9,000 particles and greater than 40 components were identified with a detection limit of -0.02% (wt/wt). This technique enables detection of both major and minor components contained within chemically defined media mixtures that are used in mammalian cell culture biopharmaceutical processes. In some embodiments, methods are applicable to complex multicomponent materials and can augment traditional chromatographic techniques.

Aspects of the disclosure relate to the use of Raman spectroscopy for evaluating dry powder cell-culture medium. Some aspects of the disclosure provide methods for evaluating a dry powder cell-culture medium, by dispersing the dry powder cell-culture medium on a substrate, and obtaining a Raman spectrum from each of a plurality of particles of the dry powder cell-culture medium. In some embodiments, the dry powder cell-culture medium is comprised of up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, or up to 70 individual components. In some embodiments, the dry powder cell-culture medium is comprised of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 individual components.

In some embodiments, at least one of the individual components of the dry powder cell-culture medium is an amino acid. In some embodiments, the amino acid may be one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, at least 2, at least 5, at least 10, at least 15, or at least 20 of the individual components are amino acids. In some embodiments, at least one of the individual components of the dry powder cell-culture medium is a vitamin. In some embodiments, the vitamin may be one or more of Biotin, Calcium Pentothenate, Choline Chloride, Cyanocobalamin, DL Alpha Lipoic Acid, Inositol, Thiamine HC1, PABA, Riboflavin, Folic Acid, and Niacinamide. In some embodiments, at least 2, at least 5, at least 10, at least 15, or at least 20 of the individual components are vitamins.

In some embodiments, at least one of the individual components of the dry powder cell-culture medium is a nutrient. In some embodiments, the nutrient may be one or more of glucose, fructose, and sucrose. In some embodiments, at least 2, at least 3, at least 4, or at least 5 of the individual components are nutrients.

In some embodiments, at least one of the individual components of the dry powder cell-culture medium is a buffer. In some embodiments, the buffer is HEPES. In other embodiments, the buffer comprises sodium bicarbonate. In some embodiments, at least 2, at least 3, at least 4, or at least 5 of the individual components are buffers.

In some embodiments, the dry powder media is Iscove's Modified Dulbecco's Media (FMDM), Dulbecco's Modified Eagle's medium (DMEM), DMEM/F12, Ham's F-10, Ham's F-12, Medium 199, Minimum Essential Medium (MEM), or Roswell Park Memorial Institute (RPMI) 1640.

In some embodiments, the dry powder cell-culture medium is dispersed on a substrate that is compatible with Raman spectroscopy. In some embodiments, the substrate comprises glass or plastic. In some embodiments, at least one of the individual components of the dry powder cell-culture medium is Raman active (e.g., detectable by Raman spectroscopy). In some embodiments, at least 2, at least 5, at least 10, at least 20, or at least 30 of the individual components of the dry powder cell-culture medium are Raman active. In some embodiments, the dry powder cell-culture medium is dispersed on a substrate at a density 80 particles per mm² or less, 60 particles per mm² or less, or 50 per mm² or less. In some embodiments, a Raman spectrum is obtained from at least 10, at least 100, at least 500, at least 1,000, at least 5,000, or at least 10,000 individual particles of the plurality of particles of the dry powder cell-culture medium. In some embodiments, at least one dimension of each of the plurality of particles is at least 1 μπι, at least 2 μπι, at least 3 μπι, at least 4 μπι, at least 5 μπι, at least 6 μπι, at least 8 μπι, at least 10 μπι, or at least 20 μπι. In some embodiments, each of the dispersed particles being analyzed is separated from other particles by a distance sufficient to allow discrete Raman spectra to be obtained for the particles being analyzed. In some embodiments, an average distance between dispersed particles is around 0.5 μπι, around 1 μηι, around 2 μηι, around 3 μηι, around 4 μηι, around 5 μηι, around 6 μηι, around 8 μηι, around 10 μηι, around 20 μηι, or more. In some embodiments, each of the plurality of particles consists essentially of one component (e.g., each individual particle consists of one individual component of the dry mixture).

In some embodiments, methods provided herein further comprise identifying at least 1 physical property of a plurality of particles of the dry powder cell-culture medium. In some embodiments, the methods comprise identifying at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 physical properties of the plurality of particles of the dry powder cell-culture media. In some embodiments, the physical property is area (e.g., surface area of a particle). In some embodiments, the physical property is circularity. In some embodiments, the physical property is volume. In some embodiments, the physical property is convexity. In some embodiments, the physical property is solidity. In some embodiments, the physical property is selected from circular equivalent (CE) diameter, high sensitivity (HS) circularity, aspect ratio, elongation, length, width, fiber elongation, fiber straightness, fiber width, perimeter, and sphere equivalent volume, or a combination of two or more thereof (e.g., in addition to, or instead of, one or more of area, circularity, volume, convexity, and/or solidity).

In some aspects, methods further comprise determining a dissolution time of the dry powder cell-culture medium. In some embodiments, methods comprise contacting the dry powder cell-culture medium with a solvent, thereby forming a liquid cell culture medium. In some embodiments, the solvent comprises water. In some embodiments, the methods comprise mixing the liquid cell culture medium until the dry powder cell-culture medium has dissolved into the solvent. In some embodiments, the liquid cell culture medium is mixed for at least 8, at least 9, at least 10, at least 11, at least 12, or at least 15 minutes.

In some aspects, methods comprise comparing the Raman spectrum obtained from each of a plurality of particles of the dry powder cell-culture medium to an appropriate standard and determining if an individual component is present in the dry powder cell-culture medium. In some embodiments, the appropriate standard is a user generated library comprising a Raman spectrum of at least one individual component that is present (e.g., expected to be present) in the dry powder cell-culture medium. In some embodiments, the appropriate standard (e.g., the user generated library comprising the Raman spectrum) can be a spectrum or spectra of one or more components expected or intended to be present in the dry powder cell culture medium. In some embodiments, the user generated library comprises a Raman spectrum of at least 2, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70 individual components of the dry powder cell-culture medium. In some embodiments, the user generated library comprises a Raman spectrum of up to 10, up to 20, up to 30, up to 40, up to 50, up to 100, up to 150, up to 200, or more individual components of the dry powder cell-culture medium. In some embodiments, at least 2, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70 Raman spectrum are obtained from each of the individual components of the dry powder cell-culture medium and where each of the individual components of the dry powder cell-culture medium are determined to be present. In some embodiments, up to 10, up to 20, up to 30, up to 40, up to 50, up to 100, up to 150, up to 200, or more Raman spectrum are obtained from each of the individual components of the dry powder cell-culture medium and where each of the individual components of the dry powder cell-culture medium are determined to be present. In some embodiments, methods further comprise using the dry powder cell-culture medium in a cell culture process if each of the individual components (e.g., components expected to be present in the dry powder cell-culture medium) of the dry powder cell-culture medium are present and not using the dry powder cell-culture medium in a cell culture process if each of the individual components of the dry powder cell-culture medium are not present.

In some aspects, the application provides methods of conducting cell culture using a dry powder cell-culture medium. In some embodiments, the methods comprise obtaining a Raman spectrum from each of a plurality of particles of the dry power cell-culture medium. In some embodiments, the methods further comprise comparing the Raman spectrum to an appropriate standard and determining if an individual component is present in the dry powder cell-culture medium. In some embodiments, the methods further comprise using the dry powder cell-culture medium in a cell culture process if the individual component is present in the dry powder cell-culture medium. In some embodiments, the dry powder cell-culture medium is comprised of up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, or up to 70 individual components. In some embodiments, the dry powder cell-culture medium is comprised of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 individual components. In some embodiments, the dry powder cell-culture medium is used in the cell culture process if it is determined that at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%), or 100% of the individual components of the dry powder cell-culture medium are present.

In some embodiments, the methods further comprise determining an amount of each individual component in the dry powder cell-culture medium. In some embodiments, the dry powder cell-culture medium is used in a cell culture process if it is determined that all of the individual components are present in the ratios expected (e.g., approximate or exact ratios expected) based on the medium being analyzed. In some embodiments, the cell culture process is conducted under conditions that permit production of a protein of interest. In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the therapeutic protein is an antibody. In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the cell culture process is conducted in a bioreactor. In some embodiments, the volume of the bioreactor cell culture is at least 0.5 L, at least 1 L, at least 10 L, at least 100 L, at least 250 L, at least 500 L, at least 500 L, at least 1,000 L, at least 2,000 L, at least 3,000 L, at least 4,000 L, at least 5,000 L, at least 7,500 L, at least 10,000 L, at least 12,500 L, at least 15,000 L, at least 20,000 L, at least 100,000 L, or more. In some embodiments, the volume of the bioreactor cell culture is in a range of 0.5 L to 10 L, 0.5 L to 100 L, 0.5 L to 500 L, 500 L to 1,000 L, 500 L to 2,500 L, 500 L to 5,000 L, 500 L to 10,000 L, 500 L to 15,000 L, 500 L to 20,000 L, 1,000 L to 4,000 L, 500 L to 100,000 L, 2,000 L to 5,000 L, 2,000 L to 10,000 L, 2,000 L to 15,000 L, 2,000 L to 20,000 L, 2,000 L to 100,000 L, 15,000 L to 20,000 L, 15,000 L to 100,000 L, 20,000 L to 50,000 L, 20,000 L to 100,000 L, or 50,000 L to 100,000 L. In some embodiments, the bioreactor cell culture produces (e.g., is capable of producing) at least 1 gram, at least 10 grams, at least 100 grams, 500 grams, 1,000 grams, 2,000 grams, 3,000 grams, or more of a recombinant protein (e.g., a protein of interest). In some embodiments, a bioreactor culture produces or is capable of producing 1 gram to 10 grams, 1 gram to 100 grams, 1 gram to 500 grams, 10 gram to 1,000 grams, 10 grams to 2,000 grams, 100 grams to 1,000 grams, 500 grams to 5,000 grams, or more of a recombinant protein (e.g., a protein of interest).

These and other aspects are described in more detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGs. 1 A-1C show a summary of CDM multi-attribute analysis by Malvern G3-ID technology (Malvern, UK). FIG. 1 A shows collected particle images, FIG. IB shows particle morphological attributes, and FIG. 1C shows particle identification by Raman spectroscopy. FIGs. 2A-2B show CDM scan time component detection optimization (FIG. 2A) and optimization experimental consistency (FIG. 2B). Consistency among certain compounds (boxed) is more prevalent than others.

FIGs. 3 A-3E show that the size of individual components, even within the same chemical class, varies. Examples of individual components tested are shown for asparagine (FIG. 3 A), proline (FIG. 3B), tryptophan (FIG. 3C), serine (FIG. 3D), and inositol (FIG. 3E).

FIGs. 4A-4B show CDM morphology differences (FIG. 4A) and dissolution properties (FIG. 4B) among suppliers.

FIGs. 5 A-5H show schematic representations of particle shapes, demonstrating the physical properties that may be measured. The schematics demonstrate measurements of convex hull (FIG. 5 A), convexity (FIG. 5B), length (FIG. 5C), major axis (FIG. 5D), SE volume (FIG. 5E), width (FIG. 5F), fiber elongation (FIG. 5G), and fiber width (FIG. 5H).

FIGs. 6A-6B show exemplary particle morphologies. FIG. 6A shows particle morphologies for histidine (3 particles), lysine-HCl (3 particles), and valine (3 particles). FIG. 6B shows particle morphologies for inositol (4 particles), biotin (3 particles), and sodium chloride (5 particles). The numbers under the particles represent CE diameter values.

FIG. 7 shows exemplary particle size distributions for phenylalanine, vitamin B 12, Serine, methionine, thiamine-HCl, Cysteine-H₂0-HCl, L-tryptophan, L-aspartic acid, and biotin. The x axis refers to the CE diameter (μπι) and the y axis refers to the frequency of particles in the sample. The morphology of more than 25,000 particles were analyzed.

DETAILED DESCRIPTION

Aspects of this disclosure are based on the surprising discovery that Raman spectroscopy is useful for confirming the presence or absence of individual components (e.g., amino acids and vitamins) of complex chemical mixtures such as chemically defined medium (CDM) used for cell culture.

In some embodiments, two different classes of CDMs can be used in fed-batch culture: basal media and feed media, and their compositions are optimized in accordance with specific cell line requirements and product quality attributes. Typically, CDMs are comprised of 5-75 components including amino acids, vitamins, inorganic salts, trace metals, and other constituents. Risks of CDM include known and unknown degradation pathways, impurities due to improper production, and deficiencies or missing components. CDMs are produced by various raw material suppliers where manufacturing control vary significantly. That is, certain manufacturers have embedded robust automated controls and electronic batch records within their processes that track the CDM component addition, whereas others rely upon manual procedures. The inherent higher risk in the latter procedure has resulted in erroneous supply of component-deficient CDM materials to biopharmaceutical

manufacturers, thereby resulting in failed cell culture production either in production efficiency or drug substance inconsistencies.

Among other aspects, the present application provides methods of evaluating a dry powder mixture (e.g., a dry powder cell-culture medium) using Raman spectroscopy to confirm the presence or absence of one or more components of the dry powder mixture. In some embodiments, methods provided herein may be useful for confirming the presence of one or more components of a dry powder cell-culture medium prior to using it for a cell culture process. For example, in some embodiments, methods are provided that involve obtaining a Raman spectrum of a dry powder mixture (e.g., obtaining discrete Raman spectra for each of a plurality of particles of a dry powder mixture) and determining, based on the Raman spectrum, the presence or absence of an individual component in the mixture. In some embodiments, the methods may further involve determining the presence or absence of the individual component in the dry powder mixture based on a comparison of the Raman spectrum to an appropriate standard (e.g., a second Raman spectrum that corresponds to the individual component, for example, from a standard from a user generated library).

In some embodiments, techniques described herein can be used to detect one or more impurities in a dry powder mixture (e.g., a dry powder cell-culture medium) using Raman spectroscopy (e.g., Raman microscopy). For example, in some embodiments, methods of the present application may be useful for detecting one or more production impurities (e.g., one or more components, whether known or unknown) present in a dry powder mixture, such as a CDM. In some embodiments, a dry powder mixture can be identified as having one or more impurities by determining that a Raman spectrum of the powder mixture (e.g., a Raman spectrum of each of one or more particles in the powder mixture) does not correspond to a Raman spectrum of the user generated library (e.g., a spectrum corresponding to that of the powder mixture is absent from the user generated library). In some embodiments, a dry powder mixture can be identified as having one or more impurities by determining that a Raman spectrum of the powder mixture corresponds to a Raman spectrum of the user generated library. For example, in some embodiments, a user generated library may include one or more Raman spectra corresponding to one or more known impurities. In some embodiments, a known impurity can be any substance that is not a desired component of the powder mixture (e.g., not a desired component of a chemically defined medium). In some embodiments, a dry powder mixture can be analyzed based on the number of unique Raman spectra obtained from the mixture. For example, in some embodiments, a defined dry powder mixture is expected to have a known amount of unique components (e.g., unique dry components). In some embodiments, where the number of unique Raman spectra obtained from the mixture is as expected, the dry powder mixture is determined to be suitable for use (e.g., suitable for use in a cell culture process). In some embodiments, where the number of unique Raman spectra obtained from the mixture is not as expected (e.g., there are one or more missing components and/or one or more additional components not expected to be present), the dry powder mixture can be determined to be unsuitable for use or potentially require additional evaluation prior to use. Accordingly, in some embodiments dry powder mixtures determined to be unsuitable for use can be subjected to additional analysis.

In some embodiments, a dry powder mixture can be analyzed based not only on the number of unique Raman spectra obtained from the mixture, but also on the relative number of particles representative of each unique spectrum. The relative number of particles having each unique spectrum can be used to evaluate (e.g., determine) the relative amount of each component in the mixture (e.g., if each spectrum can be correlated to a component of the mixture, for example by comparing the spectra to a library of reference spectra for each of the components).

In some embodiments, a particle size distribution can be determined for particles of each of one or more components within a dry powder mixture. In some embodiments, a particle size distribution can be used as an indicator of whether a dry powder mixture is suitable for use. In some embodiments, a component may be expected to be primarily present in the form of fine particles (e.g., 1 to 10 μπι in diameter). In some embodiments, a component may be expected to be primarily present in the form of coarse particles (e.g., around 100 μπι or more in diameter). In some embodiments, if a component is not present in the expected form (e.g., if it is present in a bimodal distribution in the form of both fine and coarse particles as opposed to being present in a unimodal distribution in the form of either fine or coarse particles, depending on the component), then a dry powder mixture can be identified as having a potential defect and may not be suitable for use. Accordingly, in some embodiments, even if a component is present in a correct amount (e.g., in an expected or desired percentage weight of the dry powder mixture), a dry powder mixture may not be suitable for use if the component is not present in an expected or desired size distribution.

As described herein, in some aspects, the application provides techniques related to the analysis of a dry powder mixture. In some embodiments, the dry powder mixture comprises a dry powder cell culture medium, such as a chemically defined medium. In some embodiments, the dry powder mixture comprises a pharmaceutical solid oral dosage form, such as a powder, tablet, or capsule. In some embodiments, the dry powder mixture comprises a heterogeneous excipient mixture (e.g., as used in some pharmaceutical solid oral dosage forms). In some embodiments, the dry powder mixture comprises a heterogeneous (e.g., polydispersed) material used in biopharmaceutical and/or pharmaceutical processes. In some embodiments, the dry material comprises an ancillary complex material. Examples of ancillary materials include, without limitation, resins, chromatographic stationary phases, and other analytical multicomponent reagents known in the art. In yet other embodiments, the dry powder mixture comprises non-formulated foreign particles (e.g., as found in seals, gaskets, and packaging used throughout the manufacture of a drug or substance). In some embodiments, the dry powder mixture comprises a liquid medium solution residue on a filter. In some embodiments, examples and description related to a dry powder cell culture medium also may be applied to other complex dry powder mixtures as described herein.

In some embodiments, the present disclosure presents an alternative approach to CDM characterizing utilizing technology that integrates Raman spectroscopy and static imaging, such as the Malvern G3-ID technology (Malvern, UK). However, it should be appreciated that additional and/or alternative devices and systems may be used in accordance with the disclosure. For instance, while certain embodiments are illustrated with reference to the G3-ID technology, similar or identical methods can be performed using other devices (e.g., other Raman spectrometers optionally paired with an imaging technique).

Implementing the G3ID as a CDM characterization tool was designed to: mitigate supply of component deficient CDM materials, augment laborious multiple chromatographic methods currently in use, such as amino acid analysis and LC-MS methods, and provide additional analytical attribute information where possible, such as material morphology. To this end, the emergence of multi -attribute analytical technologies on the market was leveraged to address these objectives; namely, the G3-ID technology from Malvern which uses

Morphologically Directed Raman Spectroscopy (MDRS). This technique detects individual particles, analyzes the morphology profile of each, and then collects a Raman spectra for each particle. While chromatography methods provide information about the identity of components within a sample as a bulk measurement, the G3ID provides detailed morphology and chemical identification characteristics on an individual particle basis. Although the chemical identification is essential for CDM characterization, the morphological properties can provide additional insight into the finer details of materials. This technology is advantageous in that it is automated, simple to use, less laborious than comparable chromatographic systems, requires no sample preparation, is non-solvent based and can analyze most common compounds within CDM.

Although not used widely within the biopharmaceutical industry to date, the G3-ID has demonstrated its powerful capabilities in other industries with predominant uses for compositional, impurity, and forensic analyses. It has been demonstrated to successfully detect impurities in drug products within the pharmaceutical industry, such as shedding from tubing, debris from bioreactors, and unfinished drug product. The Raman component also allows for detection of polymorphs within pharmaceutical formulations. Within the cement industry, compositional analysis allowed for characterization of six major components within formulations. In the pharmaceutical industry, a compositional analysis of particle size allows for characterization in device applications such as nasal sprays which indicate the presence of agglomerates. The G3-ID is a powerful tool for forensic investigations; studies have used this technology for identification of soil, gunshot residues, hoax powder attacks and illicit drug and diluent agent investigations. The morphology features of the instrument has demonstrated use in particle size characterizations in device applications such as nasal spray and identification of agglomerates.

This disclosure describes the development of a G3-ID characterization method capable of compositional qualitative analysis of over 40 components within CDMs present in concentrations as low as 0.02% (wt/wt). Such exhaustive compositional qualitative analysis has not been achieved nor reported with morphological directed based Raman spectroscopy technologies. Development of a suitable Raman library, optimization of analysis acquisition parameters, and method repeatability are discussed. Information gleaned from morphological studies was applied to dissolution studies. Moreover, both basal and feed CDM that vary in number of amino acids, vitamins, and miscellaneous inorganic compounds were assessed.

Dry powder cell-culture medium

Some aspects of the disclosure provide methods for evaluating a dry powder cell- culture medium. As used herein, the term "dry powder cell-culture medium" refers to a powder comprised of individual dry powder components, which may be dissolved in a solvent to produce a nutritive solution that supports the cultivation and/or growth of cells. Typically, a dry powder cell culture medium contains one or more amino acids, vitamins, buffers, inorganic salts, and nutrients {e.g., glucose). However, dry powder cell-culture medium may include additional components or additives, such as growth factors, hormones or attachment factors.

In some embodiments, the dry powder cell-culture medium is a chemically defined medium (CDM). As used herein, the term "chemically defined medium" or "CDM" refers to a cell-culture medium, compatible with the in vitro cell culture of one or more cells, in which each of the chemical components, and/or proportions thereof, of the cell culture medium are known. Accordingly, chemically defined medium does not contain animal derived products such as serum {e.g., FBS), serum derived proteins {e.g., albumin), hydrolysates, growth factors, hormones, carrier proteins or attachment factors. In some embodiments, the chemically defined medium comprises one or more of the following: an amino acid, a vitamin, an inorganic salt, a buffer, an antioxidant, or an energy source. In some

embodiments, the chemically defined medium may be supplemented with one or more components, such as a recombinant protein (e.g., recombinant albumin, recombinant transferrin and recombinant insulin), a chemically defined lipid, or an antioxidant thiol (e.g., 1-mercaptoethanol and 1-thioglycerol). In some embodiments, the chemically defined medium is in powdered form, which may be reconstituted, for example in water, for use in a cell culture process.

In some embodiments, a dry powder cell culture medium may be comprised of any suitable number of components for supporting a cell culture. In some embodiments, the dry powder cell-culture medium comprises at least 5, at least 10, at least 15, at least 20, at least

25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 75, or at least 80 individual components. In some embodiments, the dry powder cell-culture medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 individual components.

In some embodiments, as described herein, the application provides methods of analyzing a plurality of individual particles of a dry powder mixture (e.g., a dry powder cell- culture medium). In some embodiments, each of the plurality of particles consists of, or consists essentially of, one component. For example, in some embodiments, each individual particle consists of one individual component of the dry mixture. In some embodiments, one or more of these components (e.g., each component) are present in the dry powder cell- culture media as substantially homogenous particles. The term "cell culture" or "cell culture process" refers to the maintenance of cells in an artificial, e.g., an in vitro, environment. It is to be understood, however, that the term "cell culture" is a generic term and may be used to encompass the cultivation not only of individual eukaryotic {e.g., animal, plant and fungal) or prokaryotic {e.g., bacterial) cells, but also of tissues, organs, organ systems or whole organisms.

In some embodiments, a dry powder mixture (e.g., a dry powder cell-culture medium) may contain a mixture of components, such as amino acids, nutrients {e.g., glucose), salts, buffers and vitamins, as well as other additives. Dry powder cell-culture media may be formulated by mixing dry powder components in any desired proportion, for example, to grow a certain cell type {e.g., a CHO cell) in a cell culture. In addition, dry powder cell- culture media is available from commercial suppliers, such as Invitrogen or Sigma. The requirements for these components vary among cell lines, and these differences are partly responsible for the extensive number of medium formulations. Exemplary components that may be used in a dry powder cell-culture medium formulation are provided below. However, it should be appreciated that additional dry powder components (e.g., dry powder cell-culture components) would be apparent to the skilled artisan and are within the scope of this disclosure.

In some embodiments, a dry powder cell culture medium comprises one or more amino acids. Amino acids are the building blocks of proteins, and thus are typical ingredients in cell culture media. In some embodiments, the dry powder cell culture medium comprises one or more essential amino acids. An essential amino acid refers to an amino acid that cannot be derived from other compounds by an organism {e.g., a human). For example, there are nine proteinogenic amino acids in humans. In some embodiments, essential amino acids include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. L-glutamine is an essential amino acid of particular importance. L- glutamine provides nitrogen for NAD, NADPH, and nucleotides, and serves as a secondary energy source for metabolism. L-glutamine is an unstable amino acid, that, with time, converts to a form that cannot be used by cells. L-glutamine concentrations for mammalian cell culture media can vary, for example, from 0.68 mM in Medium 199 to 4 mM in

Dulbecco's Modified Eagles' s Medium. Invertebrate cell culture media can contain as much as 12.3 mM L-glutamine. Nonessential amino acids may also be added to formulations of a dry powder cell-culture medium. Supplementation of media with non-essential amino acids may stimulate growth and prolong the viability of cells in a cell culture. Exemplary amino acids that may be comprised in a dry powder cell culture medium include, without limitation, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Typically, the L-form of amino acids are found in proteins during translation in the ribosome. Accordingly, in some embodiments, the dry powder cell-culture medium comprises one or more L-isomer amino acids. In other embodiments, the dry powder cell-culture medium comprises one or more D- isomer amino acids. In some embodiments, the amino acid may be an amino acid derivative. An amino acid derivative, as used herein, refers to a molecule that is generated using an amino acid as a starting point. For example, epinephrine is derived from tyrosine in a synthesis reaction that involves enzymatic modifications and several intermediate molecules. Exemplary amino acid derivatives that may be used in a dry powder cell-culture medium have been described previously in PCT/NL201 1/050592, published on July 19, 2012 as WO2012030217 A3; the entire contents are incorporated herein by reference. However, additional amino acid derivatives would be apparent to the skilled artisan and are within the scope of this disclosure.

Dry powder amino acids and their derivatives may be provided in different forms. For example, in some embodiments, the amino acid, or derivative thereof is provided as a free base. A free base refers to the conjugate base (deprotonated) from an amine, as opposed to its conjugate acid (protonated) form. As one example, the dry powder cell-culture medium comprises a glycine free base. However, the dry powder cell-culture medium may comprise additional free base amino acid forms. In some embodiments, the amino acid, or derivative thereof is provided as a hydrochloride (HC1) form. For example, in some embodiments, the dry powder cell-culture medium comprises arginine-HCl, cystine-2HCl, histidine-HCl-H₂0, or lycine HC1. However, the dry powder cell-culture medium may comprise additional hydrochloride amino acid forms. In some embodiments, the dry powder amino acid, or derivative thereof is provided in a hydrated form, for example a monohydrated or dihydrated form. In some embodiments, the dry powder cell-culture medium comprises asparagine H₂0. However, the dry powder cell-culture medium may comprise additional hydrated amino acid forms. In some embodiments, the dry powder amino acid, or derivative thereof is provided as a salt form, for example as a sodium salt form. In some embodiments, the dry powder cell- culture medium comprises a tyrosine disodium salt (e.g., tyrosine 2Na-2H₂0). However, the dry powder cell culture medium may comprise additional amino acid salt forms. It should be appreciated that the methods provided herein may be used to distinguish between different amino acid forms, such as free base, hydrochloride, and/or hydrated forms of amino acids in a dry powder cell-culture medium, for example by using Raman spectroscopy.

In some embodiments, one or more the amino acids are glycated. It should be appreciated that the methods provided herein may be used to distinguish whether an amino acid is glycated. For example, the methods provided herein may be used to distinguish whether an amino acid is covalently bound to one or more sugar molecules. Glycation refers to the covalent attachment of a sugar molecule (e.g., fructose or glucose). Typically, glycation involves the covalent bonding of a sugar molecule to an amino acid without the controlling action of an enzyme. In some embodiments, the amino acid is glycated with one or more sugar molecules. In some embodiments, the sugar molecule is fructose. In other embodiments, the sugar molecule is glucose. However, it should be appreciated that any of the amino acids provided herein may be glycated with any sugar molecule.

The dry powder cell culture medium may be comprised of any suitable number of amino acids, or derivatives thereof, for supporting a cell culture. In some embodiments, the dry powder cell-culture medium comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40, individual amino acids, or derivatives thereof. In some embodiments, the dry powder cell-culture medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 individual amino acids, or derivatives thereof.

In some embodiments, the dry powder cell-culture medium comprises one or more vitamins. As used herein, the term vitamin refers to an organic compound that is required by a cell or organism in limited amounts for growth and/or survival. Many vitamins are essential for growth and proliferation of cells. Vitamins cannot be synthesized in sufficient quantities by cells and are therefore important supplements required in tissue culture. It should be appreciated that the vitamins present in a dry powder cell-culture medium may be based on the needs of a particular cell line to grow and/or survive in cell culture. Typically, B group vitamins are included in cell-culture media for growth stimulation. Exemplary vitamins that may be included in a dry powder cell-culture medium include, without limitation, biotin (e.g., D-biotin), choline chloride, myo-Inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxal-HCl, riboflavin, thiamine-HCl, calcium pentothenate, cyanocobalamin (vitamin Bi₂), DL alpha lipoic acid, inositol, thiamine-HCl, PABA, riboflavin (vitamin B₂), folic acid (vitamin B₉), and Niacinamide (vitamin B₃). However, additional vitamins may be comprised in a dry powder cell-culture medium and would be apparent to the skilled artisan, for example based on the needs of a particular cell grown in culture. Additionally, it should be appreciated that these and other examples of components provided herein are not intended to be limited by any salts and/or counter-ions listed. For example, in some embodiments, the components may be present in free base form, salt form, and/or as a hydrate.

The dry powder cell culture medium may be comprised of any suitable number of vitamins, for supporting a cell culture. In some embodiments, the dry powder cell-culture medium comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40, individual vitamins. In some embodiments, the dry powder cell-culture medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 vitamins.

In some embodiments, the dry powder cell-culture medium comprises one or more buffers. Regulating pH is critical for optimum culture conditions and is generally achieved by using a natural or chemical buffering system. In a natural buffering system, gaseous C0₂ can balance with the CO₃/HCO₃ content of the culture medium. Cultures with a natural buffering system need to be maintained in an air atmosphere with 5-10% C0₂, usually maintained by a C0₂ incubator. In some embodiments, the dry powder cell-culture medium comprises a chemical buffer. Exemplary chemical buffers that may be comprised in a dry powder cell-culture medium include, without limitation, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, and CABS. However, additional dry powder buffers would be apparent to the skilled artisan and are within the scope of this disclosure. In some embodiments, the dry powder cell-culture medium comprises HEPES. In some embodiments, the dry powder cell-culture medium comprises sodium bicarbonate.

The dry powder cell culture medium may be comprised of any suitable number of buffers, for supporting a cell culture. In some embodiments, the dry powder cell-culture medium comprises at least 1, at least 2, at least 3, at least 4, at least 5, individual buffers. In some embodiments, the dry powder cell-culture medium comprises 1, 2, 3, 4, or 5 individual buffers.

In some embodiments, the dry powder cell-culture media comprises a pH indicator, which allows constant monitoring of pH in a cell culture. During cell growth, the medium changes color as pH is changed due to the metabolites released by the cells. In some embodiments, the pH indicator is phenol red, however, additional dry powder pH indicators may be used. At low pH levels, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple. Medium is bright red for pH 7.4, the optimum pH value for cell culture.

In some embodiments, the dry powder cell-culture medium comprises one or more salts, (e.g., inorganic salts). Inorganic salt in the media may help to retain the osmotic balance and help in regulating membrane potential by providing sodium, potassium, and calcium ions. In some embodiments, the dry powder cell-culture medium comprises one or more inorganic salts. In some embodiments, the dry powder cell-culture medium comprises calcium chloride, magnesium sulfate, potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, sodium phosphate dibasic, sodium selenite, magnesium chloride, ferrous sulfate, sodium meta silicate, and zinc sulfate. In some embodiments, the dry powder cell-culture medium comprises one or more organic salts. In some embodiments, the dry powder cell-culture medium comprises sodium pyruvate, pyroxidine-HCl, or sodium fumarate,. However, additional organic and inorganic salts may be comprised in a dry powder cell-culture medium.

The dry powder cell culture medium may be comprised of any suitable number of salts (e.g., organic and/or inorganic salts), for supporting a cell culture. In some

embodiments, the dry powder cell-culture medium comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40, individual salts. In some embodiments, the dry powder cell-culture medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 individual salts.

In some embodiments, the dry powder cell culture medium comprises one or more nutrients. In some embodiments, the nutrient is a carbohydrate. Exemplary nutrients include, without limitation, glucose, galactose, maltose, fructose, and sucrose. However, additional nutrients may be comprised in a dry powder cell culture medium and are within the scope of this disclosure. The dry powder cell culture medium may be comprised of any suitable number of nutrients, for supporting a cell culture. In some embodiments, the dry powder cell-culture medium comprises at least 1, at least 2, at least 3, at least 4, at least 5, individual nutrients. In some embodiments, the dry powder cell-culture medium comprises 1, 2, 3, 4, or 5 individual nutrients.

In some embodiments, the dry powder mixture (e.g., the dry powder cell culture medium) comprises one or more proteins or peptides. In some embodiments, the protein or peptide is a recombinant protein or peptide. Commonly used proteins and peptides in cell culture include albumin, transferrin, aprotinin, fetuin, and fibronectin. Albumin is the main protein in blood acting to bind water, salts, free fatty acids, hormones, and vitamins, and transport them between tissues and cells. The binding capacity of albumin makes it a suitable remover of toxic substances from the cell culture media. Aprotinin is a protective agent in cell culture systems, stable at neutral and acidic pH and resistant to high temperatures and degradation by proteolytic enzymes. It has the ability to inhibit several serine proteases such as trypsin. Fetuin is a glycoprotein found in fetal and newborn serum at larger concentrations than in adult serum. It is also an inhibitor of serine proteases. Fibronectin is a key player in cell attachment. Transferrin is an iron transport protein that acts to supply iron to the cell membrane. In some embodiments, the dry powder cell-culture medium comprises recombinant albumin, transferrin, aprotinin, fetuin, and/or fibronectin. However, the dry powder cell-culture medium may comprise additional proteins (e.g., recombinant proteins), which would be apparent to the skilled artisan and are within the scope of this disclosure.

In some embodiments, the dry powder cell-culture medium comprises one or more antibiotics. Antibiotics are typically used to control the growth of bacterial and fungal contaminants. In some embodiments, the dry powder cell-culture comprises one or more penicillins, cephalosporins, carbapenems, macrolides, aminoglycosides, quinolones (e.g., fluoroquinolones), sulfonamides, or tetracyclines. However, the dry powder cell-culture medium may comprise additional antibiotics, which would be apparent to the skilled artisan and are within the scope of this disclosure.

The dry powder cell-culture medium may be formulated to support the growth of any cell, which may be a bacterial cell, a fungal cell (e.g., a yeast cell), a plant cell or an animal cell (e.g., a human cell), any of which may be a somatic cell, a germ cell, a normal cell, a diseased cell, a transformed cell, a mutant cell, a stem cell, a precursor cell or an embryonic cell. The dry powder cell-culture medium may be formulated as a bacterial cell culture medium, a plant cell culture medium, or animal cell culture medium.

Examples of dry powder cell-culture media for animal cells include, but are not limited to, DMEM, RPMI-1640, MCDB 131, MCDB 153, MDEM, LMDM, MEM, M199, McCoy's 5 A, Williams' Media E, Leibovitz's L-15 Medium, Grace's Insect Medium, IPL-41 Insect Medium, TC-100 Insect Medium, Schneider's Drosophila Medium, Wolf & Quimby's Amphibian Culture Medium, cell-specific serum-free media (SFM) such as those designed to support the culture of keratinocytes, endothelial cells, hepatocytes, melanocytes, etc., F 10 Nutrient Mixture and F12 Nutrient Mixture. Other media, media supplements and media subgroups are available commercially (e.g., from Life Technologies, Inc.; Rockville, Maryland, and Sigma; St. Louis, Missouri). Formulations for these media, media supplements and media subgroups, as well as many other commonly used animal cell culture media, media supplements and media subgroups are well-known in the art and may be found, for example in the GTBCO/BRL Catalogue and Reference Guide (Life Technologies, Inc.; Rockville, Maryland) and in the Sigma Animal Cell Catalogue (Sigma; St. Louis, Missouri).

Examples of dry powder cell-culture media for plant cells include, but are not limited to, Anderson's Plant Culture Media, CLC Basal Media, Gamborg's Media, Guillard's Marine Plant Culture Media, Provasoli's Marine Media, Kao and Michayluk's Media, Murashige and Skoog Media, McCown's Woody Plant Media, Knudson Orchid Media, Lindemann Orchid Media, and Vacin and Went Media. Formulations for these media, which are commercially available, as well as for many other commonly used plant cell culture media, are well-known in the art and may be found for example in the Sigma Plant Cell Culture Catalogue (Sigma; St. Louis, Missouri).

Examples of dry powder cell-culture media for bacterial cells include, but are not limited to, Trypticase Soy Media, Brain Heart Infusion Media, Yeast Extract Media, Peptone- Yeast Extract Media, Beef Infusion Media, Thioglycollate Media, Indole-Nitrate Media, MR- VP Media, Simmons' Citrate Media, CT A Media, Bile Esculin Media, Bordet-Gengou Media, Charcoal Yeast Extract (C YE) Media, Mannitol-salt Media, MacConkey's Media, Eosin-methylene blue (EMB) media, Thayer-Martin Media, Salmonella-Shigella Media, and Urease Media. Formulations for these media, which are commercially available, as well as for many other commonly used bacterial cell culture media, are well-known in the art and may be found for example in the DIFCO Manual (DIFCO; Norwood, Massachusetts) and in the Manual of Clinical Microbiology (American Society for Microbiology, Washington, DC).

Examples of dry powder cell-culture media for fungal cells (e.g., yeast cells) include, but are not limited to, Sabouraud Media and Yeast Morphology Media (YMA). Formulations for these media, which are commercially available, as well as for many other commonly used yeast cell culture media, are well-known in the art and may be found for example in the DIFCO Manual (DLFCO; Norwood, Massachusetts) and in the Manual of Clinical

Microbiology (American Society for Microbiology, Washington, DC).

In some embodiments, the dry powder cell-culture medium is Iscove's Modified Dulbecco's Media (FMDM). In some embodiments, the FMDM comprises the following individual components:

Amino acids: L-alanine, L-arginine-HCl, L-Asparagine-H20, L-Aspartic Acid, L- Cystine-2HC1, L-Glutamic Acid, L-Glutamine, Glycine, L-Histidine-HCl-H20, L-Isoleucine, L-Leucine, L-Lysine-HCl, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L- Threonine, L-Tryptophan, L-Tyrosine-2Na-2H20, and L- Valine.

Vitamins: D-biotin, choline chloride, folic acid, myo-Inositol, niacinamide, D- pantothenic acid (hemicalcium), pyridoxal-HCl, riboflavin, thiamine-HCl, and Vitamin B12.

Inorganic salts: calcium chloride, magnesium sulfate (anhydrous), potassium chloride, potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphate monobasic (anhydrous), and sodium selenite.

Additional components: HEPES, Phenol Red-Na, Pyruvic Acid-Na, and glucose.

Preparing dry powder cell-culture medium

Some aspects of the disclosure include treatment steps that prepare dry powder mixtures (e.g., dry powder cell-culture media) for analysis. In some embodiments, the dry powder mixture is micronized prior to dispersal on a substrate for analysis {e.g., Raman analysis and/or morphological analysis). As used herein, the term "micronize" or

"micronization" refers to the process of reducing the average diameter of a solid material's particles. Typically, techniques for micronization include mechanical techniques such as milling, bashing and grinding. As one example, the dry powder cell-culture medium may be micronized in an industrial mill, which is typically comprised of a cylindrical metallic drum that contains steel spheres. As the drum rotates, the spheres inside collide with the particles of the solid, thus crushing them towards smaller diameters. In the case of grinding, the solid particles may be formed when the grinding units of the device rub against each other while particles of the solid are trapped in between. Methods such as crushing and cutting are also used for reducing particle diameter, but produce rough particles as compared to techniques like milling. Accordingly, methods such as crushing and cutting may be performed as early steps in the micronization process. Crushing employs hammer-like tools to break the solid into smaller particles by means of impact. Cutting includes the use of sharp blades to cut larger solid particles into smaller ones.

In some embodiments, methods for micronizing dry powder mixture (e.g., dry powder cell-culture medium) include jet milling. A jet mill grinds materials by using a high speed jet of compressed air or inert gas to impact particles into each other. Jet mills can be designed to output particles below a certain size, while continue milling particles above that size, resulting in a narrow size distribution of the resulting product. Particles leaving the mill can be separated from the gas stream by cyclonic separation. As the temperature in jet milling remains relatively constant, jet milling may be compatible with heat sensitive materials. Additional micronization techniques that may be used in accordance with the disclosure include the use of supercritical fluids in the micronization process. These methods use supercritical fluids to induce a state of supersaturation, which leads to precipitation of individual particles. The most widely applied techniques of this category include the RESS process (Rapid Expansion of Supercritical Solutions), the SAS method (Supercritical Anti- Solvent) and the PGSS method (Particles from Gas Saturated Solutions). These techniques allow for greater control of the process. Parameters like relative pressure and temperature, solute concentration, and antisolvent to solvent ratio are varied to adjust the output to the producer's needs. The supercritical fluid methods result in finer control over particle diameters, distribution of particle size and consistency of morphology. Exemplary methods would be apparent to the skilled artisan and have been described previously, for example in Knez, Z., et al., "Particle Formation and Product Formulation Using Supercritical Fluids," Annual Review of Chemical and Biomolecular Engineering. 6 (1): 379-407; Tandya A., et al., "Supercritical fluid micronization techniques for gastroresistant insulin formulations," The Journal of Supercritical Fluids. 107: 9-16; and Reverchon E., et al., "Supercritical fluids based techniques to process pharmaceutical products difficult to micronize:

Palmitoylethanolamide," The Journal of Supercritical Fluids. 102: 24-31.

In the case of RESS, the supercritical fluid is used to dissolve the solid material under high pressure and temperature, thus forming a homogeneous supercritical phase. Thereafter, the mixture is expanded through a nozzle to form the smaller particles. Immediately upon exiting the nozzle, rapid expansion occurs, lowering the pressure. The pressure will drop below supercritical pressure, causing the supercritical fluid - usually carbon dioxide - to return to the gas state. This phase change severely decreases the solubility of the mixture and results in precipitation of particles. The less time it takes the solution to expand and the solute to precipitate, the narrower the particle size distribution will be. Faster precipitation times also tend to result in smaller particle diameters.

In the SAS method, the solid material is dissolved in an organic solvent. The supercritical fluid is then added as an antisolvent, which decreases the solubility of the system. As a result, particles of small diameter are formed. There are various submethods to SAS which differ in the method of introduction of the supercritical fluid into the organic solution.

In the PGSS method, the solid material is melted and the supercritical fluid is dissolved in it. However, in this case the solution is forced to expand through a nozzle, and in this way nanoparticles are formed. The PGSS method has the advantage that because of the supercritical fluid, the melting point of the solid material is reduced. Therefore, the solid melts at a lower temperature than the normal melting temperature at ambient pressure.

It should be appreciated that any of the methods for preparing dry powder mixture (e.g., dry powder cell-culture medium) may be used to obtain individual particles of a size that is suitable for obtaining a Raman spectrum from a plurality of individual particles in the dry powder mixture. In some embodiments, the methods for preparing a dry powder mixture include micronization to the point where the average circle equivalent (CE) diameter of the particles in the dry-powder cell culture medium ranges from 5 μιη to 400 μιη. The CE diameter of a particle is the diameter of a circle with the same area as a 2D image of the particle. While the shape of a particle will influence the CE diameter, this measurement may be expressed as a single number that gets larger or smaller as the particle does and is typically objective and repeatable. In some embodiments, the particles in the dry powder mixture (e.g., dry powder cell-culture medium) are micronized to the point where the average circle equivalent diameter of the particles in the dry powder cell-culture medium is from 5 μιη to 10 μπι, from 5 μιη to 20 μπι, from 5 μιη to 30 μπι, from 5 μιη to 40 μπι, from 5 μιη to 50 μπι, from 5 μπι to 100 μπι, from 5 μιη to 150 μπι, from 5 μιη to 200 μπι, from 5 μιη to 300 μπι, from 10 μπι to 20 μπι, from 10 μιη to 30 μπι, from 10 μιη to 40 μπι, from 10 μιη to 50 μπι, from 10 μπι to 100 μπι, from 10 μιη to 150 μπι, from 10 μιη to 200 μπι, from 10 μιη to 300 μπι, from 10 μιη to 400 μπι, from 20 μιη to 30 μπι, from 20 μιη to 40 μπι, from 20 μιη to 50 μπι, from 20 μιη to 100 μπι, from 20 μιη to 150 μπι, from 20 μιη to 200 μπι, from 20 μιη to 300 μπι, from 20 μιη to 400 μπι, from 30 μιη to 40 μπι, from 30 μιη to 50 μπι, from 30 μιη to 100 μπι, from 30 μιη to 150 μπι, from 30 μιη to 200 μπι, from 30 μιη to 300 μπι, from 30 μιη to 400 μπι, from 40 μιη to 50 μπι, from 40 μιη to 100 μπι, from 40 μιη to 150 μπι, from 40 μπι to 200 μπι, from 40 μιη to 300 μπι, from 40 μιη to 400 μπι, from 50 μιη to 100 μπι, from 50 μπι to 150 μπι, from 50 μιη to 200 μπι, from 50 μιη to 300 μπι, from 50 μιη to 400 μπι, from 100 μιη to 150 μπι, from 100 μιη to 200 μπι, from 100 μιη to 300 μπι, from 100 μιη to 400 μπι, from 150 μιη to 200 μπι, from 150 μιη to 300 μηι, from 150 μιη to 400 μπι, from 200 μm to 300 μιη, from 200 μιη to 400 μηι, or from 300 μιη to 400 μιη.

In some embodiments, the dry powder mixture (e.g., dry powder cell-culture medium) is micronized to the point where a significant proportion of particles of the cell culture medium are within a suitable size range for analysis. In some embodiments, the dry powder mixture is micronized to the point where at least 25% of particles of the mixture have a CE diameter ranging from 5 μπι to 200 μπι. In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of particles of the cell culture medium have a CE diameter ranging from 5 μιη to 200 μιη.

Some aspects of the disclosure provide methods for mixing the dry powder mixture (e.g., dry powder cell-culture medium) prior to dispersal on a substrate for analysis (e.g., Raman analysis and/or morphological analysis). Dry powder mixtures may be mixed using any suitable method, which may be performed to evenly disperse the individual components of the dry powder cell-culture medium. It should be appreciated that the dry powder mixture may be mixed using any suitable mixing apparatus. For example, in some embodiments, the dry powder mixture is mixed using a ball mixer, a horizontal or vertical agitated chamber, a tumbling vessel, or an air agitated mixer. It should be appreciated that additional methods and apparatuses for mixing dry powders would be apparent to the skilled artisan and are within the scope of this disclosure. In some embodiments, the mixing may be performed using acoustic methods. For example, in some embodiments, the dry powder mixture is mixed using resonant acoustic mixing. An advantage of resonant acoustic mixing is that it can be used to mix powders of various particle sizes giving uniform dispersion by fluidizing the entire dry powder mixture. In some embodiments, the dry mixture is mixed using a resonant acoustic mixer. Exemplary acoustic mixers include, without limitation a LabRam Resonant Acoustic® Mixer, such as the LabRAM, the LabRAMII, the LabRAM II H, and the pharmaRAM II. However additional acoustic mixers would be apparent to the skilled artisan and are within the scope of this disclosure. In some embodiments, the dry powder mixture is mixed at an intensity from 10 g to 100 g, For example the dry powder mixture (e.g., dry powder cell-culture medium) is mixed at an intensity from 10 g to 20 g, from 10 g to 30 g, from 10 g to 40 g, from 10 g to 50 g, from 10 g to 60 g, from 10 g to 70 g, from 10 g to 80 g, from 10 g to 90 g, from 30 g to 40 g, from 30 g to 50 g, from 30 g to 60 g, from 30 g to 70 g, from 30 g to 80 g, from 30 g to 90 g, from 30 g to 100 g, from 50 g to 60 g, from 50 g to 70 g, from 50 g to 80 g, from 50 g to 90 g, from 50 g to 100 g, from 70 g to 80 g, from 70 g to 90 g, from 70 g to 100 g, or from 90 g to 100 g. In some embodiments, the dry powder cell-culture medium is mixed at an intensity of 60 g.

The dry powder mixture (e.g., dry powder cell-culture medium) may be mixed for any suitable duration, for example a duration that provides even dispersion of the individual particles of a dry powder mixture. In some embodiments, the duration of mixing is experimentally determined to ensure that the individual components of the dry powder mixture are dispersed evenly enough to facilitate accurate analysis (e.g., Raman analysis and/or morphological analysis) of the dry powder mixture. For example, the dry powder mixture may be mixed for a plurality of durations (e.g., 30 seconds, 1 minute, 2 minutes, or 3 minutes) and analyzed at each of the durations using Raman spectroscopy and/or

morphological analysis to determine the amount of time that is suitable for dispersing the dry powder mixture evenly enough to ensure accurate analysis. In some embodiments, the dry powder mixture is mixed using any of the methods and apparatuses provided herein. In some embodiments, a dry powder mixture (e.g., dry powder cell-culture medium) is mixed from 1 second to 10 minutes. In some embodiments, the dry powder cell-culture medium is mixed from 1 second to 5 seconds, from 1 second to 10 seconds, from 1 second to 30 seconds, from 1 second to 1 minute, from 1 second to 2 minutes, from 1 second to 2 minutes, from 1 second to 3 minutes, from 1 second to 5 minutes, from 1 second to 8 minutes, from 10 seconds to 30 seconds, from 10 seconds to 1 minute, from 10 seconds to 2 minutes, from 1 second to 3 minutes, from 1 second to 5 minutes, from 10 seconds to 8 minutes, from 10 seconds to 10 minutes, from 30 seconds to 1 minute, from 30 seconds to 2 minutes, from 30 seconds to 3 minutes, from 30 seconds to 5 minutes, from 30 seconds to 8 minutes, from 30 seconds to 10 minutes, from 1 minute to 2 minutes, from 1 minute to 3 minutes, from 1 minute to 5 minutes, from 1 minute to 8 minutes, from 1 minute to 10 minutes, from 2 minutes to 3 minutes, from 2 minutes to 5 minutes, from 5 minutes to 8 minutes, from 5 minutes to 10 minutes, from 3 minutes to 5 minutes, from 3 minutes to 8 minutes, from 3 minutes to 10 minutes, from 5 minutes to 8 minutes, from 5 minutes to 10 minutes, or from 8 minutes to 10 minutes. In some embodiments, the dry powder cell-culture medium is mixed for a duration of about 1 minute.

In some embodiments, a dry powder mixture (e.g., a dry powder cell culture medium) comprises a mixture of dry components that have been combined, where the dry mixture has not been dissolved following the combining of the dry components. However, in some embodiments, a dry powder mixture comprises a dried powder resulting from a dissolved solution, e.g., a lyophilized mixture.

Dry powder dispersal

Aspects of the disclosure provide methods for evaluating a dry powder mixture (e.g., dry powder cell -culture medium) that includes a step of dispersing the dry powder mixture onto a substrate. It should be appreciated that the powder is dispersed onto the substrate in order to facilitate the analysis of individual particles, representing individual components, of the dry powder mixture. Accordingly, any manual or automated methods for dispersing dry powders to facilitate analysis of individual particles may be used and would be apparent to the skilled artisan.

In some embodiments, the dry powder mixture (e.g., dry powder cell-culture medium) is manually dispersed onto a substrate. For example, a sample of the dry powder cell-culture medium may be dispersed onto a substrate by dropping the powder onto the substrate. In some embodiments, the powder may be dropped from a certain height above the substrate. In some embodiments, the amount of, and/or height from which the powder is dropped may be determined experimentally to optimize the amount of and/or distribution of particles that are dispersed onto the substrate. However, it should be appreciated that the methods may include additional techniques for dispersing the powder onto the substrate. For example, the powder may be thrown onto the substrate, or blown onto the substrate. In addition, the substrate may be shaken or vibrated after the powder is dispersed onto the substrate to promote even dispersal of the powder on the substrate.

In some embodiments, the dry powder mixture (e.g., dry powder cell-culture medium) is dispersed onto the substrate using automated methods. Such methods may provide improved control of the dispersal process to facilitate analysis of the individual components of the dry powder cell culture medium. In some embodiments, the dry powder cell-culture medium is dispersed onto a substrate using a dry powder dispersion device. Such dispersion devices are known in the art and would be apparent to the skilled artisan. In some embodiments, the dry powder is dispersed onto the substrate using an Aero S powder dispersion device. The Aero S disperses dry samples by accelerating particles through a venturi using compressed air. The particles are then pulled through the Mastersizer 3000's measurement cell using a vacuum source. Dispersion efficiency is controlled by three variables: air pressure, sample feed rate and disperser geometry. Dry powder dispersers accelerate particles in an airstream to achieve dispersion of particles. In some embodiments, the air pressure, sample feed rate, and/or disperser geometry are adjusted to achieve a desired dispersal pattern; for example, an even dispersal pattern that allows the analysis of individual particles of the dry powder.

It should be appreciated that the dry powder mixture (e.g., dry powder cell-culture medium) may be dispersed on any suitable substrate that is compatible with Raman spectroscopy. A substrate that is "compatible with Raman spectroscopy" refers to any substrate that allows for the analysis of a particle on the substrate using Raman spectroscopy. Typically the substrate will be a material that absorbs a minimal amount of the Raman signal from the particles on the substrate. In some embodiments a substrate is compatible with Raman spectroscopy if it absorbs less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 50%, less than 60%, or less than 80% of the Raman signal from the particles on the substrate. In some embodiments, the substrate is glass. In some embodiments, the substrate is a glass plate. In some embodiments, the substrate is a fused quartz plate. In some embodiments, the substrate is a fused quartz glass plate. In some embodiments, the substrate is plastic. In some embodiments, the substrate is transparent. However, additional substrates would be apparent to the skilled artisan based on this disclosure and knowledge in the field and are within the scope of this disclosure. The substrate may be any suitable size to facilitate analysis of a plurality of particles of a dry powder cell-culture medium. In some embodiments, the substrate comprises a surface that is from 50 mm² to 50,000 mm². In some embodiments, the substrate comprises a surface that is at least 50 mm², at least 100 mm², at least 500 mm², at least 1,000 mm², at least 2,000 mm², at least 5,000 mm², at least 10,000 mm², at least 20,000 mm², at least 30,000 mm², at least 40,000 mm², or at least 50,000 mm². In some

embodiments, the substrate comprises a surface that is 180 mm x 110 mm, or 120 mm x 100 mm.

With dry powder dispersion there is the possibility of particle damage, e.g., because of the high velocity at which the particles pass through the disperser system. In some embodiments, particle damage may be minimized by avoiding impaction surfaces. In some embodiments, dispersion is achieved through the application of significant shear, rather than by impacting particles on a surface. The result is gentle but highly efficient dispersion, which can be successfully applied to even relatively friable materials. Optional impaction systems are available for strongly agglomerated samples with tough primary particles. In some embodiments, the air pressure drop across the venturi is manipulated to achieve complete sample dispersion and can be controlled to within +/-0.1 bar.

Sample feed rate through the Aero S can be closely controlled using a vibrating feeder, which maintains a suitable sample concentration for measurement. It is fitted with an interchangeable sample tray that can be configured to ensure the measurement of enough material to quantify the entire size distribution reproducibly. Such automated dispersal systems may increase the measurement robustness, and accelerate analysis of dry powder cell-culture media.

In some embodiments, dispersion of the dry powder mixture (e.g., dry powder cell- culture medium) is performed using an instrument. In some embodiments, one or more parameters of the instrument that disperses the dry powder mixture are controlled (e.g., manually controlled) by a user. In some embodiments, the one or more parameters is the pressure at which the powder is dispersed, the injection time, and/or the settling time. In some embodiments, the user may control the pressure at which the dry powder mixture is dispersed. In some embodiments, the pressure at which the powder is dispersed ranges from 0.1 to 10 bar. In some embodiments, the pressure at which the powder is dispersed ranges from 0.1 to 1 bar, from 0.1 to 2 bar, from 0.1 to 3 bar, from 0.1 to 4 bar, from 0.1 to 5 bar, from 0.1 to 8 bar, from 1 to 2 bar, from 1 to 3 bar, from 1 to 4 bar, from 1 to 5 bar, from 1 to 8 bar, from 1 bar to 10 bar, from 2 to 3 bar, from 2 to 4 bar, from 2 to 5 bar, from 2 to 8 bar, from 2 bar to 10 bar, from 3 to 4 bar, from 3 to 5 bar, from 3 to 8 bar, from 3 bar to 10 bar, from 4 to 5 bar, from 4 to 8 bar, from 4 bar to 10 bar, from 5 to 8 bar, from 5 to 10 bar, or from 8 to 10 bar. However, additional pressures may be used and may be selected depending on, inter alia, the amount of powder used, and/or the size of particles within the powder. In some embodiments, the pressure at which the powder is dispersed ranges from 3 to 5 bar.

In some embodiments, the user may control the injection time. The injection time refers to the duration for which the air pressure is applied. In some embodiments, the injection time ranges from 1 to 200 milliseconds (ms). In some embodiments, the injection time ranges from 1 ms to 5 ms, from 1 ms to 10 ms, from 1 ms to 20 ms, from 1 ms to 50 ms, from 1 ms to 100 ms, from 1 ms to 150 ms, from 1 ms to 200 ms, from 5 ms to 10 ms, from 5 ms to 20 ms, from 5 ms to 50 ms, from 5 ms to 100 ms, from 5 ms to 150 ms, from 5 ms to 200 ms, from 10 ms to 20 ms, from 10 ms to 50 ms, from 10 ms to 100 ms, from 10 ms to 150 ms, from 10 ms to 200 ms, from 20 ms to 50 ms, from 20 ms to 100 ms, from 20 ms to 150 ms, from 20 ms to 200 ms, from 50 ms to 100 ms, from 50 ms to 150 ms, from 50 ms to 200 ms, from 100 ms to 150 ms, from 100 ms to 200 ms, or from 150 ms to 200 ms. In some embodiments, the injection time ranges from 5 ms to 15 ms. In some embodiments, the injection time is 10 ms.

In some embodiments, the user may control the settling time. The settling time refers to the amount of time the particles are allowed to settle onto a substrate {e.g., a glass plate). In some embodiments, the settling time ranges from 0.5 to 600 seconds (s). However, it should be appreciated that powders comprised of small particles may take longer to settle. Accordingly, a skilled artisan may adjust {e.g., increase or decrease) the settling time based on the shape and/or size of the particles within the dry powder cell-culture medium. In some embodiments, the settling time is at least 0.5 s, at least 1 s, at least 5 s, at least 10 s, at least 20 s, at least 50 s, at least 100 s, at least 150 s, at least 200 s, at least 250 s, at least 300 s, at least 350 s, at least 400 s, at least 450 s, at least 500 s, at least 550 s, or at least 600 s. However, additional settling times may be used and would be apparent to the skilled artisan.

The dry powder dispersal methods provided herein may be used to achieve a desired dispersal density of a dry powder mixture (e.g., dry powder cell-culture medium) on a substrate. In some embodiments, the individual particles of the dry powder cell-culture medium are dispersed at a density (e.g., average number of particles per mm²) suitable for analyzing individual particles (e.g., by Raman spectroscopy or image analysis) with minimal interference from other particles in the dry powder. In some embodiments, the dry powder cell culture medium is dispersed onto the substrate to yield an average number of particles per area from 5 to 100 particles per mm². In some embodiments, the dry powder cell culture medium is dispersed onto the substrate at an average density from 5 to 10 particles/mm², from 5 to 20 particles/mm², from 5 to 10 particles/mm², from 5 to 20 particles/mm², from 5 to 30 particles/mm², from 5 to 40 particles/mm², from 5 to 50 particles/mm², from 5 to 60 partic es/mm' from 5 to 70 particles/mm , from 5 to 80 particles/mm , from 5 to 90 partic es/mm from 10 to 20 particles/mm" from 10 to 30 particles/mm , from 10 to 40 partic es/mm from 10 to 50 particles/mm from 10 to 60 particles/mm², from 10 to 70 partic es/mm from 10 to 80 particles/mm from 10 to 90 particles/mm², from 10 to 100 partic es/mm from 20 to 30 particles/mm from 20 to 40 particles/mm², from 20 to 50 partic es/mm from 20 to 60 particles/mm from 20 to 70 particles/mm², from 20 to 80 partic es/mm from 20 to 90 particles/mm from 20 to 100 particles/mm², from 30 to 40 partic es/mm from 30 to 50 particles/mm from 30 to 60 particles/mm², from 30 to 70 partic es/mm from 30 to 80 particles/mm from 30 to 90 particles/mm², from 30 to 100 partic es/mm from 40 to 50 particles/mm from 40 to 60 particles/mm², from 40 to 70 partic es/mm from 40 to 80 particles/mm from 40 to 90 particles/mm², from 40 to 100 partic es/mm from 50 to 60 particles/mm from 50 to 70 particles/mm², from 50 to 80 partic es/mm from 50 to 90 particles/mm from 50 to 100 particles/mm², from 60 to 70 partic es/mm from 60 to 80 particles/mm from 60 to 90 particles/mm², from 60 to 100 partic es/mm from 70 to 80 particles/mm from 70 to 90 particles/mm , from 70 to 100 partic es/mm from 80 to 90 particles/mm from 80 to 100 particles/mm², or from 90 to 100 partic es/mm . In some embodiments, the dry powder cell culture medium is dispersed onto the substrate at an average density from 50 to 70 particles/mm².

It should be appreciated that the average densities of particles provided herein may be for a given area of the substrate. For example, the area of the substrate on which the dry powder cell-culture media is dispersed may be any suitable size for evaluating (e.g., obtaining a Raman spectrum and/or obtaining an image) a suitable number of particles of the dry powder cell-culture medium. In some embodiments, the average densities of particles are determined for an area that is from 10 mm² to 2000 mm². In some embodiments, the average densities of particles are determined for an area that is from 10 mm² to 100 mm², from 10 mm 2 to 500 mm 2 , from 10 mm 2 to 1000 mm 2 , from 10 mm 2 to 1500 mm 2 , from 10 mm 2 to 2000 mm², from 100 mm² to 500 mm², from 100 mm² to 1000 mm², from 100 mm² to 1500 mm², from 100 mm² to 2000 mm², from 500 mm² to 1000 mm², from 500 mm² to 1500 mm², from 500 mm² to 2000 mm², from 1000 mm² to 1500 mm², from 1000 mm² to 2000 mm², or from 1500 mm² to 2000 mm². In some embodiments, the average densities of particles are determined for an area that is from 1200 mm² to 1500 mm². In some embodiments, the average densities of particles are determined for an area that is from 80 mm² to 120 mm².

Raman Spectroscopic Analysis

Aspects of the disclosure relate to analyzing individual particles of a dry powder cell- culture medium using Raman spectroscopy. Raman spectroscopy can provide information about the structure of molecules. The position and intensity of features in a Raman spectrum reflect the molecular structure and can be used to determine the chemical identity of a sample {e.g., a particle of a dry powder cell culture medium). Spectra may also show subtle changes depending on the crystalline form of the sample. With available spectral libraries and/or user generated spectral libraries, the identity of compounds by spectral library searching may be obtained. In some embodiments, the Raman spectroscopic analysis provided herein is performed using a Raman instrument (e.g., a dispersive Raman instrument or a Fourier transform Raman instrument). In some embodiments, the Raman instrument is a dispersive Raman instrument. To observe a Raman spectrum, it is necessary to separate the collected Raman scattered light into its composite wavelengths. In dispersive Raman instruments, this is accomplished by focusing the Raman scattered light {e.g., from a particle of a dry powder cell culture medium) onto a diffraction grating, which splits the beam into its constituent wavelengths, which may be directed onto a detector, such as a silicon charged-coupled device (CCD). Typically, a dispersive Raman instrument employs visible laser radiation. In some embodiments, the laser wavelengths used are 780 nm, 633 nm, 532 nm, or 473 nm.

However, additional laser wavelength may be used and would be apparent to the skilled artisan.

In some embodiments, the Raman instrument is capable of focusing light {e.g., from a laser) onto a small area on a substrate in order to analyze individual particles among a plurality of particles on the substrate. Accordingly, in some embodiments, the Raman device is configured to focus a beam of light onto the substrate. In some embodiments, the beam of light is configured to illuminate an area (e.g., an excitation spot) of less than 50 μπι² on the substrate. In some embodiments, the beam of light is configured to illuminate an area of less

2 2 2 2 2 than 0.5 μπι , less than 1 μπι , less than 1.5 μπι , less than 2 μπι , less than 2.5 μπι , less than

2 2 2 2 2 2

3 μπι , less than 3.5 μπι , less than 4 μπι , less than 4.5 μπι , less than 5 μπι , less than 6 μπι ,

2 2 2 2 2 less than 7 μπι , less than 8 μπι , less than 9 μπι , less than 10 μπι , less than 15 μπι , less

2 2 2 2 2 than 20 μπι , less than 25 μπι , less than 30 μπι , less than 35 μπι , less than 40 μπι , or less than 50 μπι². In some embodiments, the Raman spectrometer is a Raman Rxnl spectrometer (Kaiser Optical Systems, Inc. USA). However, it should be appreciated that other Raman spectrometers would be apparent to the skilled artisan and are within the scope of this disclosure.

In some embodiments, the Raman device is used with a suitable microscope to visualize the particles being excited by the Raman spectrometer. In some embodiments, the microscope is a CFI 60 brightfield/darkfield microscope (Nikon Corporation, Japan).

However additional microscopes having similar capabilities may also be used and are within the scope of this disclosure. In some embodiments, the Raman device comprises a

Morphologi G3-ID instrument (Malvern).

Some aspects of the disclosure provide methods for evaluating a dry powder cell- culture medium by obtaining a Raman spectrum from each of a plurality of particles of the dry powder cell-culture medium that are dispersed on a substrate. In some embodiments, at least one of the individual components of the cell-culture medium is Raman active. As used herein, the term "Raman active" refers to any component (e.g., molecule) that is detectable using Raman spectroscopy. In some embodiments, a molecule is Raman active if it is capable of causing a Stokes-Raman shift of light that contacts the compound. In some embodiments, a molecule is Raman active if it is comprised of one or more asymmetrical bonds. It should be appreciated that a "Raman active" molecule may be defined by the strength and/or the number of Raman peaks detected from the molecule that is exposed to light. Accordingly, in some embodiments, a molecule is Raman active if it yields a Raman spectrum having at least one unique vibrational mode (e.g., Raman peak) having a signal to noise ratio of 10 or more under standard Raman collection conditions. In some embodiments, a molecule is Raman active if it yields a Raman spectrum having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 unique vibrational modes (e.g., Raman peaks), where the weakest peak has a signal to noise ratio of 10 or more under standard Raman collection conditions. In a preferred embodiment, a molecule is Raman active if it yields a Raman spectrum having at least 3, unique vibrational modes (e.g., Raman peaks), where the weakest peak has a signal to noise ratio of 10 or more under standard Raman collection conditions.

In some embodiments, the Raman active molecule is an amino acid. In some embodiments, the Raman active molecule is alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In other embodiments, the Raman active molecule is a vitamin. In some embodiments, the Raman active molecule is biotin (e.g., D-biotin), choline chloride, myo-Inositol, niacinamide, D- pantothenic acid (hemicalcium), pyridoxal-HCl, riboflavin, thiamine-HCl, calcium

pentothenate, cyanocobalamin (vitamin Bi₂), DL alpha lipoic acid, inositol, thiamine, PABA, riboflavin (vitamin B₂), folic acid (vitamin B₉), or Niacinamide (vitamin B₃).

The dry powder cell-culture medium that is evaluated using the methods provided herein may contain one or more Raman active individual components (e.g., serine or valine). In some embodiments, the dry powder cell culture medium comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 Raman active individual components. In some embodiments, at least 40% of the individual components of a dry powder cell culture medium are Raman active. In other embodiments, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%), or at least 98%, of the individual components of a dry powder cell culture medium are Raman active.

In some embodiments, a Raman spectrum is obtained from a particle that is of a suitable size. It should be appreciated that the particle should be large enough to obtain a Raman spectrum from, which may depend on, inter alia, the area of illumination from the light source (e.g., laser) and/or the shape of the particle. In some embodiments, the methods provide obtaining Raman spectra from particles that have at least one dimension, or have a CE diameter that is 2 μιη or greater, 3 μιη or greater, 4 μιη or greater, 5 μιη or greater, 6 μιη or greater, 7 μιη or greater, 8 μιη or greater, 9 μιη or greater, 10 μιη or greater, 15 μιη or greater, 20 μιη or greater, or 30 μιη or greater. In other embodiments, the methods provide obtaining Raman spectra from particles that have at least one dimension, or have a CE diameter that is 5 μιη or greater. The methods provided herein may be used to determine the presence or absence of one or more components of a dry powder cell-culture medium. It should be appreciated that determining the presence or absence of one or more individual components of the dry powder cell-culture medium may depend on a number of factors which include, but are not limited to the Raman activity of the individual component, and the concentration of the component in the medium. For example, an individual component having a Raman signature comprising multiple unique peaks having intensities well above the signal to noise ratio will be more readily detected than components that are not Raman active, or have a weak Raman signature. In addition, when a component of the dry powder cell-culture medium is at a low concentration (e.g., 0.01%) in the culture medium, a large number of particles will need to be analyzed to reach a level of confidence that the individual component is present or absent.

Accordingly, in some embodiments, the methods include determining a number of particles of a dry powder cell-culture medium to analyze (e.g., by Raman spectroscopy) in order to determine whether an individual component of the dry powder cell-culture medium is present or absent. In some embodiments, determining a total number of particles to analyze takes into consideration one or more of the following: the uniqueness of the Raman spectrum, the Raman signal strength, the signal to noise ratio of the lowest (e.g., least intense) Raman peak, and the concentration of the individual component. Thus, the methods take into account the estimated component particles available for Raman acquisition, the analytical selectivity, and signal characteristics to identify individual components within the dry powder cell culture medium.

It should be appreciated that methods for determining the total of number particles to analyze (e.g., using Raman spectroscopy) may vary depending on a number of factors including, but not limited to, the uniqueness of the particles in the dry powder cell-culture medium, the intensity of the Raman spectrum obtained, the particle sizes, the concentration of an individual component in the medium, and the acceptable level of confidence that a component is present or absent. Accordingly, in some embodiments, the following exemplary equation may be used to determine the number of particles required to detect and identify individual components within a dry powder cell-culture medium (e.g., a chemically defined medium or a multicomponent mixture):

Total Particle Count Requ ired

Where

a is the component Raman spectrum selectivity or uniqueness index from 1 to 10 where 10 is low uniqueness and 1 is high uniqueness

β is the component Normalized Average Raman Signal Strength from 1 to 10 where 1 is high signal and 10 is low signal

Θ is the components lowest Raman peak Signal to Noise ratio within the spectral library from 1 to 10 where 1 is high S/N and 10 is low S/N

M= Total value (10) (worse case)

A= Actual value for the component

P_c is the component particle concentration or component particle count within the Raman detectable particle size range

In some embodiments, this equation accounts for the estimated component particles available for Raman acquisition (Pc), Raman spectrum selectivity or uniqueness (a) relative to the spectra comprised within a Raman library, and signal characteristics (β and Θ) to identify the target components within the material.

In some embodiments, the methods include obtaining a Raman spectrum from at least 10, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, or at least 20,000 or more individual particles of the plurality of particles of a dry powder mixture (e.g., a dry powder cell-culture medium). In some embodiments, the methods include obtaining a Raman spectrum from 10 to 10,000 individual particles of the plurality of particles of the dry powder cell-culture medium. In some embodiments, the methods include obtaining a Raman spectrum from 50 to 200, from 50 to 1,000, from 50 to 2,000, from 50 to 4,000, from 50 to 6,000, from 50 to 8,000, from 50 to 9,000, from 200 to 1,000, from 200 to 2,000, from 200 to 4,000, from 200 to 6,000, from 200 to 8,000, from 200 to 9,000, from 200 to 10,000, from 1,000 to 2,000, from 1,000 to 4,000, from 1,000 to 6,000, from 1,000 to 8,000, from 1,000 to 9,000, from 1,000 to 10,000, from 2,000 to 4,000, from 2,000 to 6,000, from 2,000 to 8,000, from 2,000 to 9,000, from 2,000 to 10,000, from 4,000 to 6,000, from 4,000 to 8,000, from 4,000 to 9,000, from 4,000 to 10,000, from 6,000 to 8,000, from 6,000 to 9,000, from 6,000 to 10,000, from 8,000 to 9,000, from 8,000 to 10,000, from 9,000 to 10,000, or from 10,000 to 20,000 or more individual particles of the plurality of particles of the dry powder mixture (e.g., dry powder cell-culture medium). In some embodiments, a suitable number of particles are analyzed (e.g., by Raman spectroscopy) to achieve a level of confidence that an individual component of the dry powder mixture (e.g., dry powder cell-culture medium) is present or absent. In some embodiments, a Raman spectrum is obtained from a suitable number of particles of a dry powder mixture (e.g., dry powder cell-culture medium) in order to achieve at least a 60% confidence level that an individual component of the dry powder mixture (e.g., an individual component expected to be present in the mixture) is present or absent. In some embodiments, a Raman spectrum is obtained from a suitable number of particles of a dry powder mixture in order to achieve at least a 65%>, at least a 70%, at least a 75%, at least an 80%>, at least an 85%), at least a 90%, at least a 95%, at least a 98%, or at least a 99% confidence level that an individual component of the dry powder mixture is present or absent.

In some aspects, the methods provided herein include comparing a Raman spectrum obtained from an individual particle of the dry powder mixture (e.g., dry powder cell-culture medium) to an appropriate standard. In some embodiments, comparing a Raman spectrum obtained from an individual particle of the dry powder mixture (e.g., dry powder cell-culture medium) to an appropriate standard is performed to determine the identity of the individual particle. In some embodiments, the appropriate standard is a Raman spectrum of a component (e.g., a component expected to be present) in the dry powder mixture. The number of and/or position of the Raman peaks for an individual component can be used to identify the particle by comparing the peaks to Raman peaks from a known component. For example, the appropriate standard may be a collection (e.g., an available Raman library) of Raman spectra of known components (e.g., dry powder cell-culture medium components). In other embodiments, the standard may be a user generated library of Raman spectrum that includes Raman spectra of each of the individual components of the dry powder mixture.

In some embodiments, the identity of the individual component is determined if it shares a threshold degree of similarity to a Raman spectrum from a known component. For example, in some embodiments, the identity of an individual component of the dry powder cell culture medium is determined if it shares at least 65%, at least 70%, at least 75%, at least 80%), at least 85%, at least 90%, at least 95%, or at least 98% similarity (e.g., correlation) with the Raman spectrum of a known component. In some embodiments, the identity of an individual component of the dry powder cell culture medium is determined if it shares at least 80%) similarity (e.g., correlation) with the Raman spectrum of a known component. Methods for determining the %> correlation between Raman spectra are known in the art and would be apparent to the skilled artisan. For example, in some embodiments, the correlation may be determined using a Bio-Rad KnowItAll® ID Expert for Raman Spectra or equivalent commercially available spectrum identification software (e.g., Omnic TQ Analyst) and/or associated Raman spectral databases.

In some embodiments, a principal component model based on the Raman library is used to determine the correlation or a hit quality index (HQ) to an unknown spectrum. In some embodiments, PCA Lack-of-fit statistic or spectrum residual (Q) is calculated where the new unknown spectrum is compared to the target known spectrum within the Raman library. In some embodiments, the Q statistic indicates how well the unknown spectrum conforms to the known library spectrum.

In some embodiments, the Raman spectrum is compared to the Raman spectra of a user generated library of Raman spectra. A "user generated library of Raman spectra" refers to a collection of Raman spectra of known components. In some embodiments, the components are components of a dry powder cell-culture medium. Such user generated libraries may be constructed using any suitable methods. In some embodiments, one or more individual components of the dry powder cell-culture media (e.g., from United States Pharmacopeia (USP) grade components) are used to obtain Raman spectrum for use in generating a library of Raman spectra. In some embodiments, a Raman spectrum and/or morphology scan is performed for each individual component of a dry powder cell-culture medium.

Some aspects of the disclosure provide methods for selecting and using a dry powder cell-culture medium to establish a cell culture. In some embodiments, the methods provided herein are used to determine whether a dry-powder cell culture medium will be used to establish a cell culture medium for growing one or more cells. In some embodiments, the dry powder cell-culture medium is dissolved in a solvent (e.g., water) and used in a cell culture if it is determined that one or more of the individual components of the dry powder cell-culture medium (e.g., a dry powder CDM) are present. In some embodiments, the dry powder cell- culture medium is dissolved in a solvent (e.g., water) and used in a cell culture if it is determined that at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 of the individual components of the dry powder cell-culture medium (e.g., a dry powder CDM) are present. In other embodiments, the dry powder cell-culture medium is dissolved in a solvent (e.g., water) and used in a cell culture if it is determined that at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the individual components of the dry powder cell-culture medium (e.g., a dry powder CDM) are present. It should be appreciated that if the certain number or percentage of components are not identified, then the dry powder cell- culture medium is not used to establish a cell culture. In some embodiments, the dry powder cell-culture medium is used in a cell culture if it is determined that all of the components of the dry powder cell-culture medium are present. In other embodiments, the dry powder cell- culture medium is used in a cell culture if it is determined that all of the Raman active components of the dry powder cell-culture medium are present.

In some embodiments, the dry powder cell-culture medium is dissolved in a solvent (e.g., water) and used in a cell culture if it is determined that all of the amino acids are present in the dry powder cell-culture medium. In some embodiments, the dry powder cell-culture medium is dissolved in a solvent (e.g., water) and used in a cell culture if it is determined that all of the vitamins are present in the dry powder cell-culture medium. It should be appreciated that certain components, such as some inorganic salts, may have a weak Raman signature. Thus, in certain embodiments, the presence or absence of individual components having a weak Raman signature do not have to be identified as present in the media to use the media in a cell culture. In some embodiments, the presence or absence of individual components having a weak Raman signature may be determined by employing

morphological analysis of the particles, as provided herein. In some embodiments, the dry powder cell-culture medium is not used in a cell culture unless a suitable number or percentage of individual components, as provided herein, are confirmed to be present in the dry powder cell -culture medium.

Image Analysis

Some aspects of the disclosure provide methods for analyzing one or more physical properties of a plurality of individual particles of a dry powder mixture (e.g., dry powder cell- culture medium). In some embodiments, analyzing a physical property of an individual particle may be performed by image analysis, for example microscopic analysis using a suitable microscope and/or image capture device (e.g., a CCD camera). In some

embodiments, the analysis is performed using a Morphologi G3-ID instrument. However, additional suitable instruments, which would be apparent to the skilled artisan, may be used to carry out the analyses provided herein. The Morphologi system provides the ability to measure the morphological characteristics (size and shape) of particles. It is an analytical tool capable of differentiating and

characterizing particulate samples derived from the Morphologi G3 hardware platforms. Additionally, it allows the user to use the Morphologi instrument as a fully featured manual microscope capable of capturing high-resolution images. Building on these capabilities, the Morphologi G3-ID also adds the ability to perform a chemical analysis of a sample using Raman spectroscopy.

In some aspects, one or more physical properties are measured. It should be appreciated that measuring and/or identifying one or more physical properties of a particle within the dry powder cell-culture medium may be used to facilitate identification of the particle and/or provide valuable performance data. For example, measuring the average particle size may be used to determine dissolution time of the dry powder cell-culture medium in a solvent (e.g., water). In some embodiments, the physical property is area, aspect ratio, circular equivalent (CE) diameter, circularity, convex hull, convexity, elongation, high sensitivity (HS) circularity, intensity mean, intensity standard deviation (SD), length, major axis, maximum distance, perimeter, spherical equivalent (SE) volume, solidity, width, fiber elongation, fiber total length, fiber straightness, and fiber width. A description of each of the physical properties is provided below.

Area - The area refers to the visual projected area of the particle. The area may be expressed in number of pixels (e.g., of an image of the particle), or an absolute size such as microns (μιη).

Aspect Ratio - The aspect ratio refers to the ratio of the width to the length of the particle. It is calculated as:

Aspect Ratio— ^'

The aspect ratio values are in the range of 0 to 1. A rod, for example, would typically have a low aspect ratio.

Circular Equivalent (CE) Diameter - The circular equivalent diameter refers the diameter of a circle with the same area as the projected area of the particle image. In some embodiments, the CE diameter is reported in μπι.

Circularity - Circularity refers to the ratio of the circumference of a circle equal to the object's projected area to the perimeter of the object. The circularity is calculated as: A perfect circle has a circularity of 1.0, while a very narrow elongated object has a circularity close to 0.

Convex Hull - The Convex hull itself is not a reported parameter but is used in calculation of some of the reported parameters. Convexity is a measurement of the surface roughness of a particle. It is calculated by dividing the convex hull perimeter by the actual particle perimeter. One way to visualize the convex hull perimeter is to imagine an elastic band placed around the particle, as shown by the dashed line in FIG. 5A. The length of this dashed line is the convex hull perimeter. The Convex hull perimeter is the smallest convex polygon that contains the region. A schematic representation of convex hull is shown in FIG. 5A.

Convexity - The convexity refers to the perimeter of the convex hull perimeter of the object divided by its Perimeter. Convexity is a measure of how 'spiky' a particle is. The convex hull can be seen as the border created by an imaginary rubber band wrapped around the object. In the schematic of FIG. 5B, B is the added 'convexity area' of the particle surrounded by the convex hull:

P ri em'afA + S

Conv xiw =— P re—r—te ^'—-ΰβτ,.—

The Convexity values are in the range 0 (least convex) to 1 (most convex).

Elongation - Elongation is 1 -Aspect Ratio. Elongation values range from 0 to 1. A rod, for example, has a high elongation value.

High Sensitivity (HS) Circularity - HS Circularity refers to the ratio of the object's projected area to the square of the perimeter of the object. This is equivalent to squaring the numerator and denominator of the circularity calculation to obtain a more sensitive measure when comparing particles of similar circularity. It is sometimes termed compactness. A perfect circle has an HS Circularity of 1.0 while a narrow rod has an HS Circularity

close to 0.

_T . .... _: , 4 .··· · Area

HS Li cii niv- -

Perimeter^

Intensity Mean - The Intensity mean refers to the average of the pixel greyscale levels in the object, where:

Ii is the intensity value of pixel (i).

N is the total number of pixels in the particle. For greyscale images the intensity mean ranges from 0 (black) to 255 (white). A plain mid-grey object has an Intensity Mean of 128.

Intensity Standard Deviation (SD) - Intensity SD refers to the standard deviation of the pixel greyscale levels in the object, where:

Ii is the intensity value of pixel (i).

N is the total number of pixels in the particle.

Intensity SD is reported in greyscale levels from 0 to 255. For example, a uniform grey object would have n intensity SD value of 0:

Intensity

Length - Lines from all points on the perimeter are projected onto the major axis (1) of the particle as shown in FIG. 5C. The longest distance between the points where two of these projections meet the axis is the length (2) of the particle.

Major Axis - The graphic in FIG. 5D shows the major axis (1) and the minor axis (2).

The major axis is the angle of the majora from a horizontal line. The major axis and minor axis are reported in degrees and can take values between 0° and 180°.

In this example the major axis = 95°.

The major axis passes through the center of mass of the object at an orientation

corresponding to the minimum rotational energy of the shape. It is also termed the orientation.

The minor axis passes through the center of mass at right angles to the major

axis.

Maximum Distance - The maximum distance is the furthest distance between any two points of the particle. Maximum distance is also known as maximal Feret diameter or caliper length. In some embodiments, the maximum distance is measured in μιη. Perimeter - Perimeter refers the total length of the object boundary, calculated by summing the length of the boundary pixels. In some embodiments, this includes an adjustment to take account of direction changes.

Spherical Equivalent (SE) Volume - The SE volume refers to the spherical equivalent (SE) volume. In some embodiments, the SE volume is measured in μιη³. This is the

volume of a sphere with the same CE diameter as the object. A schematic representation of SE volume is shown in FIG. 5E. The equation for calculating SE volume is below:

, _ , π x CEDia eter ^>

onnne =

6

Solidity - Solidity refers to the object area divided by the area enclosed by the convex hull. The equation for calculating Solidity is below:

Aoi itv—

Width - Lines from all points on the perimeter are projected onto the minor axis (1) of the particle as shown in FIG. 5F. The longest distance between the points where two of these projections meet the axis is the width (2) of the particle.

Fiber Elongation - Fiber elongation is an expression of the width to length ratio. For example, 1 = maximum elongation, and 0 = minimum elongation. A schematic representing the fiber elongation value for various shapes is shown in FIG. 5G. Fiber elongation may be calculated using the following equation:

Fiber Total Length - To assess the fiber's length, its 'skeleton' is first assessed, and the length is derived. Effectively, this gives the length of the fiber as if it were straightened out. In some embodiments, the fiber total length is measured in μπι.

Fiber Straightness - Fiber Straightness is an expression of the correlation of the shape to a straight line. For example, 1 = maximum straightness, 0 = minimum straightness. Fiber straightness may be calculated using the following equation:

Fiber Width - To determine fiber width, the fiber is analyzed and a measurement is made of its cross section, as shown in FIG. 5H, giving the width. Fiber width may be calculated using the following equation:

Fiber Width -

FiberLsiwtk

In some embodiments, each of the particles of the dry powder cell-culture media that are detected are assigned a unique identifier. As objects are detected during the scan, for example using a MorphologiG3 instrument, they may be assigned a unique identifier such as an identification number. In some embodiments, the unique identifier is a number representing the order in which the individual particle was found. For example, the unique identifier may be 1....N, where N represents the total number of particles found. In some embodiments, each frame is scanned vertically and successive frames are acquired in the Y direction, and then in the X direction, however any scanning pattern may be used.

In some embodiments, at least one physical property of a plurality of particles of a dry powder cell-culture medium is determined. In some embodiments, more than one physical property of the plurality of particles of a dry powder cell-culture medium is determined. The physical property may be any of the physical properties provided herein. For example, the property may be area, aspect ratio, circular equivalent (CE) diameter, circularity, convex hull, convexity, elongation, high sensitivity (HS) circularity, intensity mean, intensity standard deviation (SD), length, major axis, maximum distance, perimeter, spherical equivalent (SE) volume, solidity, width, fiber elongation, fiber total length, fiber straightness, or fiber width. However, additional physical properties may be obtained and would be apparent to the skilled artisan. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 physical properties for each of the plurality of particles of a dry powder cell-culture medium is determined.

In some embodiments, at least one physical property is determined for a plurality of particles of a dry powder mixture. In some embodiments, at least one physical property is determined for at least 2, at least 5, at least 10, at least 50, at least 100, at least 200, at least

400, at least 800, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least

7,000, at least 8,000, at least 9,000, or at least 10,000 individual particles of a dry powder mixture (e.g., dry powder cell-culture medium). It should be appreciated that the number of particles analyzed may depend on the goal of the analysis and or the composition of the dry powder cell culture medium. For example, a large number of particles may need to be analyzed when attempting to identify a rare particle (e.g., < 0.01%) within the dry powder cell-culture medium. Alternatively, a relatively few number of particles may need to be analyzed when attempting to identify a highly abundant particle (e.g., >5%) within the dry powder cell-culture medium. Accordingly, based on the present disclosure and knowledge in the art, a skilled artisan would be able to determine a suitable number of particles to analyze based on the goal of the analysis and/or composition of the dry powder mixture.

It should be appreciated that one or more physical properties of a particle within a dry powder mixture (e.g., dry powder cell-culture medium) may improve the ability to determine the identity of the particle. For example, the particle' s morphology may be considered, along with Raman spectroscopic data, to determine the identity of a particle of the dry powder mixture. Considering the morphology in conjunction with Raman spectroscopic data may provide improved confidence of a given particle' s identity and may also increase the speed of analysis by requiring the analysis of fewer particles of the dry powder mixture (e.g., dry powder cell-culture medium) to confirm the presence or absence of a mixture component.

A chemical class or type often exhibits disparate particle morphology characteristics. For example, inorganic components (metal salts) will likely have very different morphology characteristics in comparison to amino acids and vitamins. Moreover, smaller molecules such as amino acids may have different characteristics compared to vitamins. Thus, multivariate analysis of both Raman spectra and the particle characteristics may increase the identification quality index among several classes of materials within a complex mixture and potentially within a class of material. That is, the combination of particle characteristics with Raman spectra affords an enhanced data rich data set for identification.

In some embodiments, the uniqueness of the particle morphology is determined. The uniqueness of the particle may be based on any suitable parameter that distinguishes the particle from any other particle in the dry powder mixture (e.g., dry powder cell-culture medium). In some embodiments, the uniqueness of a particle is based on the uniqueness of the Raman spectrum obtained from the particle. In some embodiments, the uniqueness of a particle is based on the uniqueness of a physical property (e.g., morphology) of the particle. As an example of morphological uniqueness among particles of a dry powder cell-culture medium, FIGs. 6A-6B show from 3 to 5 examples of the following particles: histidine, lysine-HCl, and valine (as shown in FIG. 6A); and inositol, biotin, and sodium chloride (as shown in FIG. 6B), showing the CE diameter values for each particle. It can be seen, for example, that the morphology of biotin is unique as compared to histidine, lysine-HCl, valine, inositol, and sodium chloride. Thus, biotin may have a high uniqueness score (e.g., a score of 1), for example where a score of 1 represents high uniqueness, and where a score of 10 represents low uniqueness. The morphological uniqueness score may be considered when determining the number of particles that need to be analyzed in order to reliably confirm the presence or absence of the particle in the dry powder mixture (e.g., dry powder cell-culture medium). In some embodiments, the uniqueness may be used in the following equation to determine the number of particles that need to be analyzed in order to reliably confirm the presence or absence of the particle in the dry powder mixture:

0.5a _A + 0.7β_Α + .9Θ_Α + 0.3μ_Α

Total Particle Count Requ ired =

0.5α_Μ + 0.Ίβ_Μ + 0.9Θ_Μ + 0.3μ_Α

Where

a is the component Raman spectrum selectivity or uniqueness index from 1 to 10 where 10 is low Uniqueness and 1 is high uniqueness.

β is the component Normalized Average Raman Signal Strength from 1 to 10 where 1 is high signal and 10 is low signal.

Θ is the components lowest Raman peak Signal to Noise ratio within the spectral library from 1 to 10 where 1 is high S/N and 10 is low S/N.

μ is the component particle morphology selectivity or uniqueness index where 10 is low uniqueness and 1 is high uniqueness

M= Total value (10) (worse case).

A= Actual value for the component.

P_c is the component particle concentration or component particle count within the Raman detectable particle size range.

This equation accounts for the estimated component particles available for Raman acquisition (Pc), Raman spectrum selectivity (a) relative to the spectra comprised within a Raman library, Raman spectrum and signal characteristics (β and Θ), and the particle morphology uniqueness (μ) to identify the target components within the material.

In some embodiments, a uniqueness index may be determined by k-Nearest Neighbor (KNN) statistics via principal component analysis (PCA). In some embodiments, KNN is the mean or average distance to the k-nearest neighbors in score space for a dataset, in this case the Raman spectral library dataset and the particle characteristics dataset. In some embodiments, KNN distance provides a uniqueness estimate for a sample, such as a component library spectrum or a component particle characteristics. In some embodiments, PCA and associated output statistics (e.g., KNN) can be determined by various commercially multivariate analysis software platforms such as PLS tool box and SIMCA supplied by Eigenvector Research (Manson, WA) and Umetric (San Jose, CA), respectively. However, additional methods for measuring a uniqueness index would be apparent to the skilled artisan and are within the scope of this disclosure.

In addition to considering the morphology of individual particles when determining the uniqueness of a particle, the methods provided herein may include assessing the distribution of physical properties for an individual component of the dry powder cell culture medium. For example, an individual culture component such as serine may have a size distribution profile that aids in the determination of whether serine is present or absent in a dry powder cell culture medium. Exemplary distributions of particle size (e.g., CE diameter) are shown for phenylalanine, vitamin B 12, serine, methionine, thiamine-HCl, cysteine-FFiO- HC1, L-tryptophan, L-aspartic acid, and biotin in FIG. 7. For these analyses, the morphology of more than 25,000 particles was analyzed. The graphs shown in FIG. 7 plot the CE diameter of each particle (μπι) versus the frequency that it occurs to create the histogram.

In some embodiments, the particle size distribution provides the user a general sense of how large and/or small the particles are for a given single component. In some embodiments, when the chemically defined media are analyzed, the individual particles may be grouped based on their Raman correlation. As an example, in FIGs. 3 A-3E, when separating the chemically defined media into classes, it is may be useful to refer back to the particle size distribution of the individual component. For example, the distribution of serine (FIG. 7) shows most of the particles having a CE diameter less than 10 μπι. If after examining the chemically classed particles of serine in a CDM, the particles classified as serine are typically > 100 μπι, which is unlikely based on the individual component profile, this may be cause for additional analysis. If the media then does not dissolve sufficiently, it could be determined that the serine particles are too large, as they are usually small.

Accordingly, in some embodiments, morphological analysis of one or more particles of a dry powder cell culture medium are performed to determine the quality of the media and/or the suitability of the media for use in a cell culture process.

In some embodiments, the methods provided herein are useful for determining one or more properties of the dry powder cell culture medium. For example, identifying individual components of a dry powder cell-culture medium (e.g., using Raman spectroscopy), and/or determining one or more morphological properties (e.g., CE diameter) of the individual particles within the dry powder cell-culture medium may be useful for determining a dissolution time of the dry powder cell-culture medium in a solvent. It should be appreciated that the size and/or shape of individual particles within the dry powder cell-culture medium may be used to determine the amount of time it takes for the medium to dissolve (e.g., dissolve completely) in a solvent, such as water. Alternatively, analysis of size and/or shape of the individual particles may be used to determine the amount of agitation or mixing required to dissolve the medium in a given amount of time. For example, in FIGs. 4A and 4B (discussed in the Examples section) it can be seen that dry powder cell-culture medium having a high mean CE diameter takes longer to dissolve in water than dry powder cell- culture medium having a lower mean CE diameter. Accordingly, in some embodiments, the methods provided herein include determining a dissolution time of a dry powder cell-culture medium. In some embodiments, determining the dissolution time is based at least in part on a mean value of one or more morphological properties of particles of the dry powder cell- culture medium. In some embodiments, the one or more morphological properties is area, aspect ratio, circular equivalent (CE) diameter, circularity, convex hull, convexity, elongation, high sensitivity (HS) circularity, intensity mean, intensity standard deviation (SD), length, major axis, maximum distance, perimeter, spherical equivalent (SE) volume, solidity, width, fiber elongation, fiber total length, fiber straightness, or fiber width. In some embodiments, determining the dissolution time of the dry powder cell-culture medium is based at least in part on a mean CE diameter value of particles of the dry powder cell-culture medium.

In some embodiments, the dry powder mixture (e.g., dry powder cell-culture medium) is dissolved in a solvent (e.g., water). The solvent, typically water, may be any suitable solvent to dissolve the dry powder cell-culture medium for use in a cell culture. In some embodiments, dry powder cell-culture medium is mixed in the solvent until the dry powder cell culture medium has completely dissolved in the medium. In some embodiments, the amount of time that the cell culture medium is mixed is based on an analysis of the dry powder cell-culture medium as provided herein. In some embodiments, the dry powder cell culture medium is mixed in the solvent for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 8 minutes, at least 10 minutes, at least 12 minutes, at least 14 minutes, at least 16 minutes, at least 18 minutes, at least 20 minutes, at least 30 minutes, or at least 60 minutes. It should also be appreciated that the amount of time that the dry powder cell-culture medium is mixed into the solvent may also vary depending on other parameters such as the solvent used, the composition of the media, the temperature, the amount of dry powder cell-culture media that is being dissolved in the solvent, and the scale of the culture. Exemplary volumes of solvent per 500 grams of dry powder cell-culture medium range from about 5 mL-100 mL. In some embodiments, 500 grams of dry powder cell culture media is dissolved in from about 10 mL-50 mL, of solvent. In some embodiments, 500 grams of dry powder cell culture media is dissolved in from about 25 mL-50 mL, of solvent. In some embodiments, the scale of the culture is from 1 L to 50000 L. For example, the final volume of dry powder cell culture-medium dissolved in solvent is from 1 L to 50000 L. In some embodiments, the scale of the culture is at least 1 L, at least 5 L, at least 10 L, at least 20 L, at least 50 L, at least 100 L, at least 500 L, at least 1000 L, at least 2500 L, at least 5000 L, at least 10000 L, at least 15000 L, at least 20000 L, at least 25000 L, at least 30000 L, at least 35000 L, at least 40000 L, at least 45000 L, or at least 50000 L. Exemplary mixing times, for example to dissolve the dry powder cell culture medium into the solvent, typically range from 1 minute to 12 hours. In some embodiments, the dry powder cell culture medium is added to the solvent and mixed for at least 1 minute, at least 5 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours.

In some embodiments, the herein-described methods may be used to select cell culture medium (e.g., a CDM) for a cell culture process. The cell culture may be, for example, for protein production (e.g., for antibody production, for example for humanized antibody production). In some embodiments, the cell culture may be that of a recombinant cell (e.g., bacterial, yeast, mammalian, or other cell type) that expresses a protein of interest. In some embodiments, a protein of interest may be a therapeutic protein. In some

embodiments, a protein of interest may be an antibody (e.g., a monoclonal antibody). In some embodiments, a protein of interest may be, but is not limited to, anti-LINGO, anti- LINGO-1, interferon (e.g., interferon beta la - AVO EX), Abciximab (REOPRO®), Adalimumab (HUMIRA®), Aducanumab, Alemtuzumab (CAMPATH®), Basiliximab (SF ULECT®), Bevacizumab (AVASTIN®), Cetuximab (ERBITUX®), Certolizumab pegol (CF ZIA®), Daclizumab (ZENAPAX®), Eculizumab (SOLIRIS®), Efalizumab (RAP T A®), Gemtuzumab (MYLOTARG®), Ibritumomab tiuxetan (ZEVALIN®), Infliximab (REMICADE®), Muromonab-CD3 (ORTHOCLO E OKT3®), Natalizumab (TYSABRI®), Omalizumab (XOLAIR®), Palivizumab (SYNAGIS®), Panitumumab (VECTIBIX®), Ranibizumab (LUCENTIS®), Rituximab (RITUXAN®), Tositumomab (BEXXAR®), and/or Trastuzumab (HERCEPTIN®). In some embodiments, the protein of interest is Aducanumab. In some embodiments, the protein of interest is Natalizumab (TYSABRI®). In some embodiments, the protein of interest is a blood cascade protein. Blood cascade proteins are known in the art and include, but are not limited to, Factor VII, tissue factor, Factor IX, Factor X, Factor XI, Factor XII, Tissue factor pathway inhibitor, Factor V, prothrombin, thrombin, von WillebrandF actor, kininigen, prekallikrien, kallikrein, fribronogen, fibrin, protein C, thrombomodulin, and antithrombin. In some embodiments, the blood cascade protein is Factor IX or Factor VIII. It should be appreciated that the methods are also applicable for uses involving the production of versions of blood cascade proteins, including blood cascade proteins that are covalently bound to antibodies or antibody fragments, such as Fc. In some embodiments, the blood cascade protein is Factor IX- Fc (FIXFc) or Factor VIII - Fc (FVIIIFc). In some embodiments, one or more proteins of interest are hormones, regulatory proteins and/or neurotrophic factors. Neurotrophic factors are known in the art and include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT -4), members of the glial cell line- derived neurotrophic factor ligands (GDNF) and ciliary neurotrophic factor (CNTF). In some embodiments, the protein of interest is neublastin. It also should be appreciated, that analytical techniques described herein can be applied to other materials or samples (for example for chemical syntheses) where one or more molecules (e.g., chemical stocks) are subject to isomerization which would alter their vibrational properties.

In some embodiments, a protein of interest may be, but is not limited to, 3F8, 8H9, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, aducanumab,

afelimomab, afutuzumab, alacizumab pegol, ALD, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anifrolumab, anrukinzumab (or EVIA-638), apolizumab, arcitumomab, aselizumab, atinumab, atlizumab (or tocilizumab), atorolimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cBR-doxorubicin immunoconjugate, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, concizumab, crenezumab, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravimmab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, IMAB, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lodelcizumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, margetuximab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab,

pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab,

rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab,

rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, SGN-CD19A, SGN-CD33A, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN, ticilimumab (or tremelimumab), tildrakizumab, tigatuzumab, TNX-650, tocilizumab (or atlizumab), toralizumab, tositumomab, tovetumab, tralokinumab, trastuzumab, TRBS, tregalizumab, tremelimumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vantictumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab and zolimomab aritox.

In some aspects, cell cultures are conducted in a bioreactor. A bioreactor refers to a vessel, including an open or closed vessel, for culturing one or more cells or organisms, or for maintaining or producing cellular components, including recombinant proteins. In some embodiments, a bioreactor is used for the production of a therapeutic protein (e.g., a recombinant protein, such as an antibody) by cultured cells. In some embodiments, bioreactors are made of corrosion resistant alloys, such as stainless steel (e.g., grade-316L stainless steel). However, in some embodiments, a bioreactor may be made of glass, ceramics, plastic, or any number of materials or combinations thereof. In some

embodiments, a bioreactor is configured with one or more supply lines for supplying nutrients, glucose, 0₂, C0₂, and other components to the bioreactor. In some embodiments, a bioreactor is configured with one or more output lines for removing waste or other components from the bioreactors. In some embodiments, a bioreactor is configured with one or more spargers for bubbling a gas (e.g., 0₂, C0₂) through a culture medium. In some embodiments, a bioreactor comprises one or more agitators or mixers for mixing a culture medium. In some embodiments, a bioreactor comprises one or more heating elements and one or more thermocouples configured to permit the temperature of the bioreactor culture to be controlling. In some embodiments, a bioreactor is configured with one or more spectroscopic instruments (e.g., a Raman spectroscopic instrument) configured for obtaining spectroscopic measurements on a culture.

In some embodiments, a bioreactor has a working volume (e.g., of culture medium) of at least 0.5 L, at least 1 L, at least 10 L, at least 100 L, at least 250 L, at least 500 L, at least 500 L, at least 1000 L, at least 2000 L, at least 3000 L, at least 4000 L, at least 5000 L, at least 7500 L, at least 10000 L, at least 12500 L, at least 15000 L, at least 20000 L, at least 100000 L, or more. In some embodiments, a bioreactor has a working volume in a range of 0.5 L to 10 L, 0.5 L to 100 L, 0.5 L to 500 L, 500 L to 1000 L, 500 L to 2500 L, 500 L to 5000 L, 500 L to 10000 L, 500 L to 15000 L, 500 L to 20000 L, 1000 L to 4000 L, 500 L to 100000 L, 2000 L to 5000 L, 2000 L to 10000 L, 2000 L to 15000 L, 2000 L to 20000 L, 2000 L to 100000 L, 15000 L to 20000 L, 15000 L to 100000 L, 20000 L to 50000 L, 20000 L to 100000 L, or 50000 L to 100000 L. In some embodiments, a bioreactor comprises a culture that produces or is capable of producing at least 1 gram, at least 10 grams, at least 100 grams, 500 grams, 1000 grams, 2000 grams, 3000 grams, or more of a recombinant protein. In some embodiments, a bioreactor culture produces or is capable of producing 1 gram to 10 grams, 1 gram to 100 grams, 1 gram to 500 grams, 10 gram to 1000 grams, 10 grams to 2000 grams, 100 grams to 1000 grams, 500 grams to 5000 grams, or more of a recombinant protein.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove.

EXAMPLES

Example 1

Multicomponent Compositional Analysis of Chemically Defined Media by

Morphologically Directed Raman Spectroscopy

CDM are complex multicomponent materials that are often characterized with various traditional analytical methods. While such modalities are effective, these methods fail to provide physical and chemical characteristics of the raw material in its native state. The G3ID provides morphological data and chemical identification on an individual particle basis. Morphology characteristics include various particle attributes (area, circularity, volume, convexity, solidity, etc.) for up to 100,000 particles.

FIGs. 1 A to 1C provide insight into the information gleaned from a typical analysis. A powder sample is added to the instrument and dispersed onto a glass slide using

pressurized air. The instrument scans the slide to view the entirety of the sample, and then collects an image of each particle and compiles a morphology profile. The Raman spectra of each selected particle (FIG. 1C, top trace) is then compared to a user-created library and correlation values are used to identify the material. FIG. 1 A shows a thinly dispersed sample followed by a 1 Ox microscope image of the particles. Particles may be selected on an individual basis or using an automated procedure which can scan all particles above a specified size threshold on the glass slide. The bottom image of FIG. 1A shows the variety of morphological profiles in which some particles are elongated and others are circular. As shown in FIG. IB, individual particles can be selected and the array of morphological data is shown for each. Particles can then either be manually or automatically selected for Raman analysis based on size and shape criteria. In FIG. 1C, the Raman spectra of the particle under investigation in FIG. IB is shown. The Morphologi® software compares the features within the analyte spectrum to the spectra in the user-generated database and shows correlations to each component within the library. Throughout the data collection process, no manual user analysis is necessary, although user interpretation is required. The user-generated library contains Raman spectra of each individual Raman active component within the CDM under investigation. To create this library, pharmaceutical-grade individual components contained within typical CDM were obtained from SAFC and used as received. The materials were tested for morphological profiles and a minimum of four Raman spectra were obtained for all relevant particle sizes and shapes. The collected spectra were averaged. To validate the G3- ID acquired Raman spectra, such spectra were compared against both a KnowItAll® Raman database and other independent Raman spectral references, before being added to a user- defined library. In the analysis, a threshold correlation score of 0.8 was used to assign an identity to the analyzed particle. For example, the Raman spectra of a selected particle in FIG. 1C (top trace) has a correlation value of 0.952 with the Raman spectra amino acid Serine (second trace from the top). Thus, the identity of the particle in 1C is Serine.

With the wealth of morphologic and spectroscopic information that can be gathered on each particle, sampling time should be considered. One of the major limiting factors of traditional chromatographic methods is the time it requires to analyze a sample. For a sample consisting of 50 components, sample preparation, instrumentation, and data analysis can require days of a skilled operator's time. The G3ID technology is advantageous because powder samples can be analyzed directly without the need for solvents nor special preparation. The analysis time varies depending on the components being analyzed;

however, data collection and analysis is automated. FIG. 2A shows a chemically defined media sample (Media A) measured at various times consisting of 45 components: 17 amino acids, 11 vitamins and 17 miscellaneous components including salts and other inorganic compounds.

Several lots of the mixed media powder were analyzed at the following time points: 0.5 hours, 1 hour, 8 hours, 24 hours, and 72 hours. Samples were analyzed to determine how many components were detected and divided into three groups: amino acids, vitamins and miscellaneous. As shown in FIG. 2A, all amino acids were observed within 8 hours, the quickest of all three categories. To capture the range of vitamins, a 24 hour scan was required. The remaining miscellaneous components required up to 72 hours to reliably detect. In a mixed media powder consisting of 10 pure amino acids (Media B), a scan time of one hour reliably detected all components. FIG. 2B demonstrates the system variability which affects the collection efficiency. Although there are some distinctive features that are known to reduce efficiency, the variability of each sample can make optimizing parameters challenging. Nevertheless, amino acids and vitamins are typically consistent and thus suitable to this analysis. It is important to note that, due to the small sampling volume and variations within particle size, the number of particles chemically classified into each category is not representative of the percentage of the component in the mixed media itself. This method solely provides whether a component is present as opposed to a quantitation of each component. Verifying the presence of a component allows the analyst to confirm that all components were included during vendor preparation of the CDM. For a comprehensive raw material strategy that requires high throughput of large quantities of data, the presence or absence of a component is often sufficient to consider the risk mitigated when screening mixed media.

Three criteria were identified to qualify a sample as appropriate for G3ID analysis; namely, particle size, Raman intensity, and concentration. The first criterion is that particles are sufficiently large. In some cases, the spot size for the Raman laser is 3 μπι; however, particles that were <5 μπι in diameter were excluded from the analysis method to allow for the greatest efficiency of data collection. Small particles, even those that are particularly abundant or materials that are very Raman active, will appear as noise if their size impedes the identification. The pin mill process by which chemically defined media A and B are manufactured typically produces a material with a uniform Gaussian distribution centered at 20 μπι but particle sizes in a formulation can range from 1 μπι to 200 μπι. Raman intensity is material-dependent. Chemical identification is only possible with components that are Raman-active, although morphology profiles can be obtained for active and non-Raman- active compounds. Because chemically defined media samples frequently contain inorganics and salts that are not Raman active, these are not suitable for G3ID analysis. If these particular materials have a distinctive morphology, it is possible to approximate their presence; however, this method is not nearly as reliable as chemical identification. Finally, concentration is an important factor which is interrelated with the other two criteria. Lengthy extended acquisition times (e.g., 3 days), as shown in FIG. 2A, will contribute to identifying particles in low concentrations; however, if the less abundant components are also small or do not have distinct Raman spectra, the probably of detection drastically decreases.

Using Media B, which contained 10 amino acids that were all reliably detected, Raman active, approximately 20 μπι of CE diameter, and present in sufficient concentration, limit of detection studies were performed. The media was diluted to various concentrations and scans were run for 72 hours to ensure detection to the lowest levels. It was determined that the formulations diluted 1000-fold reduced the concentrations of several amino acids to 0.02% wt/wt and were reliably detected in all measurements. At lower concentrations, consistency was sacrificed; thus, it is recommended that this instrument is used to detect components in concentrations greater than 0.02%.

Chemically defined media are predominantly composed of amino acids and vitamins, most of which are Raman active and thus an ideal candidate for G3ID analysis. Although liquid chromatography-mass spectrometry (LCMS) can also be used to detect amino acids and vitamins, the G3ID is advantageous in that it supplements chemical characterization with morphology. Six representative amino acids and vitamins are shown in FIG. 2B. In each of three experiments, all the data showed a greater than 80% match.

Despite consistency within the percentage identified, each of the materials had a characteristic morphology profile. This level of detail can only be determined using a G3ID instrument. Within a raw material characterization strategy, key parameters such as the mean circular equivalent diameter as well as the 10^th, 50^th, and 90^th percentile of particle sizes are monitored across samples. If there is variation within any of the tracked parameters, examining the morphology data in depth as in FIGs. 3 A-3E may illuminate the differences at the particular level. As shown, particle size distributions measurements were obtained for individual components, including asparagine (FIG. 3A), proline (FIG. 3B), tryptophan (FIG. 3C), serine (FIG. 3D), and inositol (FIG. 3E). The variability within the particle size can be further extrapolated into biologically relevant factors. For example, this kind of information has been useful when determining solubility issues within the mixed media. As the basal and feed media are used in high concentrations within a biopharmaceutical production, it is essential that the raw materials are as consistent as possible. Understanding the bulk material and individual components more thoroughly allows for better process control and easier troubleshooting. To that end, media dissolution studies were performed to quantitatively examine the role of particle size in dissolution.

As shown in FIG. 4A, three CDM vendors were analyzed for morphology profiles. If the mean CE diameter and 50^th percentile overlap, the distribution is ideally Gaussian.

Vendors 2 and 3 demonstrate comparable particle size distributions of materials; however, Vendor 1 demonstrates a lower average particle size and tighter distribution. Each media sample was dissolved in the concentrations specified for cell culture and monitored using a Stratophase RI probe. The results of the dissolution are shown in FIG. 4B. As anticipated, Vendor 1 which had the smallest particle size and smallest particle size distribution was the quickest to dissolve. Vendor 3 which had the largest average particle size and largest distribution of particle sizes was the slowest to dissolve. Using morphology to relate size and dissolution information could be particularly useful when working with very concentrated chemically defined media formulations and solubility issues are a concern. Further forensic studies can be performed to extract undissolved particles which can then be chemically identified using the G3ID. Media formulations can be subsequently optimized to account for slowly dissolving particles.

As a case study, some deficient lots of the chemically defined media were tested. When testing lots of the chemically defined media with asparagine H₂0 known to be missing, zero particles of asparagine H₂0 were found and classified. Because Asparagine H₂0 has been demonstrated to consistently appear in the formulation in approximately 4% of each sample, the lot could be flagged to indicate subsequent analysis would be needed prior to use.

In another case study, even if the specific component was not one being monitored, if the component is present in a sufficiently high concentration, it could raise a red flag. When comparing glucose containing versus glucose-free media, the glucose is present at a concentration of close to 40%. As a result, if this component is missing, it drastically changes the percentages of other components. When analyzing a glucose-free lot, the other components were present in higher percentages than typically observed. As a result, an operator would most likely notice the inconsistent increase across components and be able to recognize the suspicious lot. Although these are two specific cases for the mixed media studied in this set of experiments, selecting the correct individual components to monitor can define the robustness of the analytical method.

The G3ID technology is well suited to the analysis of chemically defined media that are predominantly composed of Raman active amino acids and vitamins of sufficiently large size and concentration. The instrument is capable of chemically identifying components present in concentrations as low as 0.02% and with scan times as quick as one hour. This technique is advantageous in that in additional to chemical identification, it provides morphology information about each particle within a sample. Size and shape information can be insightful when considering the dissolution of media and can be instrumental when coupled with chemical identification when investigating solubility issues.

Materials and Methods

Samples

Two chemically defined media (CDM) (Media A and B) were provided by SAFC (Kansas, USA). Media A contained 45 components (17 amino acids, 17 vitamins, and 11 miscellaneous constituents) whereas Media B contained ten amino acids. Prior to analysis, a 200 mg solid sample was loaded into the G3-ID high pressure sample dispersion chamber. Samples were then dispersed onto a glass plate (180x110 mm) on a motorized state at 4 bar pressure with a settling dispersion time of 60 seconds.

To determine the limit of detection, blended samples of Media B were prepared with pharmaceutical grade Asparagine H₂0 (Catalogue No. : 5794-13-8 SAFC, USA) as the diluent. 200 gram samples were mixed using a LabRamll Resonant Acoustic® Mixer (Resodyn, Montana, USA) with a mixing intensity of 60g and mixing time of 60 seconds.

Instrument Configuration

The G3-ID instrument was equipped with a Raman Rxnl spectrometer (Kaiser Optical Systems, Inc. USA) and a CFI 60 brightfield/darkfield microscope (Nikon

Corporation, Japan). The Raman spectrometer contained a 785 nm excitation laser source and a CCD detector. G3-ID morphological data was acquired with a lOx objective whereas Raman spectra was obtained with the 50x objective and an excitation spot size of 3 μιη.

The data acquisition protocol was created and optimized within the Morphologi® software (ver. 8.20). Morphological characteristics were obtained with a diascopic light source with an automatic light calibration intensity of 80.0% ± 0.2. Samples were scanned using a lOx objective with manual focus, no Z stacking, plate tilt compensation, and a binary threshold to a grayscale value of 115. Morphology analysis was performed within the center of the plate and particles were sorted into categories: <5 μπι, 5-50 μπι, and >50 μιη. Raman analysis was performed on all particles with a CE diameter greater than 5 μιη. Using the XY coordinates determined in the morphology scan, a Raman spectrum of each particle was acquired. Good quality Raman spectra (100 - 2000 cm^"1) were obtained using an exposure time of 10 seconds and 1 co-adds. No additional preprocessing or background subtraction settings were applied.

Data Analysis

Chemical identification of components within a target CDM was achieved with the developed individual component Raman library. Chemical identification was performed for each particle within a range of 5 to 400 μπι and the full Raman spectrum. A software match score was assigned to each particle with 0 representing no match and 1.0 representing an identical match. A best match correlation to the specific library component greater than 0.8 grouped each particle into a chemical class. After analysis, a report was automatically generated. The report includes all morphology parameters for all particles tested and an assignment to chemical class for the particles where a Raman spectrum was obtained.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., "comprising") are also contemplated, in alternative embodiments, as "consisting of and "consisting essentially of the feature described by the open-ended transitional phrase. For example, if the disclosure describes "a composition comprising A and B", the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B".

That which is claimed is:

Claims

1. A method for evaluating a dry powder cell-culture medium, the method comprising

(i) dispersing the dry powder cell-culture medium on a substrate; and

(ii) obtaining a Raman spectrum from each of a plurality of particles of the dry powder cell-culture medium.

2. The method of claim 1, wherein the dry powder cell-culture medium is comprised of up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, or up to 70 individual components.

3. The method of claim 1 or 2, wherein the dry powder cell-culture medium is comprised of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 individual components.

4. The method of claim 2 or 3, wherein each of the plurality of particles consists essentially of a single component.

5. The method of any one of claims 2-4, wherein at least one of the individual components is an amino acid.

6. The method of claim 5, wherein the amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

7. The method of claim 5 or 6, wherein at least 2, at least 5, at least 10, at least 15, or at least 20 of the individual components are amino acids.

8. The method of any one of claims 2-7, wherein at least one of the individual components is a vitamin.

9. The method of claim 8, wherein the vitamin is selected from the group consisting of Biotin, Calcium Pentothenate, Choline Chloride, Cyanocobalamin, DL Alpha Lipoic Acid, Inositol, Thiamine HC1, PABA, Riboflavin, Folic Acid, and Niacinamide.

10. The method of claim 8 or 9, wherein at least 2, at least 5, at least 10, at least 15, or at least 20 of the individual components are vitamins.

11. The method of any one of claims 2-10, wherein at least one of the individual components is a nutrient.

12. The method of claim 11, wherein the nutrient is selected from the group consisting of glucose, fructose, and sucrose.

13. The method of claim 11 or 12, wherein at least 2, at least 3, at least 4, or at least 5 of the individual components are nutrients.

14. The method of any one of claims 2-13, wherein at least one of the individual components is a buffer.

15. The method of claim 14, wherein the buffer is HEPES.

16. The method of claim 14 or 15, wherein at least 2, at least 3, at least 4, or at least 5 of the individual components are buffers.

17. The method of any one of claims 1-16, wherein the dry powder media is IMDM, Dulbecco's Modified Eagle's medium (DMEM), DMEM/F12, Ham's F-10, Ham's F-12, Medium 199, Minimum Essential Medium (MEM), or Roswell Park Memorial Institute (RPMI) 1640.

18. The method of any one of claims 1-17, wherein the substrate is compatible with Raman spectroscopy.

19. The method of any one of claims 1-18, wherein the substrate comprises glass or plastic.

20. The method of any one of claims 2-19, wherein at least one of the individual components is Raman active.

21. The method of claim 20, wherein at least 2, at least 5, at least 10, at least 20, or at least 30 of the individual components are Raman active.

22. The method of any one of claims 1-21, wherein the dry powder cell-culture medium is dispersed on a substrate at a density 80 particles per mm² or less, 60 particles per mm² or less, or 50 particles per mm² or less.

23. The method of any one of claims 1-22, wherein a Raman spectrum is obtained from at least 10, at least 100, at least 500, at least 1000, at least 5000, or at least 10000 individual particles of the plurality of particles of the dry powder cell-culture medium.

24. The method of any one of claims 1-23, wherein at least one dimension of each of the plurality of particles is at least 3 μιτι, at least 4 μιτι, at least 5 μιτι, at least 6 μιτι, at least 8 μιτι, at least 10 μιτι, or at least 20 μιη.

25. The method of any one of claims 1-24, wherein the method further comprises

(iii) identifying at least 1 physical property of a plurality of particles of the dry powder cell-culture medium.

26. The method of claim 25, wherein step (iii) comprises identifying at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 physical properties of the plurality of particles of the dry powder cell-culture medium.

27. The method of claim 25 or 26, wherein the physical property is area.

28. The method of claim 25 or 26, wherein the physical property is circularity.

29. The method of claim 25 or 26, wherein the physical property is volume.

30. The method of claim 25 or 26, wherein the physical property is convexity.

31. The method of claim 25 or 26, wherein the physical property is solidity.

32. The method of claim 25 or 26, wherein the physical property is selected from the group consisting of circular equivalent (CE) diameter, HS circularity, aspect ratio, elongation, length, width, fiber elongation, fiber straightness, fiber width, perimeter, and sphere equivalent volume.

33. The method of any one of claims 25-32, further comprising determining a dissolution time of the dry powder cell-culture medium.

34. The method of claim 33 comprising contacting the dry powder cell-culture medium with a solvent, thereby forming a liquid cell culture medium.

35. The method of claim 34, wherein the solvent comprises water.

36. The method of claim 34 or 35 comprising mixing the liquid cell culture medium until the dry powder cell-culture medium has dissolved into the solvent.

37. The method of claim 36, wherein the liquid cell culture medium is mixed for at least 8, at least 9, at least 10, at least 11, at least 12, or at least 15 minutes.

38. The method of any one of claims 1-37, wherein the method further comprises (iv) comparing the Raman spectrum obtained in (ii) to an appropriate standard and determining if an individual component is present in the dry powder cell-culture medium.

39. The method of claim 38, wherein the appropriate standard is a user generated library comprising a Raman spectrum of at least one individual component that is present in the dry powder cell-culture medium.

40. The method of claim 39, wherein the user generated library comprises a Raman spectrum of at least 2, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70 individual components of the dry powder cell-culture medium.

41. The method of any one of claims 38-40, wherein at least 2, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70 Raman spectrum are obtained from each of the individual components of the dry powder cell-culture medium and determining whether each of the individual components of the dry powder cell-culture medium are present.

42. The method of any one of claims 38-41, further comprising using the dry powder cell- culture medium in a cell culture process if each of the individual components of the dry powder cell-culture medium are present and not using the dry powder cell-culture medium in a cell culture process if each of the individual components of the dry powder cell-culture medium are not present.

43. A method of conducting cell culture using a dry cell-culture medium, the method comprising:

obtaining a Raman spectrum from each of a plurality of particles of the dry power cell-culture medium;

comparing the Raman spectrum to an appropriate standard and determining if one or more individual components are present in the dry cell-culture medium; and

using the dry cell-culture medium in a cell culture process if the one or more individual components are present in the dry cell-culture medium.

44. The method of claim 43, wherein the dry cell-culture medium comprises up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, or up to 70 individual components.

45. The method of claim 43 or 44, wherein the dry cell-culture medium is comprised of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 individual components.

46. The method of any one of claims 43-45, wherein the dry cell-culture medium is used in the cell culture process if it is determined that at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the individual components of the dry cell-culture medium are present.

47. The method of any one of claims 43-46, wherein the cell culture process is conducted under conditions that permit production of a protein of interest.

48. The method of claim 47, wherein the protein of interest is a therapeutic protein.

49. The method of claim 48, wherein the therapeutic protein is an antibody.

50. The method of claim 49, wherein the antibody is a monoclonal antibody.