WO2014020171A1 - Capacité tampon d'anticorps - Google Patents

Capacité tampon d'anticorps Download PDF

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Publication number
WO2014020171A1
WO2014020171A1 PCT/EP2013/066330 EP2013066330W WO2014020171A1 WO 2014020171 A1 WO2014020171 A1 WO 2014020171A1 EP 2013066330 W EP2013066330 W EP 2013066330W WO 2014020171 A1 WO2014020171 A1 WO 2014020171A1
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protein
composition
buffer
antibody
concentration
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PCT/EP2013/066330
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English (en)
Inventor
Patrick Garidel
Sven BAHRENBURG
Anne KAROW
Torsten Schultz-Fademrecht
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Boehringer Ingelheim International Gmbh
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Publication of WO2014020171A1 publication Critical patent/WO2014020171A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the invention concerns the field of biomolecule formulations. It concerns a composition, preferably a liquid formulation, of a therapeutic protein, especially an antibody, at high concentration for pharmaceutical use including the purpose of storage and administration to patients. Further, the invention concerns a method of administration of a liquid formulation of a therapeutic protein, especially an antibody, to a patient, wherein the formulation to be administered to the patient is obtained by diluting a composition which contains the protein at a concentration of at least 100 mg/ml into a larger volume of a solution.
  • mAbs monoclonal antibodies
  • Numerous mAbs are in the pipelines of biotech and pharmaceutical companies nowadays and over 20 antibody drugs are already on the market (Reichert 2011).
  • the increasing interest in the development of monoclonal antibodies as therapeutics is motivated by the following major advantages.
  • antibodies inherently possess the potential to act in a very specific manner. Also, they can be used as shuttles for conjugated drug-molecules by which the drug is more efficiently delivered to a specific target. Therefore, high potency and efficacy are attributed to antibody-based therapeutics and they are generally expected to cause fewer side effects than small molecular drugs (Wang 2007).
  • monoclonal antibodies are complex molecules that are susceptible to a variety of degradation routes. Chemical degradation damages the amino acid building blocks and the backbone of the protein molecules affecting the protein primary and secondary structure. In addition, physico-chemical processes can impair the tertiary structure whose integrity is crucial for the biologically active state. Biopharmaceutical development therefore needs to address the degradation issue and seek maximal stability of the antibody therapeutic. A further challenge lies in finding the correct application form of the therapeutic. Due to their proteinogenic origin, mAbs suffer from poor bioavailability when administered via the oral route. Therefore, antibody drug products for parenteral administration are developed.
  • variable region is susceptible to aggregation at acidic pH, oxidation at elevated temperature, and in the presence of trace metals, whereas the constant region is more susceptible to deamidation at alkaline pH region and oxidation at elevated temperature (compare chapter 9.4 in Rathore AS and Mhatre R 2011).
  • asparagine deamidation rates in both the Fc and Fab regions of a model mAb on buffer type, pH and temperature was analysed in a recent study by Pace AL et al. (Pace AL 2013), as deamidation of asparagine residues in mAbs can be a major route of degradation.
  • buffering agents also referred to herein as buffer excipients
  • buffer type and buffer concentration may also have an effect on protein stability, especially aggregation (Hovgaard L 2012).
  • Asparagine deamidation of a new mAb developed by Eli Lilly was dependent on pH and buffer type, with phosphate buffer formulations having higher deamidation rates than citrate buffer formulations (Zheng and Janis 2006). Therefore, there is a need to provide a method for the solution formulation of a desired therapeutic antibody, which stabilizes the antibody molecule at its optimal pH, and which does not necessitate the addition of buffer excipients to adjust the pH of the solution formulation.
  • the invention describes a composition, especially a liquid formulation comprising a pharmaceutical protein, especially an antibody, at high concentration of at least lOOmg/ml for pharmaceutical use including the purpose of storage and administration to patients, wherein said pharmaceutical protein /antibody is in an aqueous solution that buffers changes in pH in the absence of a buffer excipient.
  • the invention relates to a method of administrating a composition to a subject comprising the following steps:
  • composition comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, having a volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably 1 ml,
  • step b Diluting said composition of step a) into a larger volume of a solution, wherein the larger volume is between 50 ml to 1 L, or between 100 ml to 500 ml, preferably 250 ml, or 500 ml, or 250 ml to 500 ml,
  • step b) administering said solution of step b) to a subject.
  • the invention relates to a method of preparing a composition for administration to a subject comprising the following steps:
  • composition comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, having a volume of 0.1 ml to 20 ml, or
  • step b Diluting said composition of step a) into a larger volume of a solution, wherein the larger volume is between 50 ml to 1 L, or between 100 ml to 500 ml, preferably 250 ml, or 500 ml, or 250 ml to 500 ml.
  • the invention specifically concerns a (pharmaceutical) composition
  • a (pharmaceutical) composition comprising a
  • composition preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient.
  • protein/antibody provides 100% of the buffer capacity of the composition.
  • a (pharmaceutical) composition comprising a (pharmaceutical or preferably therapeutical) protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml is used, wherein said composition does not contain a buffer excipient.
  • a (pharmaceutical or preferably therapeutical) protein preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml is used, wherein said composition does not contain a buffer excipient.
  • the protein/antibody provides 100% of the buffer capacity of the composition provided in method step a).
  • compositions comprising a protein at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, may be obtained by reconstitution from a lyophilisate.
  • one or more further proteins may be added as a separate composition prior to or subsequently to diluting said composition of step a) into a larger volume of a solution, or as part of the larger volume of a solution used for the dilution of the composition of step a).
  • Said one or more further proteins may be selected from the group consisting of a therapeutic protein, preferably a therapeutic antibody, an antibody fragment, an Fc-fusion protein, a full- length immunoglobulin molecule, an immunoglobulin based molecule such as an
  • the invention provides a kit comprising in one or more containers a composition according to the invention comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, having a volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably 1 ml, and a larger volume of a solution according to the invention wherein the larger volume is between 50 ml to 1 L, or between 100 ml to 500 ml, preferably 250 ml, or 500 ml, or 250 ml to 500 ml.
  • Proteins and antibodies inherently possess a considerable amount of readily ionizable and thus potential buffering groups i.e., the amino acid side chains of histidine, aspartic acid, glutamic acid, arginine, tyrosine, cysteine and lysine.
  • the buffer capacity of antibody solutions scales linearly with protein concentration up to the regime of > 200 mg/ml protein. At 220 mg/ml its buffer capacity resembles the capacity of 30 mM citrate or 50 mM histidine buffer (range of pH 5.0-6.0).
  • the study also provides information on buffer characteristics for pH ranges down to 4.0 and up to 8.0. From theoretical considerations on the buffer capacity of proteins an upper limit of buffering can be inferred.
  • conventional buffer excipients are not necessary for high concentrated protein solutions to stabilize the pH of protein solutions.
  • concentration is at least 100 mg/ml or between 100 mg/ml to 220 mg/ml.
  • FIGURE 1 is a diagrammatic representation of FIG. 1 :
  • Buffer capacity of the ionizable groups of (A) citrate and (B) histidine (dotted lines, simulated for 50 mM buffering agent) in aqueous solution versus pH and the resulting total buffer capacity (solid line).
  • the total buffer capacity also includes the contribution of the water ionization equilibrium that becomes significant at pH ⁇ 3 (dashed line). Due to three carboxyl groups with pKa values that differ around 1 unit, citrate exhibits considerable buffer capacity over a broad pH range (pH 2.5-6.0). The buffering by the amino, the carboxyl and the imidazole group in histidine hardly overlaps and practically relevant buffer capacity is only found in a narrow range around pH 6.0.
  • FIGURE 2 is a diagrammatic representation of FIGURE 1
  • FIGURE 3 is a diagrammatic representation of FIG. 1 :
  • FIGURE 4
  • FIGURE 5
  • the data values were determined with 5 ml of a given solution using 0.1 or 1 N acid or base at 25 °C.
  • the addition of titrant was normalized to micro equivalents of base or acid added per milliliter of solution.
  • the lines represent linear fits to the data points. The slopes give the buffer capacity plotted in Figure 8.
  • Titration curves for 50 mg/ml mAb3 and 25 mM histidine buffer at different ionic strength were performed at 25 °C in the range of pH 5.0-6.0 (A) and 6.0-7.0 (B). The addition of titrant was normalized to microequivalents of acid added per milliliter of solution.
  • Buffering issues here understood as buffering of pH, are omnipresent throughout the total biopharmaceutical process. Changes in proton concentration can be caused by C0 2 intake, leaching or chemical reactions (Borchert 1989). To nevertheless ensure a stable pH is essential for protecting the therapeutic (e.g. a monoclonal antibody) from degradation during manufacturing, storage and application. Moreover, profound knowledge about buffering characteristics of a system in question is also necessary when controlled pH shifts take place (e.g. in virus inactivation steps, for ion exchange chromatography). This study summarizes buffer characteristics of buffer agents widely used in the biopharmaceutical setting, i.e., acetate, citrate, histidine, succinate, phosphate.
  • the van Slyke equation was derived for the aqueous solution of the buffer agent only.
  • the ionic strength / of the buffer itself and ionization equilibria of additional substances such as NaCl are not considered but will influence the equilibrium of the buffer (cf. simplified Debye Huckel equation, equation 6, adapted from Scopes 1982).
  • This effect will play a significant role for large changes in ionic strength.
  • the effect on buffer capacity close to the pKa seems to be rather negligible.
  • uncharged excipients such as commonly used sugars (Hamada 2009), for example 10% a-trehalose in Lucentis® (Genentech, San Francisco USA), do not influence the ionization of the buffer agent via this mechanism.
  • the buffering power is in principal equal for any (monovalent) buffer, just the pH range in which a certain average buffer capacity is observed, is specific.
  • comprehensive information on buffer attributes can be found for proteins in physiological settings (vanSlyke 1922, Hainsworth 1986, Leem 1999, Lamanda 2007 and others). Findings on buffer capacity of antibodies in vitro for pH ranges down to pH 4.0 and up to 8.0 are summarized in the current study.
  • the prediction of buffering power by antibodies is not straightforward. Consistently with results of others (Gokarn 2008), models employed here that sum up the contributions of histidine; aspartate and glutamate moieties in the protein overestimate the buffer capacity. Thus, the theoretically derived values might serve as setting an upper limit.
  • the studied antibodies exhibit strong buffering in the range of pH 4.0-6.0. This range is within the pH range of maximal chemical stability of proteins (pH 4.0-7.0) and is advisable for a self-buffering formulation.
  • the buffer capacities we determined experimentally indicate the antibodies can sufficiently buffer proton changes due to leaching occurring during storage and handling (Preston 1984, Borchert 1989, White 2008).
  • An antibody solution of 50 mg/ml at pH 6 buffers around 1 mM of protons before the pH drops to pH 5.75. This amount surpasses the amount of protons leached from borosilicate glass vials by a factor of 100 (Bahrenburg & Garidel unpublished observations).
  • Biopharmaceuticals are handled at room temperature, stored at 5°C and kept at up to 40°C in stability studies thus the effect of temperature on buffer capacity needs to be considered.
  • the temperature dependence of an equilibrium reaction is factorized via the enthalpy of the ionization reaction. If the standard molar ionization enthalpy is close to zero (e.g. for carboxyl groups and phosphoric acids as well as organic derivatives of them Goldberg 2002) pKa values are hardly affected by temperature changes.
  • the buffer capacity in the tested antibodies is for the most part provided by the carboxyl side chains of aspartate and glutamate (table 2). Consistently, we found that temperature changes in the range of 5 to 40°C do not affect the buffer capacity of monoclonal antibodies in the range of pH 4.0-8.0.
  • the inverse value of the slope yields the average buffer capacity ⁇ for the chosen pH range.
  • the buffer capacity describes how many microequivalents can be added to 1 ml of the buffer or antibody system before changing the pH one unit.
  • Equation 1 is employed to buffer systems that are characterized by a single pKa value only. More complex systems are described by equation 2 adding up various pKa values to a total buffer capacity.
  • Figure 1A and IB the effect of three ionizable groups in citrate and histidine on the total buffer capacity is depicted in Figure 1A and IB respectively.
  • the pKa values of three carboxyl groups are around 1 unit apart leading to a constantly large buffer capacity over a broad range (pH 2.5-6.0) ( Figure 1 A).
  • the pKa values of the amino group, the carboxyl group and the imidazole moiety in histidine differ up to 4 units.
  • the buffer effects of these groups hardly overlap and significant buffer capacity is found in narrow pH ranges only ( Figure IB).
  • the subscript i refers to the amino acids aspartic acid, glutamic acid and histidine (relevant for the buffering within the pH range of interest)
  • N is the number of amino acids of the mAb
  • CmAb is the molar concentration of mAb
  • Ka is the acid dissociation constant of the corresponding amino acid side-chain.
  • the graphs in Figure 5 A and 5B also include the experimentally determined buffer capacities of the conventional buffer citrate and histidine respectively at different concentrations in the pH-range pH 6.0-5.0 (secondary axis).
  • the monoclonal antibodies exhibit inherent buffer capacity that at 50 mg/ml resembles the capacity of 6 mM citrate or 14 mM histidine buffer in the pH-range of pH 6.0-5.0.
  • 10 mM histidine is a commonly used buffer (e.g. end- formulation of Rituximab (Wang2007)), we conclude the self-buffering effect of a 50 mg/ml antibody solution is sufficient to replace the buffer excipient.
  • Table 5 summarizes the corresponding values from titrations of antibody solutions containing NaCl or trehalose.
  • self-buffering antibody solutions comprising a therapeutic antibody at a concentration of at least 100 mg/ml are capable of maintaining a stable pH for the purpose of stabilizing the antibody solution during long-term storage.
  • the buffer capacity is nearly identical for all temperatures. This holds true for titrations at protein concentrations of 10 mg/ml and 100 mg/ml. Also, in the range of pH 6.0-7.0 and pH 7.0-8.0 a nearly identical buffer capacity was determined from titrations at 5 °C, room temperature and 40 °C (data not shown). The experiments indicate the inherent buffer capacity of mAbs is not significantly altered upon temperature changes within the range relevant for biopharmaceutic processes.
  • a dilution application such as the preparation of a solution for administration to a patient (e.g. by infusion), wherein an aqueous solution is used for infusion into a subject / patient, which is based on a) a concentrate of the therapeutic antibody, i.e. a small volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably 1 ml of a composition provided herein (> 100 mg/ml protein concentration), and b) a larger volume of e.g. 250 ml, 500 ml or between 50 ml to 1 L, 100 ml to 500 ml, 250 ml to 500 ml of a solution.
  • This larger volume of a solution is preferably an isotonic solution.
  • protein is used interchangeably with amino acid residue sequences or polypeptide and refers to polymers of amino acids of any length. These terms also include proteins that are post-translationally modified through reactions that include, but are not limited to,
  • glycosylation, acetylation, phosphorylation or protein processing Modifications and changes, for example fusions to other proteins, amino acid sequence substitutions, deletions or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence and a protein can be obtained with like properties.
  • antibody refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant regions genes as well as the myriad immunoglobulin variable region genes.
  • antibody includes a polyclonal, monoclonal, bi-specific, multi- specific, human, humanized, or chimeric antibody.
  • antibody and “immunoglobulin” are used interchangeably.
  • antibody is used in the broadest sense.
  • antibody comprises full-length immunoglobulin molecules as well as immunoglobulin based molecules such as
  • antibody specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity.
  • antibody specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) and antibody fragments.
  • Exemplary antibodies within the scope of the present invention include but are not limited to anti-CD20, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD52, anti-HER2/neu
  • mAb monoclonal antibody
  • Monoclonal antibodies are highly specific, being direct against a single antigenic site.
  • each mAb is directed against a single determinant on the antigen.
  • the mAbs are advantageous in that they can be synthesized by cell culture (hybridomas, recombinant cells or the like) uncontaminated by other immunoglobulins.
  • the mAbs herein include for example chimeric, humanized and human antibodies.
  • Chimeric antibodies are antibodies whose light and/or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions belonging to identical or homologous to corresponding sequences of different species, such as mouse and human. Or alternatively, whose heavy chain genes are belonging to a particular antibody class or subclass while the remainder of the chain is from another antibody class or subclass of the same or from another species. It covers also fragments of such antibodies.
  • the variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3.
  • a typical therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody (e.g. ATCC Accession No. CRL 9688 secretes an anti-Tac chimeric antibody), although other mammalian species may be used.
  • humanized antibodies refers to specific chimeric antibodies, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ' , F(ab)2 or other antigen-binding subsequences of antibodies), and which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary - determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary - determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all off the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin.
  • Humanized antibody comprising a human framework region and one or more CDRs from a non-human (usually a mouse or rat) antibody. Adjustments in framework amino acids might be required to keep antigen binding specificity, affinity and or structure of domain.
  • immunoglobulins are generally heterotetrameric glycoproteins of about 150 kDa, composed of two identical light and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulins isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has an amino terminal variable domain (VH) followed by carboxy terminal constant domains (CH). Each light chain has a variable N-terminal domain (VL) and a C-terminal constant domain (CL).
  • VH amino terminal variable domain
  • CH carboxy terminal constant domains
  • CL C-terminal constant domain
  • antibodies can be assigned to different classes. There are five major classes: IgA, IgD, IgE, IgG and IgM.
  • the heavy chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma and mu domains, respectively.
  • the mu chain of IgM contains five domains (VH, CHmul, CHmu2, CHmu3 and CHmu4).
  • the heavy chain of IgE also contains five domains while the heavy chain of IgA has four domains.
  • the immunoglobulin class can be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • the Fc region of a full antibody usually comprises two CH2 domains and two CH3 domains.
  • the amino acid sequences of immunoglobulin CH2 domains are known or are generally available to the skilled artisan (Kabat et al, 1991).
  • a preferred immunoglobulin CH2 domain within the context of the present invention is a human IgG and preferably from IgGl, IgG2, IgG3, IgG4, more preferably a human IgGl and IgG3 and even more preferred a human IgGl .
  • the immunoglobulin CH2 domain preferably begins at amino acid position equivalent to glutamine 233 of human IgGl and extends through amino acid equivalent to lysine 340 (Ellison and Hood, 1982).
  • the CH2 domain is a CH2 domain of an immunoglobulin having a single N-linked oligosaccharide of a human IgG CH2 domain.
  • the CH2 domain is preferably the CH2 domain of human IgGl .
  • desired proteins/polypeptides are for example, but not limited to insulin, insulinlike growth factor, hGH, tPA, cytokines, such as interleukines (IL), e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta, TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1, VEGF, and single domain antibodies (camelid derived antibodies). Also included is the production of erythropoietin or any other hormone growth factors and any other polypeptides that can serve as agonist
  • Fc-fusion proteins are defined as proteins which contain or are modified to contain at least the portion of the CH2 domain of the heavy chain immunoglobulin constant region comprising the single N-linked glycosylation site. According to the Kabat EU nomenclature (Kabat et al, 1991) this position is Asn297 in an IgGl, IgG2, IgG3 or IgG4 antibody.
  • the other part of the fusion protein can be the complete sequence or any part of the sequence of a natural or modified heterologous protein or a composition of complete sequences or any part of the sequence of natural or modified heterologous protein proteins.
  • immunoglobulin constant domain sequences may be obtained from any immunoglobulin subtypes, such as IgGl, IgG2, IgG3, IgG4, IgAl or IgA2 subtypes or classes such as IgA, IgE, IgD or IgM. Preferentially they are derived from human immunoglobulin, more preferred from human IgG and even more preferred from human IgGl and IgG3.
  • immunoglobulin subtypes such as IgGl, IgG2, IgG3, IgG4, IgAl or IgA2 subtypes or classes such as IgA, IgE, IgD or IgM.
  • Fc fusion proteins comprise MCPl-Fc, ICAM-Fc, EPO-Fc, scFv fragments or the like coupled to the CH2 domain of the heavy chain
  • Fc-fusion proteins can be constructed by genetic engineering approaches by introducing the CH2 domain of the heavy chain immunoglobulin constant region comprising the N-linked glycosylation site into another expression construct comprising for example other immunoglobulin domains, enzymatically active protein portions, or effector domains.
  • an Fc fusion protein according to the present invention comprises also a single chain Fv (scFv) fragment linked to the CH2 domain of the heavy chain
  • immunoglobulin constant region comprising e.g. the N-linked glycosylation site.
  • Fab fragments consist of the variable regions of both chains which are held together by the adjacent constant region. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced in the mean time by genetic engineering.
  • Further antibody fragments include F(ab')2 fragments, which may be prepared by proteolytic cleaving with pepsin.
  • Antibody fragments also include Fc fragments, and scFv fragments, as well as other antibody derivatives described further herein below.
  • scFv single- chain- Fv
  • scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and bio distribution properties as well as with increased binding avidity.
  • scFv were prepared as fusion proteins with multimerisation domains.
  • the multimerisation domains may be, e.g. the CH3 region of an IgG or coiled coil structure (helix structures) such as Leucin-zipper domains.
  • the interaction between the VH/VL regions of the scFv are used for the multimerisation (e.g. dia-, tri- and pentabodies).
  • diabody By diabody the skilled person means a bivalent homodimeric scFv derivative.
  • the shortening of the Linker in a scFv molecule to 5- 10 amino acids leads to the formation of homodimers in which an inter-chain VH/VL- superimposition takes place.
  • Diabodies may additionally be stabilised by the incorporation of disulphide bridges.
  • minibody By minibody the skilled person means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerisation region which is connected to the scFv via a Hinge region (e.g. also from IgGl) and a Linker region.
  • an immunoglobulin preferably IgG
  • IgGl the dimerisation region which is connected to the scFv via a Hinge region (e.g. also from IgGl) and a Linker region.
  • triabody By triabody the skilled person means a: trivalent homotrimeric scFv derivative. ScFv derivatives wherein VH-VL are fused directly without a linker sequence lead to the formation of trimers.
  • miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv.
  • the multimerisation is carried out by di-, trior tetrameric coiled coil structures.
  • sinaffold proteins a skilled person means any functional domain of a protein that is coupled by genetic cloning or by co-translational processes with another protein or part of a protein that has another function.
  • water means liquid hydrogen oxide in its meaning in the chemical arts.
  • WFI water for injection
  • composition means a mixture of substances.
  • composition means a mixture of substances including the therapeutically active substance for pharmaceutical use.
  • compositions/ pharmaceutical compositions of the present invention may be used to treat cancer or other abnormal proliferative diseases.
  • Cancers are classified in two ways: by the type of tissue in which the cancer originates (histological type) and by primary site, or the location in the body where the cancer first developed.
  • the most common sites in which cancer develops include the skin, lungs, female breasts, prostate, colon and rectum, the lymphoid system, cervix and uterus.
  • compositions are thus useful in the treatment of a variety of cancers, including but not limited to the following: AIDS-related cancer such as Kaposi's sarcoma; bone related cancer such as Ewing's family of tumors and osteosarcoma; brain related cancer such as adult brain tumor, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood cerebral astro cytoma/malignant glioma, childhood ependymoma, childhood medulloblastoma, childhood supratentorial primitive neuroectodermal tumors, childhood visual pathway and hypothalamic glioma and other childhood brain tumors; breast cancer;
  • AIDS-related cancer such as Kaposi's sarcoma
  • bone related cancer such as Ewing's family of tumors and osteosarcoma
  • brain related cancer such as adult brain tumor, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood cerebral astro cytoma/malignant glioma,
  • digestive/gastrointestinal related cancer such as anal cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumor, colon cancer, esophageal cancer, gallbladder cancer, adult primary liver cancer, childhood liver cancer, pancreatic cancer, rectal cancer, small intestine cancer and stomach (gastric) cancer; endocrine related cancer such as adrenocortical carcinoma, gastrointestinal carcinoid tumor, islet cell carcinoma (endocrine pancreas), parathyroid cancer, pheochromocytoma, pituitary tumor and thyroid cancer; eye related cancer such as intraocular melanoma, and retinoblastoma; genitourinary related cancer such as bladder cancer, kidney (renal cell) cancer, penile cancer, prostate cancer, transitional cell renal pelvis and ureter cancer, testicular cancer, urethral cancer, Wilms' tumor and other childhood kidney tumors; germ cell related cancer such as childhood extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ
  • gynecologic related cancer such as cervical cancer, endometrial cancer, gestational trophoblastic tumor, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, uterine sarcoma, vaginal cancer and vulvar cancer; head and neck related cancer such as hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, metastatic squamous neck cancer with occult primary, nasopharyngeal cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer and salivary gland cancer; hematologic/blood related cancer such as leukemias, such as adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia and hairy cell leukemia; and lymphomas, such as AIDS-related lymphom
  • lung related cancer such as non- small cell lung cancer and small cell lung cancer musculoskeletal related cancer such as Ewing's family of tumors, osteosarcoma, malignant fibrous histiocytoma of bone, childhood rhabdomyosarcoma, adult soft tissue sarcoma, childhood soft tissue sarcoma and uterine sarcoma
  • neurologic related cancer such as adult brain tumor, childhood brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependmoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma and other brain tumors such as neuroblastoma, pituitary tumor and primary
  • compositions/ pharmaceutical compositions of the present invention may be administered in a therapeutically effective amount in any conventional dosage form in any conventional manner.
  • parenteral routes of administration are useful in various embodiments of the invention: administration by intravenous, intraarterial, intracardiac, intraspinal, intrathecal, intraosseous, intraarticular, intrasynovial, intracutaneous, intradermal, subcutaneous, peritoneal, and/or intramuscular injection.
  • a therapeutically effective amount can be determined by a skilled artisan based upon such factors as weight, metabolism, and severity of the affliction etc.
  • the active compound is dosed at about 1 mg to about 500 mg per kilogram of body weight on a daily basis. More preferably the active compound is dosed at about 1 mg to about 100 mg per kilogram of body weight on a daily basis.
  • the invention provides a method of administrating a composition to a subject comprising the following steps:
  • composition comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, having a volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably 1ml,
  • step b Diluting said composition of step a) into a larger volume of a solution, wherein the larger volume is between 50 ml to 1 L, or between 100 ml to 500 ml, preferably 250 ml, or 500 ml, or 250 ml to 500 ml,
  • the invention also provides a method of preparing a composition for administration to a subject comprising the following steps:
  • composition comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, having a volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably 1 ml,
  • step b Diluting said composition of step a) into a larger volume of a solution, wherein the larger volume is between 50 ml to 1 L, or between 100 ml to 500 ml, preferably 250 ml, or 500 ml, or 250 ml to 500 ml.
  • the invention concerns a (pharmaceutical) composition
  • a (pharmaceutical and/or therapeutical) protein preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient.
  • a (pharmaceutical and/or therapeutical) protein preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient.
  • a (pharmaceutical and/or therapeutical) protein preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient.
  • said protein is a pharmaceutical protein.
  • said protein is a therapeutic protein.
  • the invention concerns a (pharmaceutical) composition
  • a (pharmaceutical) composition comprising a (pharmaceutical) protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein the buffer concentration in the composition is 0 mg/ml.
  • the invention concerns a (pharmaceutical) composition
  • a (pharmaceutical) composition comprising:
  • a (pharmaceutical) protein preferably an antibody or antibody fragment or Fc- fusion protein, at a concentration of at least 100 mg/ml and
  • composition comprising:
  • a (pharmaceutical) protein preferably an antibody or antibody fragment or Fc- fusion protein, at a concentration of at least 100 mg/ml and
  • said composition is an isotonic solution for example comprising salt like NaCl and/or sugar components like trehalose or saccharose.
  • said composition is an isotonic solution which does not contain buffer excipients.
  • said isotonic solution has a volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably 1 ml.
  • said composition has an osmolarity of approximately 308 mosmol/1 / an osmolality of around 300 mosmol/kg.
  • said composition is a liquid.
  • said composition is a liquid formulation.
  • said composition is an aqueous solution.
  • said aqueous solution is an isotonic solution such as 0.9% m/V NaCl in water.
  • said liquid isotonic solution such as 0.9% m/V NaCl in water.
  • compositions and methods of the invention changes in pH in the absence of a buffer excipient.
  • protein/antibody provides 100% of the buffer capacity of said composition.
  • the composition is non-isotonic.
  • said non-isotonic composition is a non-isotonic solution.
  • said non-isotonic solution has a volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably 1 ml.
  • the only other component of the composition besides the protein component is water (H 2 0), preferably water for injection (WFI).
  • WFI water for injection
  • said composition comprising only protein (e.g.
  • antibody and water has a volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably 1 ml.
  • said non-isotonic composition and/or said composition comprising only protein and water is diluted into an excess volume / a larger volume of a buffer solution and/or an isotonic solution.
  • said non-isotonic composition and/or said composition comprising only protein and water is diluted into an excess volume / a larger volume of an isotonic solution which does not contain a buffer excipient.
  • the composition of the invention is diluted into an excess volume / a larger volume of an isotonic solution, preferably with an osmolarity of around 308 mosmol/1 / an osmolality of around 300 mosmol/kg, which does not contain a buffer excipient.
  • said isotonic solution is a physiological salt solution (9 g NaCl in 1 Liter (0.9 % m/V) of water, preferably with an osmolarity of around
  • said excess volume / larger volume of a buffer solution and/or an isotonic solution has a volume of 250 ml, 500 ml or between 50 ml to 1 L, 100 ml to 500 ml, or 250 ml to 500 ml. In a specific embodiment of the present invention said excess volume / larger volume of a solution does not contain a buffer excipient.
  • the protein is a therapeutic protein, preferably a therapeutic antibody, an antibody fragment, an Fc-fusion protein, a full-length immunoglobulin molecule, an immunoglobulin based molecule such as an immunoglobulin fragment, an immunoglobulin isoform, a fusion protein comprising at least one immunoglobulin chain, or an
  • immunoglobulin conjugated to a non-proteinaceous moiety conjugated to a non-proteinaceous moiety.
  • the protein (preferably an antibody) concentration is at least 110 mg/ml, at least 115 mg/ml, at least 120 mg/ml, at least 150 mg/ml, at least 200 mg/ml, at least 220 mg/ml, at least 300 mg/ml or within the range of 100 mg/ml to 220 mg/ml or 100 mg/ml to 300 mg/ml.
  • the protein has a buffer capacity of at least that of approximately 4.0 mM citrate buffer in the pH range of pH 4.0 to 7.0 under the same conditions.
  • the protein concentration is approximately 100 mg/ml.
  • composition has been approved for pharmaceutical use by an authority legally empowered to grant such approval.
  • the protein has a buffer capacity of approximately 60 mM citrate buffer in the pH range of pH 4.0 to 5.0 under the same conditions.
  • the protein concentration is approximately 220 mg/ml.
  • the composition has been approved for pharmaceutical use by an authority legally empowered to grant such approval.
  • the protein/antibody has a buffer capacity as listed below: pH-range cone. protein/mAb [mg/ml] cone. Citrate [mM] buffer capacity [ ⁇ / ⁇ ]
  • the antibody solution of 220 mg/ml protein in trehalose or NaCl exhibits a buffer capacity resembling the capacity of around 60 mM citrate buffer.
  • the antibody solution of 220 mg/ml protein in trehalose or NaCl exhibits a buffer capacity resembling the capacity of around 50 mM histidine buffer or around 30 mM citrate buffer.
  • the pH of the composition maintained by the buffering action of the protein is between approximately pH 4.0 and pH 8.0 (see table 4), preferably pH 4.0 to pH 7.0 , or pH 4.0 to pH 6.0, or pH 5.0 to 7.0. More preferably, the pH of the composition maintained by the buffering action of the protein is pH 4.0 to pH 5.0, pH 5.0 to pH 6.0, or pH 6.0 to pH 7.0. In specific embodiments the pH of the solution obtained in step b) is between approximately pH 4.0 and pH 8.0, preferably pH 4.0 to pH 7.0, or pH 4.0 to pH 6.0, or pH 5.0 to 7.0. More preferably, the pH of the solution obtained in step b) is pH 4.0 to pH 5.0, pH 5.0 to pH 6.0, or pH 6.0 to pH 7.0.
  • the antibody is a full-length immunoglobulin molecule, an immunoglobulin based molecule such as an immunoglobulin fragment, an immunoglobulin isoform, a fusion protein comprising at least one immunoglobulin chain, or an immunoglobulin conjugated to a non-proteinaceous moiety.
  • composition comprising a protein at a concentration of at least 100 mg/ml is provided by reconstitution of a lyophilisate and/or by removing residual buffer excipients, preferably via dialysis or ultrafiltration diafiltration (UF/DF) .
  • UF/DF ultrafiltration diafiltration
  • one or more further proteins may be added as a separate composition prior to or subsequently to diluting said composition of step a) of the methods of the invention into a larger volume of a solution, or as part of the larger volume of a solution used for the dilution of the composition of step a).
  • Said one or more further proteins may be selected from the group consisting of a therapeutic protein, preferably a therapeutic antibody, an antibody fragment, an Fc-fusion protein, a full-length immunoglobulin molecule, an immunoglobulin based molecule such as an immunoglobulin fragment, an immunoglobulin isoform, a fusion protein comprising at least one immunoglobulin chain, or an immunoglobulin conjugated to a non-proteinaceous moiety.
  • the one or more further proteins are added as a separate composition comprising the one or more further proteins at a (total) concentration of at least 100 mg/ml.
  • the one or more further proteins may provide 100% of the buffer capacity of the separate composition comprising the one or more further proteins.
  • the protein, or the protein and the one or more further proteins provide/s 100% of the buffer capacity of the solution obtained in step b) of the methods according to the invention.
  • the (total) concentration of the one or more further proteins in the separate composition may be at least 110 mg/ml, at least 115 mg/ml, at least 120 mg/ml, at least 150 mg/ml, at least 200 mg/ml, at least 220 mg/ml, at least 300 mg/ml or within the range of 100 mg/ml to 220 mg/ml or 100 mg/ml to 300 mg/ml.
  • the invention furthermore provides a kit comprising in one or more containers a composition according to the invention comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, having a volume of 0.1 ml to 20 ml, or 1 ml to 10 ml, preferably
  • the kit may optionally also comprise instructions.
  • composition obtained by the methods according to the present invention.
  • compositions described herein are for use as a medicament.
  • compositions described herein or the kit described herein are for use in a method for the treatment of a patient suffering from any disease, preferably cancer.
  • compositions comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, comprising removing residual buffer excipients, preferably via dialysis or ultrafiltration/diafiltration (UF/DF).
  • a buffer excipient comprising removing residual buffer excipients, preferably via dialysis or ultrafiltration/diafiltration (UF/DF).
  • the invention also relates to the following items:
  • a method of administrating a composition to a subject comprising the following steps: a) Providing a composition comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient,
  • step b) Diluting said composition of step a) into a larger volume of a solution
  • step b) Administrating said solution of step b) to a subject.
  • a method of preparing a composition for administration to a subject comprising the following steps:
  • composition comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient,
  • step b) Diluting said composition of step a) into a larger volume of a solution.
  • a kit comprising in one or more containers
  • composition according to the invention comprising a protein, preferably an antibody or antibody fragment or Fc-fusion protein, at a concentration of at least 100 mg/ml, wherein said composition does not contain a buffer excipient, having a certain volume, and
  • mAbl monoclonal antibodies
  • mAb2 monoclonal antibodies
  • mAb3 monoclonal antibodies
  • Base and acid standard solutions (1 N or 0.1 N HC1 and accordingly 1 N or 0.1 N NaOH; volumetric) used for the titration studies were purchased from Sigma- Aldrich (St. Louis, MO, USA). All solutions were prepared using water for injection (WFI).
  • the buffer systems being investigated in terms of buffer capacity were acetate, citrate, succinate, phosphate and histidine buffers. Chemicals used in the preparation thereof were of analytical grade. The buffers were prepared at concentrations of 10, 25 and 50 mM. To simulate conditions close to the situation in the development of sub-cutaneous formulations, the solutions also contained 130 mM NaCl (acetate buffer), 125 mM NaCl (citrate and succinate buffer), 135 mM NaCl (phosphate), 140 mM NaCl (histidine buffer). These saline contents each plus the contribution of 25 mM buffer salt adjust the solutions to isotonicity. Antibody solutions were prepared as follows.
  • the bulk was first ultrafiltered over a tangential flow filtration system (Sartorius, Goettingen, Germany) to a final concentration of approximately 100 mg/ml.
  • the concentrated solution was diafiltered against a solution containing 160 mM NaCl (or 10 mM as indicated).
  • the minimum of diavolume exchanges was at least eight exclusion volumes.
  • Protein dilutions were prepared in 160 mM NaCl (or 10 mM as indicated). mAb4 was ultrafiltered against water for injection obtaining a protein concentration > 200 mg/ml. The solution was then adjusted to isotonicity ( ⁇ 300 mosmol/kg) by spiking with NaCl or trehalose (as indicated). Dilutions of mAb4 were prepared with aqueous NaCl or trehalose retaining around 300 mosmol/kg of the solution being titrated.
  • Titration curves were generated by adding HC1 or NaOH standard solutions to 5 ml of each buffer system in a clean lOR-glass vial (Fiolax Clear Glass Vials, Schott forma vitrum, Mullheim, Germany), mixing and measuring the pH value (WTW pH 340,ticianlich- Technische performanceen, Weilheim, Germany).
  • the pH meter had been calibrated via a two point calibration using standard buffer solutions pH 4.0 and 7.0 (Mettler-Toledo,
  • the pH was plotted versus the microequivalents ⁇ Eq) of acid or base added for each titration normalized for the solution volume.
  • ⁇ Eq is equal to the micromoles of titrant added.
  • Titration curves were dissected in segments and the data points of each segment were fitted to a linear equation. The inverse value of the slope yields the average buffer capacity ⁇ for the chosen pH range.
  • the buffer capacity describes how many micro equivalents can be added to 1 ml of the buffer or antibody system before changing the pH one unit.
  • buffer capacity of a certain buffer system was plotted versus buffer concentration and the linear fit thereof yields the 'slope of capacity'.
  • buffer capacities of different buffer systems can be compared and the buffer capacities at further concentrations can be extrapolated.
  • Equation 1 is employed to buffer systems that are characterized by a single pKa value only. More complex systems are described by equation 2 adding up various pKa values to a total buffer capacity.
  • Figure 1A and IB the effect of three ionizable groups in citrate and histidine on the total buffer capacity is depicted in Figure 1A and IB respectively.
  • the pKa values of three carboxyl groups are around 1 unit apart leading to a constantly large buffer capacity over a broad range (pH 2.5-6.0) ( Figure 1 A).
  • the pKa values of the amino group, the carboxyl group and the imidazole moiety in histidine differ up to 4 units.
  • the buffer effects of these groups hardly overlap and significant buffer capacity is found in narrow pH ranges only ( Figure IB).
  • Table 1 summarizes the pKa values used for the calculations and Table 2 the number of the amino acids in niAbsl-3 contributing to the buffer capacity in the pH range of relevance.
  • Table 1 pKa values used for calculating theoretical buffer capacities (Lide2004 (at zero ionic strength, 25 °C), Hastingsl922; Thurlkill2006a (0.1 M KC1 at 25 °C))
  • the buffer capacity can only be determined for a discrete value by differentiating pH value with respect to acid/base equivalents added (equation 4, exemplarily B for base equivalents).
  • Table 3 summarizes the experimentally derived parameters for the buffer excipients acetate, citrate, succinate, phosphate and histidine which are widely used in protein science.
  • the parameters allow for the computation of the average buffer capacity for these buffers in a certain pH range at any desired concentration by multiplying parameter a with the buffer concentration of interest. This procedure is successfully applied in our lab. It allows for an estimate how much acid/base needs to be added to a buffered solution of known concentration to achieve a certain pH shift.
  • Table 3 lists the corresponding parameters for values simulated employing the van Slyke equation. Except for phosphate buffer, we find a good correlation between experimentally derived values and predicted values. Larger deviations can be seen for pH ranges in which the corresponding buffer exhibits comparably low buffer capacity (pH far from pKa). Here, a larger susceptibility of the parameters to experimental errors is anticipated.
  • the graphs in Figure 5 A and 5B also include the experimentally determined buffer capacities of the conventional buffer citrate and histidine respectively at different concentrations in the pH-range pH 6.0-5.0 (secondary axis).
  • the monoclonal antibodies exhibit inherent buffer capacity that at 50 mg/ml resembles the capacity of 6 mM citrate or 14 mM histidine buffer in the pH-range of pH 6.0-5.0.
  • 10 mM histidine is a commonly used buffer (e.g. end- formulation of Rituximab (Wang2007)), we conclude the self-buffering effect of a 50 mg/ml antibody solution is sufficient to replace the buffer excipient.
  • Table 4 additionally lists the parameters for mAbl, mAb2 and mAb3 determined via the theoretical models (equations 2 and 3).
  • the buffer capacities of all buffering amino acids cf. Tables 1, 2) are regarded as independently summing up to a total buffer capacity.
  • the slopes of capacity differ by a factor of 2. This might be due to the fact that the pKa values of the amino acid side chains can shift in the native protein environment compared to the theoretical pKa values for the ionizable side chain in isolation. As a result, they buffer in a pH range different from the predicted. We therefore suggest that the buffering characteristics should be derived experimentally for the antibody of interest.
  • Table 5 summarizes the corresponding values from titrations of antibody solutions containing NaCl or trehalose.
  • EXAMPLE 4 BUFFER CAPACITY OF ANTIBODIES AT VARYING TEMPERATURE
  • antibodies are commonly stored and shipped at 2-8 °C and for accelerated high temperature stability studies temperatures up to 40 °C are chosen.
  • any equilibrium constant (and therefore the pKa) varies with temperature, the buffer capacity in a defined pH range is also subject to change.
  • a temperature change has further consequences (e.g. changing conformational equilibria) and an estimation of the total effect of a temperature shift on the self-buffering characteristics of proteins is not straight-forward.
  • the monoclonal antibody (mAb) solution of more than 110 mg/ml not containing buffer excipient is used for storage.
  • a solution for infusion is prepared by diluting this solution into physiological salt solution in an infusion bag resulting in pH values of the composition as tabulated.
  • a first concentrate of the mAb which had a pH of 4.67 was diluted to the concentration of mAb indicated in the table with a physiological salt solution in an infusion bag.
  • the volume of the infusion bag was 50 ml.
  • the pH in the infusion bag was different from the apparent pH of the physiological salt solution in the infusion bag, which was pH 5.5.
  • the data demonstrate that, in the absence of a buffer in the infusion bag, the pH of the solution for infusion varies depending on the concentration of mAb in the solution for infusion. Therefore, the concentration of mAb in the solution for infusion may be selected such as to provide the optimal pH for the mAb.
  • the data shown here illustrate the procedure according to the methods of the invention for preparing a composition for administration to a subject, e.g., by infusion, wherein an isotonic solution compatible with administration to a patient was used for the dilution.
  • the mAb has a significant buffering capacity, which may be used to adjust the pH of the solution for infusion, especially in the range of pH 4.0 to 7.0, and more particularly, in the range of pH 4.0 to 6.0 or in the range of pH 5.0 to 7.0.

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Abstract

La présente invention concerne le domaine des formulations de biomolécules. Elle concerne une formulation liquide d'anticorps thérapeutiques à des concentrations élevées pour une utilisation pharmaceutique y compris des fins de stockage et d'administration aux patients. Sur la base de cette formulation, les anticorps sont dans une solution aqueuse qui tamponne les modifications de pH en l'absence d'un excipient tampon.
PCT/EP2013/066330 2012-08-03 2013-08-02 Capacité tampon d'anticorps WO2014020171A1 (fr)

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