Process for concentration of antibodies and therapeutic products thereof

DESCRIPTION

PROCESS FOR CONCENTRATION OF ANTIBODIES AND THERAPEUTIC PRODUCTS THEREOF

Background

Methods for isolating, purifying and concentrating biological materials are known and include, for example, chromatography, ultrafiltration and lyophilization, see generally R. Hatti-Kaul et al., Downstream Processing in Biotechnology, Basic Biotechnology, Cap 9, pages 187-211, 2^The ed., Cambridge University Press (2001). Processes for making concentrated monoclonal antibody preparations for administration to humans are known, see, for example, US Patent No. 6,252,055, which uses ultrafiltration and recirculating the resulting filtrate.

Some challenges associated with available antibody concentration methods include, for example, low fluxes, long process times, large membrane areas, mechanical recovery yield and losses, intensive operator intervention or manipulation, and low mass transfer rates, inefficiencies. and pressure limits on the concentration equipment. These and other challenges can contribute to a high total cost of manufacture and ultimately to a high cost to therapeutic drug users.

There is a need to improve processes for the preparation of highly concentrated protein formulations, such as antibody liquid preparations and therapeutic products thereof.

ΕΡ1786830Β1

summary

In general terms, the present disclosure generally relates to processes for concentrating proteins, such as processes for concentrating an antibody preparation, pharmaceutical formulations containing such a preparation and their use in human or animal therapy.

In embodiments, the present disclosure provides processes for preparing highly concentrated proteins, such as antibody preparations according to the invention as defined in the claims; and therapeutic products prepared by the process, such as therapeutic antibody products. Accordingly, the present disclosure provides a process for protein concentration which comprises

<td>agreement c</td><td>om invention</td><td colspan="2">: a first</td><td colspan="2">ultrafiltration of</td><td>an</td>

<td>first</td><td>preparation</td><td>in</td><td>antibody</td><td>for</td><td>provide</td><td>an</td>

<td>Monday</td><td>preparation</td><td>in</td><td>antibody;</td><td>an</td><td>diafiltration</td><td>gives</td>

<td>Monday</td><td>preparation</td><td>in</td><td>antibody</td><td>for</td><td>provide</td><td>an</td>

diafiltered intermediate antibody preparation; and a second ultrafiltration of the diafiltered intermediate antibody preparation to provide a third antibody preparation, wherein one or more of the first ultrafiltration, second ultrafiltration, and diafiltration are accomplished at elevated temperatures, for example, about 30 ° C. at about 50 ° C, and as defined in the claims.

The present disclosure also provides, in embodiments of the invention, a process for protein concentration comprising: a first ultrafiltration of a first protein mixture to provide a second protein mixture; diafiltration of the second protein mixture to provide a diafiltered protein mixture; and a second ultrafiltration of the diafiltered protein mixture to provide a

This is a third protein blend, whereby one or more of the first ultrafiltration, diafiltration, and second ultrafiltration are achieved, for example, at about 45 ° C as defined in the claims.

The present disclosure also relates to a highly concentrated antibody composition prepared by the above processes.

Brief Description of the Figures

FIG. 1 illustrates an apparatus for performing the preparative process in the embodiments of the present disclosure.

FIGS. 2 through 17 illustrate various process values

<td>measured</td><td>or</td><td>observed from</td><td>multiple stages</td><td>or modes</td><td>in</td>

<td>process,</td><td colspan="2">in the ways of</td><td>realization</td><td colspan="2">of the present</td>

<td>disclosure</td><td> •</td><td></td><td></td><td></td><td></td>

<td>FIGS.</td><td> 18</td><td>and 19 illustrate</td><td>the effect of</td><td colspan="2">temperature</td>

<td colspan="2">high in</td><td>quality of</td><td>product in</td><td>forms</td><td>in</td>

<td>realization</td><td>gives</td><td colspan="2">present disclosure.</td><td></td><td></td>

<td>FIGS.</td><td> 20</td><td>and 21 illustrate</td><td>the effect of</td><td colspan="2">temperature</td>

<td colspan="2">high over</td><td>the control of</td><td colspan="2">bioburden, in the forms</td><td>in</td>

<td>realization</td><td>gives</td><td colspan="2">present disclosure.</td><td></td><td></td>

FIG. 22 illustrates the effect of elevated temperature on the flow process and the time process on the embodiments of the present disclosure.

FIGS. 23 to 25 illustrate various measured or observed multi-step process or scaling process values ​​in the embodiments of the present disclosure.

Detailed Description

The various embodiments of the present disclosed herein will be described in detail with

ΕΡ1786830Β1 reference to drawings, if any. Reference to the various invention forms, which claims the scope of the scope of the document.

achievement is not limited only by the attached to this

Additionally, any example set forth in this specification is not intended to be limiting and merely determines some of the many possible embodiments for the claimed invention.

The following are used unless otherwise described:

Ultrafiltrate, 'UF, ultrafiltration, ultrafiltrate,' and similar terms refer to, for example, discriminating between molecules in the mixture, primarily based on size and molecular form, and separating different or performing concentration using synthetic membranes physical and chemical properties molecules appropriate semipermeable molecules. Diafiltering, diafiltering, for example, concentration of similar with diafiltered, diafiltered

DF, and similar terms refer to the removal, substitution or reduction of salts or solvents of solutions or mixtures containing proteins, peptides, nucleic acids or other biomolecules using an ultrafiltration membrane.

Transmembrane Pressure or PTM refers to the mean applied pressure from the feed to the membrane filtrate side calculated as PTM [bar] = [(P_F + P_R ) / 2] - P_f where P_F is the supply pressure, the P_R is the back pressure and Pf is the filtrate pressure.

Tangential flow filtration, cross flow filtration, TFF, as terms refer to a filtration mode wherein the solute-containing solution passes tangentially through the UF membrane and low molecular weight salts or solutes pass completely through.

ΕΡ1786830Β1 the application of pressure.

The term antibody is used in the broadest sense and specifically covers intact monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragment as long as they exhibit desired biological activity. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. Described in terms of its structure, an antibody is a Y-shaped protein consisting of four amino acid chains, two light chains and two heavy chains. In a sufficiently simplified model for this appeal, each antibody first has two regions: a variable region and a constant region. The variable region, located at the ends of the Y arms, binds and interacts with the target antigen. This variable region includes a complementarity determining region (CDR) that recognizes and binds the antibody to a specific binding site on a particular antigen. The constant region, located at the tail of Y, is recognized by and interacts with the immune system (Janeway, C., Travers, P., Walport, M., Shlomchik (2001). Immuno Biology, 5^The Ed, Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody.

The basic unit of the 4 chain antibody is a heterotetrameric glycoprotein composed of two identical light chains (1) and two identical heavy (H) chains (an IgM antibody consists of 5 of the heterotetrameric basic units together with an additional polypeptide called the J chain, and therefore contains 10 sites of

Antigen binding, while secreted IgA antibodies can polymerize to form pooled polyvalents containing from 2 to 5 basic 4-chain units along with the J-chain). In the case of IgGs, the 4-chain unit generally has about 150,000 Daltons. Each 1 chain is linked to one H chain by a covalent disulfide bond, while the two H chains are linked by one or more disulfide bonds depending on the H chain isotype. Each light and heavy chain also contains regularly spaced intrachain disulfide bridges. Each H string contains at the N-terminal a variable domain (V_H) followed by a series of constant domains (C_H) for each of the α and γ chains and four constant domains C_H for the isotypes μ and ε. Each chain 1 contains, at the N-terminal, a variable domain (V_L) followed by a constant domain (C_L) at its other end. OV_L is aligned with the V_H and the C_Laligned with the first heavy chain constant domain (C_H1) . Particular amino acid residues are believed to form a relationship between the light chain and the heavy chain variable domains. Pairing a V_H and V_Ltogether form a single antigen binding site. For the structure and properties of the different antibody classes, see, for example, Basic and Clinical Immunology, 8^The edition, D. Stites, A. Terr and T. Parslow (eds.), Appleton &amp; Lange, Norwalk, CT, 1994, page 71 Chapter 6.

Chain 1 of any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C_H), immunoglobulins may be assigned to different classes or isotypes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM having the heavy chain named α, δ, ε, γ and μ,

ΕΡ1786830Β1 respectively. Classes γ and α are further subclassed on the basis of relatively minor differences in sequence and C function._H, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2.

The term variable refers to the fact that certain segments of the variable domains have vastly different sequences between antibodies. Domain V mediates antigen binding and defines the specificity of a specific antibody for its specific antigen. However, variability is not evenly distributed over the 110 amino acid series of the variable domains. In contrast, V regions consist of relatively invariable segments called 15- to 30-amino acid framework regions (FRs) separated by shorter regions of extreme variability called hypervariable regions that are 9 to 12 amino acids in length. Native light and heavy chain variable domains comprise 4 FRs each, largely adopting β-leaf configuration, connected over 3 hypervariable regions, which form loops that connect and, in some cases, form part of the β-leaf structure. . The hypervariable regions of each chain are held together in close proximity by the FRs and, with the hypervariable regions of the other chain, contribute to the formation of antibody antigen binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in the binding of an antibody to the antigen, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC).

The term hypervariable region, when used herein, refers to the amino acid residues of a

ΕΡ1786830Β1 antibodies that are responsible for binding of antigens. The hypervariable region generally comprises amino acid residues of a complementarity determining region or CDR (e.g., around residues 24 to 34 (L1), 50 to 56 (L2) and 89 to 97 (L3) in V_L, and about 31 to 35 (Hl), 50 to 65 (H2) and 95 to 102 (H3) at V_H (see Kabat et al., supra) and / or those hypervariable loop residues (e.g., around Chothia 26-32 (L1), 50-52 (L2) and 91-96 residues). (L3) on V_L, and 26 to 32 (H1), 52A to 55 (H2) and 96 to 101 (H3) at V_H (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)).

The term monoclonal antibody as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, individual antibodies comprising the population are identical and / or binding to the same epitope (s) except for possible variations that may occur during monoclonal antibody production, which may be present in minor amounts. Such a monoclonal antibody typically includes an antibody consisting of a target binding polypeptide sequence, wherein the target binding polypeptide sequence has been obtained by a process that includes selecting a single target binding polypeptide sequence from a target. a variety of polypeptide sequences. For example, the selection process may be selecting a single clone from a variety of clones, such as a set of hybridoma clones, phage clone or recombinant DNA clone. It should be understood that the selected target binding sequence may additionally be altered, for example, to improve target affinity, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vitro. in vivo to create a multispecific antibody, etc .; and that an antibody that

No. 1786830Β1 in the altered target binding sequence is also a monoclonal antibody in the present invention. In contrast, to polyclonal antibody preparations that typically include antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically not contaminated by other immunoglobulins. The monoclonal adjective indicates the character of the antibody to be obtained from a substantially homogeneous antibody population and should not be construed as requiring antibody production by any specific method. For example, monoclonal antibodies to be used according to the present invention may be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256: 495 (1975)). Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2^The Ed. 1988); Hammerling et al. In: Monoclonal Antibodies and T-Cell Hybridomas, 563-681, (Elsevier, NY, 1981)), recombinant DNA methods (See, e.g., US Patent 4,816,567), phage library technology. (see, for example, Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol Biol, 222: 581-597 (1991); Sidhu et al., J. Mol. Biol 338 (2): 299-310 (2004), Lee et al., J. Mol. Biol.340 (5): 1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284 (1-2): 119-122 (2004), and technologies for producing or similar human antibodies in animals having parts or all of the loci or genes encoding human immunoglobulin sequences (see, for example) WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits, et al.

ΕΡ1786830Β1

Proc. Nail Acad. Know. USA, 90: 2551 (1993); Jakobovits, et al., Nature, 362: 255-258 (1993); Bruggemann, et al. , Year in Immuno., 7:33 (1993); US Patent 5,545,806; US Patent 5,569,825; US Patent 5,591,669 (all to GenPharm); US Patent 5,545,807; WO 1997/17852; US patent

5,569,825; US Patent 779-783 (1994); et al.,

5.661.016;

(1992); Lonberg, Morrison, Nature.

5,545,807; US Patent 5,545,806; US Patent US Patent 5,625.26; US Patent 5,633,425; and

Marks, et al., Bio / Technology, 10:

et al., Nature. 368: 856-859 368: 812-813 (1994); Fishwild,

Nature Biotechnology. 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intem. Rev. Immunol., 13: 65-93 (1995).

Chimeric antibodies (immunoglobulins) wherein a portion of the light and / or heavy chain is identical to or homologous to corresponding sequences in antibodies derived from a specific species, or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from other species or belonging to another antibody class or subclass, as well as fragments of such antibodies, to exhibit the desired biological activity (US Patent 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Humanized antibody as used herein is a subset of chimeric antibodies.

Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues from a hypervariable region of the patient are replaced by residues from a hypervariable region from a nonhuman species (donor antibody) such as mouse, rat, rabbit or primate. human being that has the specificity, affinity and

Capacidade1786830Β1 desired capacity. In some cases, framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the receptor antibody or donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are to one. human immunoglobulin sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. 0 The number of these amino acid substitutions in the FR region is typically no more than 6 in the H chain, and no more than 3 in the 1 chain. The optionally humanized antibody will also consist of at least a portion of an immunoglobulin constant region (Fc), typically that of an human immunoglobulin Jones, et al.

For more details, see

Nature 321

522-525 (1986), Reichmann, et., And Presta, Curr. Op. Struct al., Nature 332: 323-329 (1988);

Biol. 2: 593-596 (1992).

Antibody fragments comprise an intact antibody moiety, preferably the antigen binder or the variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; diabodies; linear antibodies; (see US Patent 5,641,870, Example 2; Zapata, et al., Protein Eng., 8 (10): 1057-1062 (1995)); single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen binding fragments called

861786830Β1 Fab fragments, each with a single antigen binding site, and a residual Fc fragment, the name of which reflects its ability to rapidly crystallize. The Fab fragment consists of an entire strand 1 together with the H chain variable domain region (V_H), and the first heavy chain constant domain (C_H1) . Each Fab fragment is monovalent with respect to antigen binding, that is, it has a single antigen binding site. Pepsin treatment generates an F (ab ') 2 fragment which corresponds to about two disulfide-linked Fab fragments which have divalent antigen binding activity and is still capable of cross-linking antigen. Fab fragments differ from Fab fragments having additionally few residues in the carboxy-terminal region of the C domain._H1 including one or more cisterns of the antibody hinge region. Fab'SH is the term for Fab 'in which the cistern residue (s) of the constant domains carry a free thiol group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cisterns between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxyterminal portions of both H-chains joined by disulfide bridges. Antibody effector functions are determined by sequences in the Fc region, which is also recognized in part by Fc receptors (FcR) found in certain cell types.

Fv is the minimal antibody fragment that contains a complete antigen recognition and binding site. Said fragment consists of a dimer of a light chain and heavy chain variable region domain in strong, non-covalent association. From the fold of these 2 domains emanates 6 hypervariable loops (3 loops of the H chain and

ΕΡ1786830Β1 L chain loops) which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen specific CDRs) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

Single-chain Fv or scFv antibody fragments comprise antibody VH and VL domains, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V domains._H and V_L which allows scFv to form the desired antigen binding structure. For scFv analysis, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, United States, p. 269-315 (1994).

It modifies, for example, the amount of an ingredient in the compositions, concentration of an asset, buffer volume, diavolumes, pore size, molecular appearance, decrease in molecular weight, process temperature, process time, yields, rates. flow rates, pressures, bioburden, and values ​​and scales thereof used in the methods of the invention refer to the variation in the numerical amount that may occur, for example, with typical measurement and handling procedures used in the production of concentrates or solutions for use; through inadvertent error in these procedures; by differences in the manufacture, source, or purity of the ingredients used to make the compositions or to perform the methods; and as considerations. The term also encompasses amounts that differ over time from a composition with a particular initial concentration or mixture. 0

The term about also encompasses the amounts that differ due to mixing or processing a composition with an initial concentration or mixture. Whether or not modified by the term about the claims include equivalents to the amounts.

It consists essentially of the process of obtaining a concentrated protein composition or antibody composition comprising steps and ingredients listed in the claim, and other steps and ingredients that do not substantially affect the basic and original properties of the composition, such as as the multiplicity of steps or buffer medium. Ingredients that substantially affect the basic properties of the composition and method of the present disclosure give undesirable characteristics including, for example, bioburden, such as undesirable toxicity or irritability associated with contaminants.

Undefined article one or one and its definitive article corresponding to it as used herein are understood to designate at least one or more, unless otherwise specified.

The present disclosure provides, in embodiments, the above-mentioned processes of the invention as defined in the claims and the concentrated antibody products thereof.

In the embodiments of the present disclosure, the preparative processes and products thereof may be used in making highly concentrated antibody preparations and similar preparations, such as purifying and concentrating proteins or similar substances from natural or synthetic sources, and such Products may be useful in treating pathological conditions such as asthma, cancer, psoriasis, inhibiting angiogenesis and unknown pathological conditions.

In the embodiments of the above process

No. 1786830Β1 for preparing highly concentrated antibody compositions of the disclosure, follow additional examples of how to make and use the preparation processes and products of the disclosure.

In embodiments of the present disclosure, a process is provided for preparing highly concentrated antibody compositions, for example, according to performing the following steps in the listed order, comprising:

a first ultrafiltration of a first antibody preparation having a concentration of, for example, about 0.1 to 10 grams per liter (g / l) to provide a second antibody preparation such as retentate having a higher concentration of antibody, for example, from about 10 to 50 grams per liter;

a diafiltration of the resulting second antibody preparation to provide a diafiltered intermediate antibody preparation such as the retentate having a concentration about the same as the resulting second antibody preparation retentate, i.e. diafiltration to perform a volume buffer exchange constant; and a second ultrafiltration of the diafiltered intermediate antibody preparation to provide a third antibody preparation such as the retentate having a higher antibody concentration, for example from about 150 to 200 grams per liter.

The process for preparing the disclosure may further comprise an optional product recovery step or steps, for example as disclosed and illustrated herein.

In embodiments of the above-mentioned process of the disclosure, one or more of the first ultrafiltration, diafiltration, and second ultrafiltration may be performed, for example, from about 30 ° C to 70 ° C. Nas

In embodiments, these steps may also be performed, for example, from about 30 ° C to 50 ° C. In embodiments, these steps may also be performed, for example, from about 35 ° C to 50 ° C. In embodiments, these steps may also be performed, for example, at about 45 ° C, such as about 45 ° C or so at about 5 ° C. Depending on the type of antibody preparation above, for processes performed at temperatures above about 70 ° C, the preparation may show signs of deterioration, such as denaturation, agglomeration and similar phenomena. For processes performed at temperatures below about 30 ° C to 35 ° C, the flow rates are undesirably low and the process time is undesirably long, making the low temperature process less attractive for efficient commercial production.

The first antibody preparation may have an antibody concentration of, for example, from about 0.1 to about 100 grams per liter (g / l). Antibody concentration is, for example, a common concentration typically available from other steps or preliminary methods of protein or antibody purification, such as centrifugation, filtration, chromatography and the like. The resulting second antibody preparation obtained from the first ultrafiltration may have an antibody concentration, for example from about 10 to about 50 grams per liter, for example from about 20 to 40 grams per liter, such as 30 grams per liter. liter. A scale for the antibody concentration of the intermediate antibody preparation may depend, for example, on a balance of factors such as sample volume and sample flow obtained with a special buffer containing the second antibody preparation. The intermediate antibody preparation may have an antibody concentration of, for example, from about 25 to about 35 grams per liter and the

The third antibody preparation may have an antibody concentration, for example, from about 170 to about 200 grams per liter. The third antibody preparation, in embodiments, may have a concentration, for example, of from about 50 to about 250 grams per liter of antibody, such as from about 100 to 230 grams per liter, and from about 170 to about 250 grams per liter. 200 grams per liter, such as

<td>185 grams</td><td>per liter.</td><td></td><td></td><td></td>

<td>It will be</td><td>evident to</td><td>an expert on</td><td colspan="2">specialty,</td>

<td>understand</td><td>at present</td><td>disclosure which</td><td>the preparation</td><td>in</td>

<td>antibodies</td><td>intermediate</td><td>and the third</td><td>preparation</td><td>in</td>

<td>antibodies</td><td>understand the</td><td>even retained</td><td>ultrafiltered</td><td>The</td>

<td>exception of</td><td>, for example,</td><td>differences in</td><td>concentration</td><td>of</td>

antibody resulting from first and second concentration by ultrafiltration, and differences in buffer suspension medium resulting from buffer exchange on diafiltration. Thus, there is little, if any, change in composition, such as degradation of the target protein or antibody product in the embodiments of the present disclosure.

Conventional ultrafiltration concentration methods can generally have a longer time and few inefficient through-puts having a considerably long process, such as a process from several days to several weeks, a process with volume pretty small, or both.

In embodiments, the process of concentrating the protein of the disclosure may be carried out within, for example, from about 1 to 10 hours, preferably from about 2 to 5 hours, and more preferably from about 3 hours. Preferences favor higher production flow and smaller membrane areas.

The first ultrafiltration can be performed, for example, at about 35% of the total process time. Thus, for example, in a purification process and concentration of the disclosure with about 3 hours time

Total1786830Β1 total process, the first ultrafiltration can be performed in about 45 minutes. The second ultrafiltration can be performed, for example, at about 15% of the total process time. Thus, for example, in a purification process and concentration of the disclosure with about 3 hours of total process time, the second ultrafiltration may be performed in about 15 minutes. Diafiltration can be performed, for example, in about 50% of the total process time. Thus, for example, in the process of disclosure with about 3 hours of total process time, the diafiltration may be performed in about 90 to about 120 minutes.

In embodiments, the first and second ultrafiltration may be performed, for example, with an ultrafiltration membrane having a nominal pore size, or molecular weight cutoff, of about 5 to about 50 kiloDaltons. Another suitable nominal pore size is, for example, from about 10 to 40 kiloDaltons. However another suitable nominal pore size or molecular weight cutoff is about 30 kiloDaltons.

In embodiments, the first antibody preparation may contain, for example, an antibody that has an apparent molecular weight of, for example, about 100 to 200 kiloDaltons. In other embodiments, the first antibody preparation may contain an antibody that has an apparent molecular weight of, for example, about 150 kiloDaltons such as, for example, when the antibody preparation comprises anti-IgE or IgE antibody, e.g. e.g. US Patent 6,172,213 issued to Genentech, Inc.

Other antibodies for use in the present disclosure include antibodies for cancer treatment, see, for example: PCT / US02 / 19592; PCT / US01 / 20118; PCT / US01 / 25464; PCT / US01 / 26626; PCT / US02 / 28859; document

ΕΡ1786830Β1

PCT / US02 / 41798; PCT / USΟ3 / 11148;

PCT / US02 / 12206; PCT / US02 / 12619; and PCT / US02 / 33050. Still other antibodies suitable for use in the present disclosure include an anti-CD20 antibody and similar antibodies including human, non-human, murine, hybrid and chimeric forms. See, for example, US Patent 6,582,959 (VEGF) and US Patent Application 2002/0122797 A1 (human VEGF).

In embodiments, antibodies within the scope of the disclosure include hybrid and recombinant antibodies (e.g., humanized and human antibodies) without regard to the species of origin or class of the immunoglobulin or designation subclass, as well as antibody fragments. (e.g. Fab, F (ab ') 2 / · and F_v). See, US Patent 4,816,567; Mage and Lamoyi, Monoclonal Antibody Production Techniques and Applications, 79-97, Mareei Dekker, Inc., New York, (1987).

Monoclonal antibodies may be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352: 624-628 and Marks et al., 1991, J. Mol Biol. 222: 581-597, for example.

Monoclonal antibodies of the present invention specifically include chimeric antibodies wherein a portion of the light and / or heavy chain is identical to or homologous to corresponding sequences in antibodies derived from a specific species, or belonging to a specific antibody class or subclass, while that the rest of the chain is identical, or homologous, corresponding sequences in antibodies derived from other species or belonging to another antibody class or subclass, as well as fragments of such antibodies, to exhibit desired biological activity (US Patent 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 68516855). Antibodies may include primatized antibodies that comprise antibody binding sequences.

861786830Β1 variable domain antigens derived from non-human primates (eg, Old World Monkeys, Chipanzé, etc.) and human constant region sequences.

Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, unlike polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinant. In addition to their specificity, monoclonal antibodies are advantageous in that they are not contaminated with other antibodies. 0 monoclonal adjective indicates the character of the antibody as being obtained from a substantially homogeneous antibody population of antibodies, that is, the individual antibodies comprising the population are identical except for the possible natural mutations that may be present in smaller amounts, and not should be construed as a requirement for antibody production by any particular method. For example, monoclonal antibodies to be used for use in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by methods of Recombinant DNA. Other known methods for antibody production are described, for example, in Goding, Monoclonal Antibodies: Principles and Practice, 59-103, Academic Press (1986); Kozbor, J. Immunol, 133: 3001 (1984). Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, 51-63, Maree Dekker, Inc., New York (1987).

Several methods have been used to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses cells from various

ΕΡ1786830Β1 species including mouse (murine), hamster, mice and humans. Another method for preparing MAbs is genetic engineering including recombinant DNA techniques. Monoclonal antibodies made by these techniques include, but are not limited to, chimeric antibodies and humanized antibodies. A chimeric antibody combines regions of DNA encoding more than one kind of species. For example, a chimeric antibody may derive the variable region from a mouse and the constant region from a human. A humanized antibody comes predominantly from a human, even if it contains nonhuman portions. As a chimeric antibody, a humanized antibody may contain a complete human constant region. But unlike a chimeric antibody, the variable region may be partially derived from a human. Non-human, synthetic portions of a humanized antibody often come from CDRs in murine antibodies. In any event, these regions are crucial in allowing the antibody to recognize and bind to a specific antigen.

As noted, murine antibodies play an important role in antibody technology. While useful for short-term diagnosis and therapy, murine antibodies cannot be administered for a long term without increasing the risk of a deleterious immunogenic response. This response, called the human anti-mouse antibody (HAMA), occurs when a human immune system recognizes the murine antibody as foreign and attacks it. A HAMA response can cause toxic shock or even death. Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing non-human portions of the administered antibodies. In addition, chimeric and humanized antibodies have the added benefit of activating secondary human immune responses, such as antibody dependent cellular cytotoxicity.

An intact antibody is one which comprises

ΕΡ1786830Β1 antigen binding variable region as well as a light chain constant domain (C_L) and heavy chain constant domains CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or variant amino acid sequence thereof. The intact antibody may have one or more effector functions that refer to those biological activities assigned to the Fc region (a native Fc sequence region or Fc variant amino acid sequence region) of an antibody. Examples of antibody effector functions include Clq binding; complement-dependent cytotoxicity, Fc receptor binding; antibody-dependent cell cytotoxicity (ADCC); phagocytosis; low regulation of cell surface receptors (eg, B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of their heavy chains, the intact antibody may be assigned different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and many of these can be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the classes of different antibodies are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known.

In embodiments, the first ultrafiltration concentrates the first antibody preparation to provide the second antibody preparation that has a concentration of about 30 grams of antibody per liter, and the second ultrafiltration concentrates the intermediate antibody preparation (obtained by diafiltration). to provide the third antibody preparation having a concentration of, for example, from about 170 to about

ΕΡ1786830Β1

200 grams of antibody per liter. The first and second ultrafiltration may be performed with the same ultrafilter membrane, and if desired, within the same process container or circuit, for example, to minimize handling, loss, spillover, impact on efficiency. , and in the economy. The first and second ultrafiltration can be performed with any appropriate ultrafilter apparatus or membrane. Many suitable ultrafilter apparatuses and membranes, which are capable of performing tangential flow filtration (TFF) operation, ultrafiltration, and diafiltration, are commercially available, such as from Millipore, Pall Corp. In embodiments, a suitable ultrafilter membrane may be, for example, any regenerated cellulose compound which has a relatively low protein absorption profile compared to other available ultrafilter membranes such as polyethersulfone.

The diafiltration operation exchanges a first buffer composition present in the first and second antibody preparation with a desired second buffer in the third antibody preparation. In embodiments, the first buffer may comprise, for example, a mixture of aqueous sodium chloride and a TRIS buffer, and the second buffer may comprise, for example, a mixture of aqueous histidine chloride and arginine chloride. Diafiltration can be performed by a buffer exchange at constant volume, constant concentration or both. In embodiments, diafiltration performs a buffer exchange at a constant volume and constant concentration. The diafiltration may perform a buffer exchange, for example, from about 5 to 15 times the volume of (i.e. diavolumes). The diafiltration may perform a buffer exchange, for example, about 8 times the volume (8 diavolumes), ie 8 times the sample volume.

ΕΡ1786830Β1 containing the antibody preparation to be exchanged. For example, a 10 1 antibody preparation may be diafiltered in 5 (diavolumes) or the 50 1 volume of exchange buffer. Swap volume and swap volume preferences take into account a balance of factors, for example transfer process efficiency, product purity, patient and government acceptance standards, and similar standards, and may depend, for example. , concentration and type of buffer (e.g., the first buffer) in the first antibody preparation and similar considerations.

The first, second ultrafiltration and diafiltration are preferably performed with tangential flow filtration (TFF mode) through an ultrafilter membrane, and the ultrafilter membrane is preferably the same membrane for each step. The yield of the final assembly product (i.e. the third antibody preparation) can be, for example, greater than about 70 wt%, for example, about 80 to 100 wt% based on the weight of the antibodies. of the first antibody preparation. The yield of the third antibody preparation may be, in embodiments, greater than about 90% by weight, in embodiments, greater than about 95% by weight, and in embodiments, greater than about 98%. by weight based on the weight of the antibodies of the first antibody preparation.

The first ultrafiltration may have a recirculation rate, for example, from about 50 to 1000 ml / min, and preferably from about 100 to 1000 ml / min. The recirculation rate can be scaled according to the available membrane area, for example, the membrane areas of

0.46, 1.85, 18.58, 92, 90 m^z Similar areas allow for an increasingly higher rate of recirculation. Thus, an appropriate recirculation rate scale, in embodiments, may be, for example, from about 5.38

ΕΡ1786830Β1

1 / min / m² at about 53.82 1 / min / m². Ultrafiltration and diafiltration may be performed, for example, at transmembrane pressures of about 34.47 to about 344.74 kPa. Ultrafiltration and diafiltration may be performed, for example, at transmembrane pressures of about 68.95 to about 344.74 kPa. In the embodiments of the present disclosure there is provided a process of preparing a concentrate for a more dilute antibody formulation, the antibody concentrate having a minimum biocarbon, for example, below or below the detectable limit, such as less that about 100 CFU / ml.

The antibody compositions of the disclosure may be, for example, concentrated monoclonal antibody preparation for administration to humans, such as at a concentration greater than or equal to about 100 g / 1 (mg / ml) such as, for example, about from 120 to about 170 g / l.

background disclosure humanized recombinant antibody

Antibody compositions of the disclosure may be, for example, immunoglobulins, such as IgA, IgD, IgE, IgG, and IgM, subclasses thereof; recombinants thereof; fragments thereof; and mixtures thereof or any. In a preferred composition of includes anti-IgE antibodies

Antibody compositions of the disclosure may include a buffer. A preferred buffer may be, for example, an aqueous mixture of histidine chloride and arginine chloride.

The preparative processes of the disclosure are preferably carried out on the same apparatus and without intervention or with minimal operator intervention, for example as illustrated in FIG.

The first antibody preparation may be provided or prepared using a variety of chemical, physical, mechanical or non-mechanical methods, or biochemical methods such as grinding, ultrasound,

Homogenization, enzymatic digestion, solvent extraction, centrifugation, chromatography and the like, and combinations thereof, see, for example, the aforementioned R. Hatti-Kaul et al., Downstream Processing in Biotechnology, in Basic Biotechnology, Cap 9 The third antibody preparation may be further processed, if desired, using, for example, nanofiltration (to remove, for example, divalent ions), reverse osmosis (to remove, for example, monovalent ions), and similar methods. of liquid purification. The third antibody preparation of the present disclosure may be packaged, stored, or directly used. The third antibody preparation may be further processed using, for example, an additional step and concentration such as drying, lyophilization, lyophilization and reconstitution methods and the like. The resulting third concentrated antibody product may be further reconstituted, if desired, with an appropriate liquid.

With reference to the figures, FIG. 1 illustrates an apparatus in the embodiments of the present disclosure for carrying out the preparative process including an ultrafiltration and diafiltration system (100) having a TFF ultrafiltration-difiltration unit (UF-DF) (110) having a UF membrane -DF (115) which is in communication with the recirculation tank (120) this tank serves as a main feed and a retentate reservoir. In embodiments, the tank (120) may have a temperature control system comprising, for example, an insulating coating (125), a thermostat or temperature controlled heating element (126), such as a heat exchanger element. heater or a circulating heated liquid system including a heater (not shown), a flow regulator (127) such as a recirculation pump, and a suitable liquid for transfer of

Calor1786830óis1 heat, such as water, glycols, or mixtures thereof. All components in the circuit or component in the circuit that contribute to or flow, such as piping, valves, pumps, tanks, and the like, may optionally be insulated or optionally adapted so that the external heater maintains strict control over flow specifications. temperature and prevents temperature changes in the recirculating fluid handle within and between the filter chamber (110) and the recirculation tank (120). In embodiments, for example, when system (100) is performing first ultrafiltration or first ultrafiltration, such as in batch feed mode, the system may include an optional feed tank (128) which is in fluid communication with the system. recirculating feed tank (120) and can be used, for example, to compensate, replenish or exhaust liquid from the liquid feed tank (handle 110-120) For example, supplementing the recirculation phase (120).

A pump (130) pumps the tank (120) through the UF / DF unit (110) and recirculates thereafter the resulting retentate (the unfiltered portion or portion of the membrane-excluded feed liquid) to the recirculating tank (120). ). A second tank (140) stores and optionally pumps (not shown) a buffer in the main circuit during constant volume diafiltration, addition rate and the volume of buffer introduced into the main circuit is preferably at the same rate and volume as the filtrate leaves. the main circuit through the membrane (115). Buffer tank (140) may optionally be shell-insulated (143) and may include the equivalent of the above heating element and a recirculation pump (not shown). An optional inert gas source (145) such as nitrogen or other compressed gas sources may be used, for example for the recovery of

ΕΡ1786830Β1 test the similar.

product, to pressurize retentate return, to exclude oxygen, to level, to clean, to membrane integrity, and to operations A third tank (160) is used to collect and recover filtrate by withdrawing unit (110). Valves (150,170) may be used as appropriate to regulate direction and optionally the net flow rate in the system. All values ​​and pumps can be actuated manually, coordinated by computer control, or both. An optional fourth tank (190) and outlet stream that may provide a subordinate waste discharge, product recovery, or monitoring system, for example when equipment with an optional monitoring device (180), such as an optical density meter , optional filter (s) (185) as a protective filter, product filter and optional subsystems. In embodiments, the main fluid circuit (loop 110-120) may optionally be equipped with an inline monitoring system.

Concentrated antibody preparations prepared by processes of the present disclosure may be used for human therapeutic administration, including immunoglobulin products, for intramuscular (IVIG) or intravenous (IMIG) administration. Concentrated antibody preparations of the disclosure may include a stabilizer, for example, buffered amino acid saline, simple sugars, or as stabilizers, appropriate ion chelators such as EDTA or citrate ion, and combinations thereof, see see, for example, Wang, Y.-CJ et al., Parenteral formulations of proteins and peptides: stability and stabilizers, J. Parenteral Sci. Technol, 42, Suppl. S3-S26 (1988). Derwent Summary of the Document

JP01268646A (AN89-359879) reports that the application describes an IgG antibody injection preparation₃ which has a concentration of 0.1 micrograms / ml to 100 mg / ml.

ΕΡ1786830Β1

The subject matter of these publications is believed to be outside the scope of this disclosure.

The preparations according to the disclosure may be substantially free of aggregates. Acceptable levels of aggregate contaminants would be less than, for example, about 5 wt%, and ideally less than 2 wt%. Levels as low as 0.2 wt% can be obtained, although aggregate contaminants of about 1 wt% are more typical. The preparation in the embodiments may also preferably be free of excipients traditionally used to stabilize the polyclonal formulation, for example glycine and / or maltose.

The present disclosure may provide a monoclonal antibody preparation for administration to a human characterized in that the antibody of the preparation is a recombinant antibody and may be at a concentration of 100 mg / ml or greater, preferably greater than 150 mg / ml. The preparation is preferably substantially free of any protein aggregation.

The pH of the pharmaceutical formulation of the disclosure will depend in particular on the route of administration. However, in order to maximize antibody solubility in the concentrated solution, the pH of the solution should be different from the isoelectric point (pi) pH of the antibody.

In the embodiments of the disclosure, the monoclonal preparation may be provided for use in human therapy. Various human disorders can be treated such as cancer or infectious diseases, for example those mentioned above, and immune disorders such as T-cell mediated disorders including severe vasculitis, rheumatoid arthritis, systemic lupus, also autoimmune disorders such as multiple sclerosis, graft versus host disease, psoriasis, juvenile diabetes, Sjogren's disease, thyroid disease, myasthenia gravis, transplant rejection,

Do1786830Β1 inflammatory bowel disease, asthma, IgE-mediated disorders, and similar disorders or conditions, or combinations thereof.

The disclosure therefore provides, in embodiments, the use of a concentrated monoclonal antibody preparation as described herein in the manufacture of medicament for the treatment of some of the above disorders and similar disorders. Also provided is a method of treating a human having a disorder comprising administering to the subject a therapeutically effective amount of a preparation according to the disclosure. Dosages of such antibody preparations will vary with the circumstances being treated and the recipient of the treatment, but may, for example, be in the range of about 50 to about 2000 mg for an adult patient, preferably about 100 to 1000 mg administered daily or weekly for 1 to 30 days and repeated as needed. Doses may be administered as single or multiple doses.

Process description. The formulation step typically exchanges a large volume of purified drug substance, for example, resulting from ion exchange chromatography, composition and final concentrations of the excipient. There was typically no purification obtained at this step except for the removal of small molecules. The emphasis was on yield, buffer exchange, and high robustness at the formulation stage. During formulation by TFF (tangential flow filtration), the protein contained in the feed solution was pumped through the membrane system and returned to the recycle bin (recirculation). The TFF membrane retained the protein (as part of the retentate) when the filtrate (or permeate) was passed through the membrane.

861786830Β1 by the pressure. Pressure is called transmembrane pressure (PTM) and is typically controlled by the use of a retentate pressure control valve. The process was achieved by following a first ultrafiltration (concentration), diafiltration (buffer volume exchange), and a second (additional concentration). 0 Number of diavolumes (volumetric equivalents) required to remove components The process buffer can readily be calculated or determined experimentally.

ultrafiltration constant

UF / DF process in general for Anti-IgE. The pH of an anion exchange chromatography kit was adjusted to a pH of about 6 using 0.5 M aqueous phosphoric acid. The adjusted pH of the anion exchange assembly was formulated by the ultrafiltration / diafiltration (UF / DF) process of the present disclosure using a membrane having a nominal molecular elimination of 10,000 - 30,000 Daltons. Prior to processing, the UF membrane was equilibrated with diafiltration buffer (0.02 M histidine, 0.02 M HCl-arginine, pH 6).

The anion exchange product (anion exchange assembly) was then loaded into the system and concentrated to an intermediate concentration by the first ultrafiltration. The set was then diafiltered (8 X or 8 diavolumes) into its formulation (0.02 M histidine, 0.02 M HCl-arginine, pH 6). The pool was then concentrated by the second ultrafiltration to a final bulk concentration greater than 170 g / l and recovered through a sterile 0.22 micrometer filter. The entire UF / DF process was performed at an adjusted temperature point of about 45 ° C. This temperature control was obtained using the incoming assembly anion exchange temperature, diafiltration buffer temperature control and the use of an insulating recirculation tank coating for the

861786830Β1 UF / DF process as illustrated herein.

After UF / DF, the recovered set was diluted (i.e. conditioned) to a concentration of about 150 g / l in 0.02 M histidine and 0.2 M HCl-arginine, 0.04% polysorbate. -20 at pH 6 (final formulation). During the conditioning steps the temperature of the formulated volume was allowed to return to room temperature. After conditioning, the formulated volume was recovered again through a sterile 0.22 micrometer filter.

The UF / DF system can be regenerated with 0.1 N sodium hydroxide and sanitized with 1.4% Minncare®. When not used the system can be stored in 0.1 N aqueous sodium hydroxide. UF / DF membranes can be stored, for example, in 0.1% Roccal® / 20% water-glycerol solution between campaigns.

Ultrafiltration / Diafiltration Process General Procedures

Operating Parameters: Feed flow rate to 5.38 1 / min / m². A constant retentate pressure control (eg 68.95 kPa) was used for pre-use cleaning and balancing as C_Stop· The constant retentate pressure or constant PTM was used for the process.

Pre-Use Balancing: The following preparations were performed on clean Pellicon-2 cassette membranes prior to use to ensure that the membranes were properly equilibrated.

ΕΡ1786830Β1

<td>Volume (1 / m²)</td><td>Solution (room temperature)</td><td>Mode</td>

<td></td><td></td><td>SPFO</td>

<td> 10,76</td><td>WFI</td><td>SPFO</td>

<td> 10,76</td><td>DF cap</td><td>SPFO</td>

<td> 5,38</td><td>DF cap</td><td>TRFO, 10 minutes</td>

<td></td><td> —</td><td>SPFO</td>

Process Usage: The following was performed on the resulting initial anion exchange set (set Q) obtained from an earlier separation step, for example, a Q-Sepharose chromatography step:

first or first ultrafiltration (UF1) at a concentration of about 5 g / 1 at a concentration for diafiltration (C_Df);

diafiltration or diafiltration (DF1) with four (4) volumes of diafiltration (DV) with DF buffer;

diafiltrate continuously (DF2) with four (4) diafiltration (DV) volumes of DF buffer;

ultrafiltrate a second or second ultrafiltration (UF2) to a final concentration (C_Fmai); and optional product recovery.

The previous steps were typically performed at low dP Recycling (mixing), for example 15 minutes.

Post-Use Cleaning: The following sequence and tabulated conditions were used for cleaning Pellicon-2 cassette membranes immediately after use.

ΕΡ1786830Β1

<td>Volume (1 / m²)</td><td>Solution (temperature environment)</td><td>Mode</td>

<td> 10,76</td><td>0.1 N NaOH</td><td>SPFO</td>

<td> 5,38</td><td>0.1 N NaOH</td><td>TRFO, 30 minutes</td>

<td> -</td><td></td><td>SPFO</td>

<td> 10,76</td><td>WFI</td><td>SPFO</td>

<td> 5,38</td><td>300 ppm of Minncare®</td><td>TRFO, 30 minutes</td>

<td> -</td><td></td><td>SPFO</td>

<td> 10,76</td><td>WFI</td><td>SPFO</td>

<td></td><td> -</td><td>Test of integrity to 206.84 kPa</td>

<td> 5,38</td><td>0.1 N NaOH</td><td>TRFO, storage of 15 minutes</td>

Settings for TFF Operating Modes.

Single pass with open filtrate (SPFO). The retentate and filtrate are directed to drain. Filtered valve open.

Total recycling with open filtrate (TRFO). The retentate and filtrate are directed to the recycling container. Filtered valve open.

Staggered Ultrafiltration (FB-UF). The retentate is directed to the recycling tank, the filtrate directed to the drain, and the incoming assembly is transferred to the recycling tank.

Discontinued Ultrafiltration (B-UF). The retentate is directed to the recycling tank and the filtrate is directed

ΕΡ1786830Β1 for the drain.

Diafiltration (DF). The retentate is directed to the recycling tank, the filtrate is directed to the drain, and the diafiltration plug is transferred to the recycling tank. dP refers to the differential pressure.

Product transfer. The ultrafilter membrane and recycling tank unit is open to the assembly tank. The nitrogen pressure is controlled. The assembly is first transferred using the recycling pump and then a manual peristaltic pump is used.

Feed transfer. The incoming assembly is pumped to the recycling tank.

Total Recycling with Closed Filtrate (TRFC). The retentate is directed to the recycling container. Filtered valve closed.

Q set refers to the protein set resulting from, for example, an earlier Q-Sepharose chromatography step that is buffer conditioned, also referred to as the conditioned set.

WFI refers to water for injection.

EXAMPLES

The following examples serve to more fully describe the modes of use of the disclosure described above, as well as to determine the best contemplated modes for carrying out various aspects of the disclosure. It is understood that these examples are in no way intended to limit the true scope of this disclosure, but is preferably presented for illustrative purposes.

Example 1

RhuMAb E25 High Concentration Formulation

A pilot scale production UF system was used to concentrate / formulate rhuMAb E25 (an antibody

Recombinant Human IgE Monoclonal (1786830Β1, US Patent 6,172,213). A Millipore Pelicon Ultrafiltration / Diafiltration system was assembled with a 0.52 square meter membrane composed of 10,000 Dalton regenerated cellulose. The system consisted of a membrane holder, a Model 6 Waukeskaw rotary lobe feed pump, 1.27 cm 316L stainless steel recirculation pipeline, and a recirculation vessel. The pressure indicators / transmitters (Anderson) were located at the inlet (FEED), the drainage (RETAINED) and the permeate (FILTERED) membrane holder. Flow meters (Yokogawa ADMAG) were located at the inlet (FEED) and permeate (FILTERED) of the membrane holder. A back pressure regulating valve (Mikroseal) was located in the membrane support flow to control the retentate pressure and to effect transmembrane pressure (PTM). A 40 l 316L stainless steel wrapped tank was used for the recirculation vessel. This tank was placed with a level indicator, a top-mounted agitator (Lightnin), a circuit breaker and a vortex bottom valve (NovAseptic). Temperature control was achieved by using the glycol feed modulated temperature to the tank liner.

During this operation, the feed flow rate was set at a constant rate of 2.85 1 / min (5.38 1 / min / m²). During all pre-use and post-use operation the retentate pressure control was set to a constant of 68.95 kPa. During ultrafiltration and diafiltration operations the system used a C control scheme._pairIn order to control the flow through the membrane, see, for example, Reis, et al. Constant C_wallUltrafiltration Process Control, J. of Membrane Science, 130 (1997), 123-140.

Prior to the process, the storage solution of the

ΕΡ1786830Β1 system (0.1 N NaOH) was discharged in a single pass into drainage mode, first purified with water 21.53 1 / m² (AP) and then with 10.76 1 / m² diafiltration buffer (50 mM histidine / pH 6.0). Once discharged, the system was equilibrated by re-circulating the 5.38 1 / m diafiltration buffer² for 10 minutes. The pH of the recirculated solution was checked to confirm equilibrium. The level in the tank was then reduced to a minimum measurable value to minimize dilution of the incoming protein pool. The protein pool from an earlier Q-Sepharose chromatography step was measured as 3.2 g of E25 / 1 and had a volume of 43.1. The protein was in a 25 mM solution of TRIS buffer and about 200 mM NaCl and pH adjusted to 6.2. To begin operation the protein assembly was transferred to the recirculation vessel. In the vessel the assembly was shaken through a top mounted impeller and the temperature was kept at room temperature (20-25 ° C).

During the process the whole was concentrated in the UF1 mode to 50 g of E25 / 1 (about 2.81). At the start of diafiltration the set temperature point in the container was increased to 40 ° C. The increase in temperature and control was affected by hot glycol flow through the outer shell of the tank. The pool was then diafiltered with 8 diavolumes of diafiltration buffer. Diafiltration was performed at a constant volume, which was achieved by combining the flow rate of the protected solution being transferred to the recirculation tank at the flow rate of the filtrate being removed from the system. At the end of diafiltration, the pool was further concentrated in the UF2 modality. This phase was also performed by setting the temperature point to 40 ° C. The target for this final concentration was 110 g / 1. This was achieved without the need to reduce the flow rate.

1786830Β1 of the power supply. Then mixing a low pressure reduction was performed where the feed pump was controlled to maintain a minimum pressure of 34.74-68.95 kPa through the feed channel. A sample was pulled from the recirculation tank and a final volume concentration of about 120 g / l was measured. Table 1 summarizes the production and flow results of UF1, DF (DF1 + DF2), and UF2.

Table 1

<td>Phase of process</td><td>Production Normalized (g / m²/H)</td><td>Flow Normalized (LMH / kPa)</td>

<td>UF1</td><td> 148,54</td><td> 0,72</td>

<td>DF</td><td> 148,54</td><td> 0,42</td>

<td>UF2</td><td> 1.952,58</td><td> 0,24</td>

FIG. 2 shows the observed or measured process values ​​over time parameters for feed flow rate (210), tank temperature (220), feed dP (230), PTM (240), filtrate flow rate ( 250) during the various stages or process mode including UF1 (10), DF (20), UF2 (30).

FIG. 3 shows the observed or measured process values ​​over time for concentration E25 (310), flow (320) and PTM (240).

FIG. 4 shows the observed or measured process values ​​over time for decreasing the pressure against the observed protein concentration for UFl (410) and UF2 (420) at 37 ° C.

The protein pool was recovered by a series of steps. First the recirculation assembly was pumped into the tank through a Millipac 200 with a 0.22 micron filter sterilized using the

ΕΡ1786830Β1 Swiveling lobe feed. The protein solution was then displaced from the pipeline and membrane unit with a 34.47 kPa nitrogen gas flow applied down from the highest point on the retentate line. The final phase was a flow below the tank and the feed line, also using nitrogen gas at 34.47 kPa.

Product recovery is believed to be improved compared to Example 1 when conducted at room temperature because the elevated temperature used in one or more of the ultrafiltration, diafiltration or recovery steps reduced the viscous effect. For example, when temperature control was turned off during product recovery, the system slowly cooled during this operation which caused difficulties in membrane unit recovery. Alternatively, recovery may be performed first by the membrane holder and then by the recirculation vessel.

To determine mass loss during recovery, 1.74 L of DF buffer was added to the system and recirculated for about 5 minutes and recovered using the same sequence as described above. This volume was then analyzed for protein concentration with the other pools. Table 2 summarizes the results.

ΕΡ1786830Β1

Table 2

<td></td><td>Volume (D</td><td>Concentration (g / D</td><td>Pasta (g)</td><td>Income or {Loss} (%)</td>

<td>Q set</td><td> 43, 1</td><td> 3,2</td><td> 137, 9</td><td> 100</td>

<td>Set recovered</td><td> 0, 99</td><td> 120</td><td> 118, 8</td><td> 86, 1</td>

<td>Cap cleaning</td><td> 1,74</td><td> 9,8</td><td> 17, 1</td><td> 12,4</td>

<td>Filtered</td><td> 65,3</td><td> 0,04</td><td> 2, 6</td><td> 1, 9</td>

Postprocessing, the membrane was regenerated using 0.1 N NaOH, 10.76 1 / m² pass wash followed by total recirculation of 5.38 1 / m² for 30 minutes. This was followed by washing at 10.76 1 / m² of AP (pure water). Followed by a total recirculation of the 300 ppm Minncare® solution for 30 minutes. The system was again flushed with 10.76 l / m² AP and finally recirculated for 15 minutes with 0.1 N NaOH and stored. The recovered pool was diluted to 80 g of E25 / 1 and conditioned to the final formulation of 50 mM histidine / 150 mM trehalose / 0.02% polysorbate 20 / pH 6.0. Product quality was assessed by size exclusion chromatography (SEC) for incoming Set Q and final recovered volume. These data are summarized in Table 3.

Table 3

<td>Set</td><td>ESA Results (% of monomer)</td>

<td>Q set</td><td> 99, 8</td>

<td>Final Volume</td><td> 99, 8</td>

ΕΡ1786830Β1

Comparative Example 2

RhuMAb E25 High Concentration Formulation at Room Temperature

Example 1 was performed with the following exceptions. Prior to the process, the system storage solution (0.1 N NaOH) was washed in a single pass to the first drainage mode at 21.53 1 / m² of purified water (AP) and then with 10.76 l / m² diafiltration buffer (20 mM histidine / pH 6.0). After washes, the system was equilibrated by recirculation of 5.38 1 / m² of diafiltration buffer for 10 minutes. The pH of the recirculated solution was checked to confirm equilibrium. The level in the tank was then reduced to a minimum measurable value to minimize dilution of the incoming protein pool.

The protein pool resulting from the Q-Sepharose chromatography step was measured to be 3.3 g E25 / 1 and have a volume of 33.3 1. The protein was in a 25 mM TRIS buffer solution and about 200 mM NaCl and pH adjusted to 6.2. To begin operation the protein pool was transferred to the recirculation vessel. In the vessel the assembly was shaken through a top mounted impeller and the temperature was kept at room temperature (20-25 ° C). During the process the whole was concentrated downwards in the UF1 mode at 50 g E25 / 1 (about 2.2 L). The pool was then diafiltered with 8 diavolumes of the diafiltration buffer. Diafiltration was performed at a constant volume, where the volume was obtained by combining the buffer flow rate that is being transferred into the recirculation tank to the flow rate of the filtrate being removed from the system. The diafiltration was also performed at room temperature. At the end of diafiltration, the pool was more concentrated in UF2 mode. The target for this final concentration was 110 g / 1. However, due to a reduction in high pressure through the

ΕΡ1786830Β1 feed channel, this concentration was not obtained. In an attempt to achieve this concentration the feed flow rate was reduced to 1.4 l / min at an additional concentration of about 80 g of E25 / 1 due to a pressure drop across the feed channel which reached 344.74. kPa. UF2 continued until the high pressure drop of 344.74 kPa was reached again and the process was stopped. Then a low pressure drop was attempted with a feed pump to maintain a 34.47 kPa pressure drop across the feed channel. Again, the slimy nature of the protein solution made it difficult to get the rotary lobe pump to reach additional pressures. A sample was taken from the recirculation tank and a final concentration of about 104 g / l was measured. O

Table 4 summarizes the production and flow measured during the UF1, DF (DF1 + DF2), and UF2 phases.

Table 4

<td>Process phase</td><td>Production Normalized (g / m²/H)</td><td>Flow Normalized (LMH / kPa)</td>

<td>UF1</td><td> 156,78</td><td> 0,77</td>

<td>DF</td><td> 102,26</td><td> 0,21</td>

<td>UF2</td><td> 1.556,47</td><td> 0, 11</td>

FIG. 5 shows the observed or measured over time process values ​​for the feed flow rate (210), tank temperature (220), feed dP (230), PTM (240), and feed rate parameters. flow of the filtrate (250) during the various process phases or mode including UF1 (10), DF (20), UF2 (30).

FIG. 6 shows the observed or measured process values ​​over time for the E25 concentration (310),

861786830Β1 stream (320) and PTM (240).

FIG. 7 shows the observed or measured process values ​​over time for decreasing the pressure against the observed protein concentration for UFl (410) and UF2 (420) at 24 ° C.

The protein pool was recovered by a series of steps. First, the recirculation assembly was pumped into the tank through a Millipac 200 with a 0.22 micron filter sterilized using the rotary lobe feed pump. The protein solution was then displaced from the pipeline and membrane unit with a 34.47 kPa nitrogen gas flow applied down from the highest point on the retentate line. The final phase was a flow below the tank and the feed line, also using nitrogen gas at 34.47 kPa.

To determine mass loss during recovery, 1.85 l of DF buffer was added to the system and recirculated for about 5 minutes and recovered using the same sequence as Example 1. This volume was then analyzed for protein concentration with the other sets. Table 5 summarizes the results.

Table 5

<td></td><td>Volume 1)</td><td>Concentration (g / D</td><td>Mass (g)</td><td>Yield or {Loss} (%)</td>

<td>Q set</td><td> 33, 3</td><td> 3, 3</td><td> 109, 9</td><td> 100</td>

<td>Set recovered</td><td> 0,77</td><td> 104,4</td><td> 80, 4</td><td> 73,1</td>

<td>Cleaning cap</td><td> 1,85</td><td> 14,7</td><td> 27,2</td><td> 24,7</td>

<td>Filtered</td><td> 52,2</td><td> 0, 03</td><td> 1,6</td><td> 1,5</td>

ΕΡ1786830Β1

After the process, the membrane was regenerated using 0.1 N NaOH with a single pass wash of 10.76 1 / m²followed by the total recirculation of 5.38 1 / m² for 30 minutes. This was followed by washing at 10.76 1 / m² of AP (pure water). Followed by a total recirculation of the 300 ppm Minncare® solution for 30 minutes. The system was again finally stored washed with 10.76 l / m² AP was recirculated for 15 minutes with 0.1 N NaOH and The recovered set was diluted to 80 g of E25 / 1 and conditioned to the final formulation of 20 mM histidine / 250 mM sucrose / 0.02% polysorbate 20 / pH 6.0. Product quality was assessed by size exclusion chromatography (SEC) for incoming Set Q and final recovered volume. These data are summarized in Table 6.

Table 6

<td>Set</td><td>ESA Results (% of monomer)</td>

<td>Q set</td><td> 99, 8</td>

<td>Volume Final</td><td> 99, 8</td>

Example 3

RhuMAb E26 High Concentration Formulation with Initial Staggered Mode

Example 1 was repeated with the following exceptions. The concentrate / formula was rhuMAb E26 (a recombinant human IgE monoclonal antibody). The products in this example were used in the toxicological evaluation. A Millipore Pelicon Ultrafiltration / Diafiltration system was assembled with a 1.06 square meters, 30,000 Daltons membrane composed of regenerated cellulose. The flow rate

1786830Β1 feed set at a constant rate of 5.0 1 / min (4.30 1 / min / m²). During ultrafiltration and diafiltration operations the retentate pressure was maintained between about 41.37-55.16 kPa. The protein pool that resulted from the previous step by Q-Sepharose chromatography was measured to be 6.7 g E26 / 1 and had a volume of 59.31.

Because the incoming set was larger than the recirculation container, the UF1 process began in batch feed mode. In this mode, Set Q was added to the recirculation vessel at approximately the same rate as the filtrate passed through the TFF membrane to the drain. After the remaining Set Q was transferred to the recirculation vessel, the UFl process continued in batch mode. During the FLU, the pool was concentrated to 50 g of E26 / 1 (about 7.91 l). At the beginning of diafiltration the recirculation vessel temperature set point was increased to 40 ° C. The increase in temperature and control was affected by the hot flow of glycol through the outer shell of the tank. The pool was then diafiltered with 8 diavolumes of the diafiltration buffer. Diafiltration was performed at a constant volume, which was achieved by combining the flow rate of the protected solution being transferred to the recirculation tank at the flow rate of the filtrate being removed from the system. At the end of diafiltration, the pool was further concentrated in UF2 mode to a final concentration of 109 g of E26 / 1 (3.6

1) . This phase was also performed using a high temperature point of 40 ° C. Then a low pressure drop mixture was performed where the feed pump was controlled to maintain a minimum pressure of 34.47-68.95 kPa through the feed channel. Table 7 summarizes the production and flow results of UFl, DF (DF1 + DF2), and UF2.

ΕΡ1786830Β1

Table 7

<td>Process phase</td><td>Production Normalized (g / m²/H)</td><td>Flow Normalized (LMH / kPa)</td>

<td>UF1</td><td> 280,94</td><td> 0,54</td>

<td>DF</td><td> 206,67</td><td> 0,34</td>

<td>UF2</td><td> 1.874,09</td><td> 0,26</td>

FIG. 8 shows the observed or measured over time process values ​​for the feed flow rate (210), tank temperature (220), feed dP (230), PTM (240), and feed rate parameters. filtrate flow (250 ° C).

FIG. 9 shows the observed or measured process values ​​over time for concentration E26 (910), flow (920) and PTM (940).

FIG. 10 shows the observed or measured process values ​​over time for the pressure drop against the observed protein concentration for UF1 (1010) and UF2 (1020).

Just prior to product recovery, a 10 ml sample was analyzed for detection and a bioburden titration. A typical rejection limit is 1000 colony forming units (CFU) per ml. The results of this test were 1.8 CFU / ml, an appropriate value for this step and well below the rejection limit. To determine mass loss during recovery, 908.1 ml of DF buffer was added to the system and recirculated for about 5 minutes and recovered using the same sequence as described above. This volume was then analyzed for protein concentration with the other sets. Table 8 summarizes the results.

ΕΡ1786830Β1

Table 8

<td></td><td>Volume 1)</td><td>Concentration (g / D</td><td>Mass (g)</td><td>Yield or {Loss} (%)</td>

<td>Q set</td><td> 59,3</td><td> 6,7</td><td> 397, 3</td><td> 100</td>

<td>Set recovered</td><td> 3, 41</td><td> 109,1</td><td> 372, 0</td><td> 93, 6</td>

<td>Cap cleaning</td><td> 0, 908</td><td> 20,4</td><td> 18,5</td><td> 4,7</td>

<td>Filtered</td><td> 120</td><td>n / a</td><td>n / a</td><td>n / a</td>

The recovered pool was diluted to 80 g of E25 / 1 and conditioned to the final formulation of 50 mM histidine / 150 mM trehalose / 0.02% polysorbate 20 / pH 6.0. Product quality was assessed by size exclusion chromatography (SEC) for the incoming Q Set, the retentate set after UF1, the retentate set after DF and for the final volume recovered. These data are summarized in Table 9.

Table 9

<td>Set</td><td>ESA Results (% of monomers)</td>

<td>Q set</td><td> 99, 8</td>

<td>UFl end</td><td> 99, 8</td>

<td>DF Final</td><td> 99, 8</td>

<td>Final Volume</td><td> 99, 8</td>

ΕΡ1786830Β1

Example 4

RhuMAb E26 High Concentration Formulation for Toxicological Evaluation - Comparison of 10kD and 30kD

Example 3 was repeated with the following exceptions. Two UF systems for scale production were used to concentrate / formulate rhuMAb E26. Two Millipore Pelicon Ultrafiltration / Diafiltration systems were assembled with all 1.06 square meters in a membrane composed of regenerated cellulose with a pore size of 10,000 Daltons and another with a pore size of 30,000 Daltons. Retentate pressures were maintained at about 41.37-62.05 kPa.

10 kDa process

The protein pool resulting from the previous Q-Sepharose chromatography step was measured as 5.85 g E26 / 1 and had a volume of 62.4 1. During the UFI, the pool was concentrated to 50 g E26 / L (about 7.3 l). At the end of diafiltration, the pool was further concentrated in the UF2 mode to a final concentration of 107.5 g of E26 / 1 (3.41). Table 10 summarizes the production and flow results of UFI, DF and UF2.

Table 10

<td>Process phase</td><td>Production Normalized (g / m²/H)</td><td>Flow Normalized (LMH / kPa)</td>

<td>UFI</td><td> 234,66</td><td> 0,52</td>

<td>DF</td><td> 171,15</td><td> 0,38</td>

<td>UF2</td><td> 1.478,97</td><td> 0,28</td>

To determine mass loss during recovery, 987 ml of DF buffer was added to the

ΕΡ1786830Β1 system and recirculated for about 5 minutes and recovered using the same sequence as described above. This volume was then analyzed for protein concentration with the other pools. Table 11 summarizes the results.

Table 11

<td></td><td>Volume 1)</td><td>Concentration (g / D</td><td>Pasta (g)</td><td>Yield or {Loss} (%)</td>

<td>Q set</td><td> 62,4</td><td> 5, 85</td><td> 365,4</td><td> 100</td>

<td>Set recovered</td><td> 3,38</td><td> 107,5</td><td> 361,7</td><td> 98, 9</td>

<td>Cap cleaning</td><td> 0, 987</td><td> 19, 9</td><td> 19, 6</td><td> 5,4</td>

<td>Filtered</td><td> 125</td><td>n / a</td><td>n / a</td><td>n / a</td>

FIG. 11 shows the observed or measured over time process values ​​for the feed flow rate (210), tank temperature (220), feed dP (230), PTM (240), and feed rate parameters. filtrate flow (250) during the various process phases or mode including UF1 (10), DF (20), UF2 (30) and low dP (40) for the 10 kDa process.

FIG. 12 shows the observed or measured process values ​​over time for concentration E26 (1210), flow (1020) and PTM (1240) during the various process phases or mode including UF1 (10), DF (20), UF2 (30) and low dP (40) for the 10 kDa process.

FIG. 13 shows the observed or measured process values ​​over time for the drop in pressure against the observed protein concentration for UFl (1310) and UF2 (1320) for the 10 kDa process.

ΕΡ1786830Β1

30 kDa process

The protein pool resulting from the previous Q-Sepharose chromatography step was measured to be 5.85 g E26 / 1 and had a volume of 64.5 1. During the UFI, the pool was concentrated to 50 g E26 / 1. / 1 (about 7.5 l). At the end of diafiltration, the pool was further concentrated in the UF2 mode to a final concentration of 117.5 g of E26 / 1 (3.21). Table 12 summarizes the production and flow results of UFI, DF and UF2.

Table 12

<td>Phase of process</td><td>Standard Production (g / m²/H)</td><td>Normalized Flow (LMH / kPa)</td>

<td>UFI</td><td> 274,48</td><td> 0,58</td>

<td>DF</td><td> 189,44</td><td> 0,35</td>

<td>UF2</td><td> 1.942,90</td><td> 0,23</td>

To determine mass loss during recovery, 918 ml of DF buffer was added to the system and recirculated for about 5 minutes and recovered using the same sequence as described above. The recovered pool was diluted to 80 g of E25 / 1 and conditioned to the final formulation of 50 mM histidine / 150 mM trehalose / 0.02% polysorbate 20 / pH 6.0. Table 13 summarizes the results.

ΕΡ1786830Β1

Table 13

<td></td><td>Volume (D</td><td>Concentration (g / D</td><td>Pasta (g)</td><td>Yield or {Loss} (%)</td>

<td>Q set</td><td> 64,5</td><td> 5, 85</td><td> 377,3</td><td> 100</td>

<td>Set recovered</td><td> 3,20</td><td> 117,5</td><td> 376, 0</td><td> 9 9,6</td>

<td>Cap cleaning</td><td> 0,918</td><td> 22,7</td><td> 20,8</td><td> 5,5</td>

<td>Filtered</td><td> 125</td><td>n / a</td><td>n / a</td><td>n / a</td>

FIG. 14 shows the observed or measured over time process values ​​for the feed flow rate (210), tank temperature (220), feed dP (230), PTM (240), and feed rate parameters. filtrate flow (250) during various process phases or mode including UF1 (10), DF (20), UF2 (30) and low dP (40) for the 30 kD process.

FIG. 15 shows the observed or measured process values ​​over time for concentration E26 (1510), flow (1520) and PTM (1540) during the various process phases or mode including UF1 (10), DF (20), UF2 (30) and low dP (40) for the 30 kD process.

FIG. 16 shows the observed or measured process values ​​over time for the pressure drop against the observed protein concentration for UF1 (1610) and UF2 (1620) for the 30 kD process.

Example 5

Scaled Increase for rhuMAb E25 Liquid

Example 1 was repeated with the following exceptions.

A UF system for scale production was used to concentrate / formulate a liquid rhuMAb E25 (a recombinant human IgE monoclonal antibody). The product may

ΕΡ1786830Β1 be used for therapeutic application and research on bioequivalence in humans. A 0.52 square meter Millipore Pelicon Ultrafiltration / Diafiltration system was assembled with a membrane composed of regenerated cellulose with a pore size of 30,000 Daltons. Each system consisted of a membrane holder, a Viking S3S rotary feed pump, 3.83 cm 316L stainless steel recirculation piping and a 250 1 recirculation container.

A 250 l 316L stainless steel lined tank was used for the recirculation vessel. Temperature control of this tank was achieved by temperature modulation by glycol feed in the tank liner. The glycol feed temperature in the tank liner has been increased or decreased using steam heat exchange or cold glycol source respectively.

For this operation, the feed flow rate was set at a constant rate of 114 l / min (0.5 l / min / hr). Diafiltration buffer (20 mM histidine / 200 mM arginine chloride / pH 6.0) was prepared in a separate tank. The temperature of this buffer was adjusted to 45 ° C before the start of the process. This allowed accurate temperature control throughout the process.

Prior to processing, the system storage solution (0.1 N NaOH) was equilibrated in a single pass to first drain with 10.76 l / m² water for injection (WFI) and then at 10.76 1 / m² of diafiltration buffer. After washes, the system was equilibrated by recirculation of 5.38 1 / m² of diafiltration buffer for 10 minutes. The pH of the recirculated solution was checked to confirm equilibrium.

The protein pool resulting from the Q-Sepharose chromatography step was measured to be 5.2562 g of E25 / 1 and having a volume of 1,141 1. The protein was

861786830Β1 in a 25 mM solution of TRIS buffer and about 200 mM NaCl and the pH adjusted to 6.2. Just before execution the set temperature set point was set to 45 ° C. To begin operation the protein pool was transferred to the recirculation vessel through a 0.22 micron pore filter at a level of about 200 l in the tank. Because the incoming set was larger than the recirculation container, the UF1 process started in staggered mode. In this mode, Set Q was added to the recirculation vessel at about the same rate as the filtrate passed through the TFF membrane to the drain. After the remaining Set Q was transferred to the recirculation vessel, process UF1 continued in batch mode. During the UFl mode, the pool was concentrated to about 30 g of E25 / 1 (about 200 l). The set was then diafiltered with about 8 diavolumes of diafiltration buffer, the temperature was³C. Diafiltration was performed at a constant volume, which was achieved by combining the flow rate of the protected solution being transferred to the recirculation tank at the flow rate of the filtrate being removed from the system. At the end of diafiltration, the pool was further concentrated in the UF2 mode to a final concentration&gt; 170 g of E25 / 1 (35 l).

This phase of the UF2 mode was also performed at a high temperature point of 45 ° C +/- 5 ° C. Then a low pressure drop mixture was performed where the feed pump was controlled to maintain a pressure drop of 34.47-68.95 kPa through the feed channel. A sample was pulled and an examination was performed to confirm the recovery. The concentration of this sample was 219 g of E25 / 1. Table 14 summarizes the production and flow results measured during the UFl, DF (DF1 + DF2) and UF2 phases.

diafiltration. During maintained between 40 and 50 spectrophotometric concentration before

ΕΡ1786830Β1

Table 14

<td>Process phase</td><td>Production Normalized (g / m²/H)</td><td>Flow Normalized (LMH / kPa)</td>

<td>UF1</td><td> 471,46</td><td> 0,48</td>

<td>DF</td><td> 278,78</td><td> 0,36</td>

<td>UF2</td><td> 849,27</td><td> 0,09</td>

Just before product recovery, a 30 ml sample was analyzed for detection and a bioburden titration. The result of this test was &lt;0.13 CFU / ml. The protein pool was recovered by a series of steps. First, the product was displaced from the membrane in a single pass mode using 5 l of DF buffer added to the retentate line. The product was filtered into a recovery tank through a 0.22 micron 0.69 m pore class sterile filter.²followed by a sterile filter of 0.22 micron pore class 0.19 m². The assembly in the recirculation tank was then pumped into the tank using a rotary lobe feed pump. The residual protein solution was then displaced below the tank line and the feed with a 34.47 kPa nitrogen gas flow. The final phase was a downward flow of the membrane unit, which now consisted mostly of DF buffer from the initial displacement of the product. This phase also used the 34.47 kPa nitrogen gas applied at the highest point on the retentate line. The recovered pool was first diluted to about 153 g of E25 / 1 using DF buffer. Finally, the pool was conditioned to the final form of 20 mM histidine / 200 mM HCl-arginine / 0.04% polysorbate 20 / pH 6.0. The volumes from the recovered set, from the set

861786830Β1, and the conditioned set (set Q) were then individually analyzed for protein concentration. Table 15 summarizes the results.

Table 15

<td></td><td>Volume (D</td><td>Concentration (g / 1)</td><td>Pasta (g)</td><td>Income or {Loss} (%)</td>

<td>Q set</td><td> 1.141</td><td> 5,2562</td><td> 5.997,3</td><td> 100</td>

<td>Set recovered</td><td> 35, 0</td><td> 170, 0</td><td> 5.950,0</td><td> 99,2</td>

<td>Set Diluted</td><td> 39, 0</td><td> 147, 0</td><td> 5.726</td><td> 95,5</td>

FIG. 17 shows parameters of feed flow (210), tank temperature (220), feed dP (230), PTM (240), filtrate flow rate (250) during various process phases or mode including UF1 ( 10), DFl (20), DF2 (25), UF2 (30) and low dP (50).

Example 6

Preparation of rhuMAb E25 Liquid

Example 5 was repeated with the following exceptions. A UF system for scale production was used to concentrate / formulate a liquid rhuMAb E25 (E25, a recombinant human IgE monoclonal antibody). A Millipore Pelicon Ultrafiltration / Diafiltration system was assembled with a 20.99 square meter regenerated cellulose composite membrane with a pore size of 30,000 Daltons. Each system consisted of a membrane holder, a Viking S3S rotary feed pump, 3.81 cm 316L stainless steel recirculation piping and a 250 1 recirculation container. A 250 1 316L coated stainless steel tank was used. to the recirculation container. The feed flow rate

ΕΡ1786830Β1 was adjusted at a constant rate of 114 1 / min (5.38 1 / min / m²). Throughout the pre-use and post-use operation the retentate pressure control was set to a constant of 68.95 kPa. During ultrafiltration and diafiltration operations the system used the control C scheme_Stopto control the flow through the membrane. Diafiltration buffer (20 mM histidine / 200 mM arginine chloride / pH 6.0) was prepared in a separate tank. The temperature of this buffer was adjusted to 45 ° C before the start of the process. This allowed accurate temperature control throughout the process. Temperature control of this tank was achieved by temperature modulation by glycol feed in the tank liner. The protein pool resulting from the Q-Sepharose chromatography step was measured to be 5.5438 g E25 / 1 and have a volume of 1082 1. The protein was in a 25 mM solution of TRIS buffer and about 200 mM. NaCl and pH adjusted to 6.2. Just before operation, the temperature of this set was set to 45 ° C. To begin operation the protein pool was transferred to the recirculation vessel through a 0.22 micron pore filter; at a level of about 200 1 in the tank. In the vessel the assembly was shaken by a top mounted stirrer and the temperature was maintained at (40-50 ° C). Because the incoming set was larger than the recirculation container, the UF1 process began in batch feed mode. In this mode, Set Q was added to the recirculation vessel at about the same rate as filtrate passed through the TFF membrane to the drain. After the remaining Set Q was transferred to the recirculation vessel, process UF1 continued in batch mode. During UF1 the pool was concentrated to 30 g E25 / 1 (about 200 l). The pool was then diafiltered with 8 diavolumes of the diafiltration buffer. During diafiltration the set temperature point of the

ΕΡ1786830Β1 recirculation container was kept at 40 to 50 ° C. The diafiltration was performed at a constant volume, which was achieved by combining the flow rate of the protected solution being transferred to the recirculation chamber at the flow rate of the filtrate being removed from the system. At the end of diafiltration, the pool was further concentrated in the UF2 mode to a final concentration&gt; 170 g of E25 / 1 (35 l). This phase of the UF2 mode was also performed by adjusting the temperature rise to 45 ° C +/- 5 ° C. Then maintaining a pressure drop of 34.47-68.95 kPa through the supply channel. A sample was pulled and a spectrophotometric examination was performed to confirm the concentration before recovery. The concentration of this sample was 191 g of E25 / 1 and the pool volume was 31.9 l. A process graph of the time over parameter was comparable to those noted above and summarized in FIG. 17

Table 14

<td>Process phase</td><td>Production Normalized (g / m²/H)</td><td>Normalized Flow (LMH / kPa)</td>

<td>UF1</td><td> 485,46</td><td> 0,46</td>

<td>DF</td><td> 278,79</td><td> 0,36</td>

<td>UF2</td><td> 1.306,74</td><td> 0, 11</td>

Just before product recovery, a 30 ml sample was analyzed for detection and a bioburden titration. The result of this test was below the limit of detection (&lt;0.13 CFU / ml).

The protein pool was recovered by a series of

861786830Β1 steps. First, the product was displaced from the membrane in a single pass mode using 5 l of DF buffer added to the retentate line. The product was filtered into a recovery tank through a 0.22 micron 0.69 m pore class sterile filter.²followed by a sterile filter of 0.22 micron pore class 0.19 m². The assembly in the recirculation tank was then pumped into the tank using a rotary lobe feed pump. The residual protein solution was then displaced below the tank line and the feed with a 34.47 kPa nitrogen gas flow. The final phase was a downward flow of the membrane unit, which now consisted mostly of DF buffer from the initial displacement of the product. This phase also used the 34.47 kPa nitrogen gas applied at the highest point on the retentate line. The recovered pool was first diluted to about 153 g of E25 / 1 using DF buffer. Finally, the set was conditioned to the final form of 20 mM histidine / 200 mM HCl-arginine / 0.04% polysorbate 20 / pH 6.0. The volumes of the recovered set, diluted set, and conditioned set (set Q) were then analyzed individually for protein concentration. Table 15 summarizes the results.

Table 15

the of

<td></td><td>Volume (D</td><td>Concentration O (g / D</td><td>Pasta (g)</td><td>Income or {Loss} (%)</td>

<td>Q set</td><td> 1,082</td><td> 5,5438</td><td> 5.998,4</td><td> 100</td>

<td>Set recovered</td><td> 34, 95</td><td> 167,08</td><td> 5.839,8</td><td> 97, 4</td>

<td>Set Diluted</td><td> 38,2</td><td> 152,14</td><td> 5.810,3</td><td> 96, 7</td>

ΕΡ1786830Β1

Example 7

Effect of High Temperature on Product Quality

E25 samples at 30 g / l and 150 g / l in histidine and Q buffers were kept at various temperatures for 24 hours. The samples were subjected to turbidimetry measurement and SEC tests. Turbidimetry results against temperature for E25 at 30 g / 1 in buffer Q are shown in FIG. 18. FIG. 19 shows the amount of soluble E25 aggregate (150 g / l in 50 mM histidine buffer, pH 6.0) observed over time and the temperature of 23 ° C, 40 ° C, 50 ° C, 60 ° C and 70 ° C. The four time intervals (0 hour, 4 hours, 7.5 hours, and 24 hours) for each of these temperatures are shown as the set of four bars from left to right as 1810 and 1910 in FIGS. 18 and 19. The turbidimetry of the solution was essentially unchanged after 24 hours at 60 ° C. No significant soluble E25 aggregates were observed below 70 ° C, which suggests that product samples were substantially stable up to at least 60 ° C and at least 24 hours.

Example 8

Effect of High Temperature on Bioburden

E25 samples at 30 g / 1 in arginine and histidine buffer were inoculated with 10³ colony forming units per ml for two test organisms: one Gram positive (Staphylococcus aureus); and a negative Gram (Pseudomonas chlororaphis). Samples were taken after 1.5 hours and 6 hours. The results shown in the bar graphs of FIGS. 20 and 21 indicate that these organisms are both decreased with increasing temperature. The three temperature ranges (25 ° C, 40 ° C, and 50 ° C) for each observed time interval are shown as a set of three left-to-right bars as 2010 and 2110 in FIGS. 20 and 21. The inoculations shown were

861786830Β1 conducted in arginine buffer with protein concentrations at 30 g / 1.

Example 9

Effect of Temperature on Process Flow

Samples of 10 g / 1 E25 in 0.2 M arginine, 25 mM histidine buffer, pH 6.0 were evaluated for their influence on flow against transmembrane pressure (PTM). FIG. 22 shows that rising temperatures in the system also increased process flow during UF / DF operations. Flow tours at various volumes of concentrations and at three different temperatures of 23 ° C (2210), 40 ° C (2220) and 46 ° C (2230) were performed. The mass transfer coefficient and filtrate flow increased by about 2 to about 3 times providing considerably reduced process time.

Example 10

RhuMAB anti-CP20 High Concentration Formulation (2H7)

A pilot scale UF pilot system was used to concentrate / formulate anti-CD20 rhuMAb (2H7, a recombinant human monoclonal antibody). Example 1 was repeated with the following exceptions. A Millipore Pelicon Ultrafiltration / Diafiltration system was assembled with a 1.63 square meter membrane composed of cellulose regenerated to 30,000 Daltons. Each system consisted of a membrane holder, a Viking S3S rotary feed pump, 1.27 cm 316L stainless steel recirculation pipeline and a 40 1 recirculation vessel. Backpressure regulating valves were HD Baumann, Inc. The temperature Glycol feed in the tank liner has been raised or decreased regulated as needed using an electric heat exchanger, a cold glycol source, or both.

During this operation, the flow rate of the

Alimentação1786830Β1 feed was adjusted at a constant rate of 8.5 l / min (about 5.38 l / min / m²). FIG. 23 states that the value over time tends to the feed flow rate (210) 0 to 20 range, pH (212) range 2 to 12, filtrate flow rate (250) range 0 0 5, recycle tank (2320) scale 0 to 45, dP of retentate (2350) scale 0 to 100 during various process steps or mode including UF1 (10), DF1 (20), and UF2 (30).

During ultrafiltration and diafiltration operations the system used constant retentate pressure followed by a constant feed / retentate delta pressure control scheme to control flow through the membrane. Diafiltration buffer (30 mM sodium acetate / pH 4.9) was prepared in a separate tank. The temperature of this buffer was adjusted to 45 ° C prior to the process for accurate temperature control in the entire process. Prior to processing, the system storage solution (0.1 N NaOH) was equilibrated in a single pass to first drain with 10.76 l / m² water for injection (WFI) and then at 10.76 1 / m² of diafiltration buffer. After washes, the system was equilibrated by recirculation of 5.38 1 / m² of diafiltration buffer for 10 minutes. The pH of the recirculated solution was checked to confirm equilibrium.

The set of proteins that resulted from the above Q-Sepharose chromatography step was measured to be 2.31 g of 2H7 / 1 and had a volume of 356 l. The protein was in a 6 mM acid free HEPES / 19 solution. mM HEPES sodium salt and 25 mM sodium acetate which had been adjusted to pH 5.3 with 0.5 M acetic acid. Just prior to operation, the temperature of this set was adjusted to 45 ° C. To begin operation the protein pool was transferred to the recirculation vessel through a 0.22 micron pore filter at a level of about 40 l in the tank. In the container the set was

861786830Β1 stirred by top-mounted stirrer and the temperature was maintained at 40-50 ° C.

Because the incoming assembly is larger than the recirculation container, the UF1 process began in batch feed mode (see FIG. 23). In this mode, Set Q was added to the recirculation vessel at about the same rate as filtrate passed through the TFF membrane to the drain. After the remaining Set Q was transferred to the recirculation vessel, process UF1 continued in batch mode. During UF1 the pool was concentrated to 50 g of 2H7 / 1 (about 16 L). The pool was then diafiltered with 10 diavolumes of the diafiltration buffer. During diafiltration the recirculation vessel temperature set point was maintained at 40 to 50 ° C. Diafiltration was performed at a constant volume, which was achieved by combining the flow rate of the protected solution being transferred to the recirculation tank at the flow rate of the filtrate being removed from the system. At the end of diafiltration, the pool was further concentrated in the UF2 mode to a final concentration&gt; 190 g of 2H7 / 1 (4.31). See FIG. 23 incorporating the constant dP control at 344.74 kPa at the end of this phase. This phase was also performed by adjusting the temperature rise to 45 ° C +/- 5 ° C. Then maintaining a drop in pressure where the feed pump was controlled to maintain 137.9 kPa through the feed channel. A sample was drawn and a density measurement was performed to confirm concentration prior to recovery. The concentration of this sample was 189 g of 2H7 / 1. Table 16 summarizes the production and flow results.

ΕΡ1786830Β1

I love 16.

<td>Phase of process</td><td>Standard Production (g / m²/H)</td><td>Flow Normalized (LMH / kPa)</td>

<td>UF1</td><td> 344,45</td><td> 0, 69</td>

<td>DF</td><td> 602,78</td><td> 0,34</td>

<td>UF2</td><td> 2.873,99</td><td> 0,23</td>

Protein set was recovered by a series of steps. First, the product was displaced from the membrane in a single pass mode using 0.21 L of DF buffer added to the retentate line. The product was filtered into a recovery tank through a sterile 0.22 micron pore filter. The assembly in the recirculation tank was then pumped into the tank using a rotary lobe feed pump. The residual protein solution was then displaced below the tank line and the feed with a 34.47 kPa nitrogen gas flow. The final phase was a downward flow of the membrane unit, which now consisted mostly of DF buffer from the initial displacement of the product. This phase also used the 34.47 kPa nitrogen gas applied at the highest point on the retentate line.

If necessary, the recovered pool was first diluted to about 175 g of 2H7 / 1 using dilution buffer (30 mM sodium acetate, pH 5.3). Finally, the pool was diluted to a concentration of 150 g of 2H7 / 1 and conditioned to the final form of 30 mM sodium acetate, 49% trehalose, 0.21% polysorbate, pH 5.3.

The volumes of the recovered set, the diluted set, and the conditioned set were then analyzed for

ΕΡ1786830Β1 protein concentration. Table 17 presents the results.

Table 17

<td></td><td>Volume 1)</td><td>Concentration (g / D</td><td>Mass (g)</td><td>Yield or {Loss} (%)</td>

<td>Q set</td><td> 355,81</td><td> 2, 31</td><td> 821,92</td><td> 100, 0</td>

<td>Set Recovered</td><td> 4, 64</td><td> 180,02</td><td> 835, 3</td><td> 101, 6</td>

<td>Set Diluted</td><td> 4, 871</td><td> 149,40</td><td> 727, 7</td><td> 88,5</td>

<td colspan="5">Note: Yields include loss due to sampling. 0 volume and concentration recovered from the set include the addition of buffer offset.</td>

After the process, the membrane was regenerated using 0.1 N NaOH with a single pass wash of 10.76 1 / m²followed by total recirculation 5.38 1 / m² for 30 minutes. This was followed by washing at 10.76 1 / m² of AP (pure water). Followed by a total recirculation of the Minncare® solution for 30 minutes. The system was again washed with 10.76 l / m² of AP and finally recirculated for 15 minutes with 0.1 N NaOH and stored.

Example 11

RhuMAb Anti-CD20 High Concentration Formulation

A pilot scale UF pilot system was used to concentrate / formulate rhuMAb anti-CD20 (2H7) for use in a phase I clinical study in humans at a GMP manufacturing facility. Example 10 was repeated with the following exceptions.

The protein pool that resulted from the previous Q-Sepharose chromatography step was measured as

861786830Β1 being 3.729 g of 2H7 / 1 and had a volume of 262 1. The protein was in a solution of 6 mM acid free HEPES / 19 mM HEPES sodium salt and 25 mM sodium acetate which had been adjusted to a pH 5.3 with 0.5 M acetic acid. Just before operation, the temperature of this set was adjusted to 45 ° C. To begin operation the protein pool was transferred to the recirculation vessel through a 0.22 micron pore filter; at a level of about 40 1 in the tank. In the vessel the assembly was shaken by a top mounted stirrer and the temperature was maintained at 40-50 ° C.

During UF1 the pool was concentrated to 50 g of 2H7 / 1 (about 20 L). FIG. 24 demonstrates that the hourly value trend for the recycling tank (210) range from -0.713963 to 295.989, dP of retentate (2420), range from -0.237899 to 98.6629, feed flow rate (250) scale from -0.356981 to 147.994 and filtrate flow rate (2450) scale from -0.118994 to 49.3315 during the process. The pool was then diafiltered with 10 diavolumes of the diafiltration buffer. During diafiltration the recirculation vessel temperature set point was maintained at 40 to 50 ° C. Diafiltration was performed at a constant volume, which was achieved by combining the flow rate of the protected solution being transferred to the recirculation tank at the flow rate of the filtrate being removed from the system. At the end of diafiltration, the pool was further concentrated in the UF2 mode to a final concentration&gt; 190 g of 2H7 / 1 (5.25 l). See yourself at

FIG. 24 incorporating the constant dP control at 275.79 kPa at the end of this phase. This phase was also performed by adjusting the temperature rise to 45 ° C +/- 5 ° C. Then maintaining a drop in pressure where the feed pump was controlled to maintain 137.9 kPa through the feed channel. A sample was pulled and

ΕΡ1786830Β1 a density measurement was performed to confirm concentration before recovery. The concentration of this sample was 194 g of 2H7 / 1. Table 18 summarizes the production and flow results.

Table 18

<td>Process phase</td><td>Production Normalized (g / m²/H)</td><td>Flow Normalized (LMH / kPa)</td>

<td>UFI</td><td> 548,96</td><td> 0,55</td>

<td>DF</td><td> 495,14</td><td> 0,31</td>

<td>UF2</td><td> 3.078,50</td><td> 0,23</td>

Just before product recovery, a 30 ml sample was analyzed for detection and a bioburden titration. The result of this test was below the limit of detection (ie &lt;0.13 CFU / ml). The protein pool was recovered by a series of steps from Example 10. The volumes of the recovered pool, the diluted pool, and the conditioned pool were then analyzed for protein concentration. Table 19 presents the results. The membrane was regenerated as in Example 10.

ΕΡ1786830Β1

Table 19

<td></td><td>Volume 1)</td><td>Concentration (g / D</td><td>Mass (g)</td><td>Yield or {Loss} (%)</td>

<td>Q set</td><td> 262</td><td> 3,72</td><td> 977</td><td> 100</td>

<td>Set recovered</td><td> 5, 0</td><td> 174, 0</td><td> 863, 0</td><td> 88,3</td>

<td>Set Diluted</td><td> 5,421</td><td> 149, 6</td><td> 811, 0</td><td> 83, 0</td>

Example 12

RhuMAb Anti-CD20 GMP High Concentration Formulation

Example 11 was repeated with the following exceptions. The protein pool resulting from the above Q-Sepharose chromatography step was measured to be 5.106 g of 2H7 / 1 and had a volume of 196 1. The protein was in a 6 mM acid free HEPES / 19 mM solution. HEPES sodium salt and 25 mM sodium acetate which had been adjusted to pH 5.3 with 0.5 M acetic acid. Just prior to operation, the temperature of this set was adjusted to 45 ° C. To begin operation the protein pool was transferred to the recirculation vessel through a 0.22 micron pore filter at a level of about 40 l in the tank. In the vessel the assembly was shaken by a top mounted stirrer and the temperature was maintained at 40-50 ° C.

During the UFI the pool was concentrated to 50 g of 2H7 / 1 (about 20 L). FIG. 24 demonstrates that the hourly value trend for the recycling tank (210) scale from 0 to 150, dP of retentate (2520), scale from 0 to 100, feed flow rate (250) scale from 0 to 100 and filtrate flow rate (2550) scale from 0 to 50 during the process. The pool was then diafiltered with 10 diavolumes of the diafiltration buffer. During the

F1786830Β1 diafiltration The recirculation vessel temperature set point was maintained between 40 and 50 ° C. Diafiltration was performed at a constant volume, which was achieved by combining the flow rate of the protected solution being transferred to the recirculation tank at the flow rate of the filtrate being removed from the system. At the end of diafiltration, the pool was further concentrated in UF2 mode to a final concentration&gt; 190 g of 2H7 / 1 (5.26 L) again using the constant dP control at the end of this phase (see, FIG. 25 ). This phase was also performed by adjusting the temperature rise to 45 ° C +/- 5 ° C. Then maintaining a drop in pressure where the feed pump was controlled to maintain 137.9 kPa through the feed channel. A sample was drawn and a density measurement was performed to confirm concentration prior to recovery. The concentration of this sample was 191 g of 2H7 / 1. Table 20 summarizes the production and flow results.

Table 20

<td>Process phase</td><td>Production Normalized (g / m²/H)</td><td>Flow Normalized (LMH / kPa)</td>

<td>UF1</td><td> 721,19</td><td> 0,52</td>

<td>DF</td><td> 505,91</td><td> 0,30</td>

<td>UF2</td><td> 3.143,09</td><td> 0,26</td>

Just before product recovery, a 30 ml sample was analyzed for detection and a bioburden titration. The result of this test was below the limit of detection (ie &lt;0.13 CFU / ml). The protein pool was

861786830Β1 recovered by a series of the steps of Example 11. The volumes of the recovered set, diluted set, and conditioned set were then analyzed for protein concentration. Table 21 presents the results. The membrane was regenerated as in Example 11.

Table 21

<td></td><td>Volume 1)</td><td>Concentration (g / D</td><td>Mass (g)</td><td>Yield or {Loss} (%)</td>

<td>Q set</td><td> 19 6</td><td> 5,106</td><td> 1000</td><td> 100</td>

<td>Set Recovered</td><td> 4, 9</td><td> 187,1</td><td> 918, 0</td><td> 91,8</td>

<td>Set Diluted</td><td> 6,075</td><td> 150,9</td><td> 916, 9</td><td> 91,7</td>

The disclosure has been described with reference to various specific and preferred embodiments and techniques.

ΕΡ1786830Β1

DOCUMENTS REFERRED TO IN THE DESCRIPTION

This list of documents referred to by the author of this patent application is for the reader's information only. It is not an integral part of the European patent document. Notwithstanding its careful preparation, IEP assumes no responsibility for any errors or omissions.

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