Methods for preparing compositions comprising protein d polypeptides

Abstract

The present invention relates to a method for preparing an immunogenic composition. More particularly, the present invention relates to a method for preparing a liquid composition of a protein D polypeptide and its use in the preparation of an immunogenic composition comprising a protein D polypeptide, which can be used for the treatment or prevention of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in a subject, e.g. a human.

Description

Technical Field

[0001] This invention relates to a method for preparing an immunogenic composition. More particularly, this invention relates to a method for preparing a liquid composition of a protein D peptide and its use in preparing an immunogenic composition comprising the protein D peptide, said immunogenic composition being used to treat or prevent acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in a subject (e.g., a human). Background of the Invention

[0003] Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory condition that causes irreversible decline in lung function due to the inhalation of tobacco smoke or other irritants. COPD is considered to encompass several often co-occurring conditions (airflow obstruction, chronic bronchitis, bronchiolitis or small airway disease, and emphysema) (Wilson et al., Eur. Respir. J. 2001; 17:995–1007). Exacerbations are typically associated with increased wheezing and often include an increased cough that may produce mucus or purulent sputum (Wilson, Eur. Respir. J 2001 17:995-1007). COPD is physiologically defined as irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (Criteria for Diagnosis and Care of Patients with Chronic Obstructive Pulmonary Disease: American Thoracic Society. Am J Respir Crit Care Med. 1995 Nov; 152(5Pt 2): S77-121).

[0004] COPD is a leading cause of morbidity and mortality worldwide. In 2005, approximately one in 20 deaths in the United States had COPD as the underlying cause (Drugs and Aging 26:985–999 (2009)). It is projected that by 2020, COPD will rise to become the fifth leading cause of disability-adjusted life years (DAW), the fifth leading cause of chronic ineffective disease, and the third leading cause of mortality (Lancet 349:1498–1504 (1997)). The course of COPD is characterized by progressive worsening of airflow limitation and decline in lung function. COPD can be complicated by frequent and recurrent acute exacerbations (AEs), which are associated with significant healthcare costs and high morbidity (Proceedings of the American Thoracic Society 4:554–564 (2007)). A study showed that approximately 50% of acute exacerbations of COPD are caused by non-typed Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa (Drugs and Aging 26:985-999 (2009)). Haemophilus influenzae has been found in 20-30% of COPD exacerbations; Streptococcus pneumoniae in 10-15%; and Moraxella catarrhalis in 10-15% of COPD exacerbations (New England Journal of Medicine 359:2355-2365 (2008)). In Bangladesh, 20% of patients with COPD showed positive sputum cultures for Pseudomonas, Klebsiella, Streptococcus pneumoniae, and Haemophilus influenzae, while 65% of patients with AECOPD (acute exacerbation of COPD) showed positive cultures for Pseudomonas, Klebsiella, Acinetobacter, Enterobacter spp., Moraxella catarrhalis, and combinations thereof (Mymensingh Medical Journal 19:576-585 (2010)). However, the two most important measures proposed for preventing COPD exacerbations are active immunization and long-term maintenance drug therapy (Proceedings of the American Thoracic Society 4:554–564 (2007)).

[0005] One of the challenges in treating and managing COPD is the heterogeneity of this complex disease in terms of severity, progression, exercise tolerance, and the nature of symptoms. This complexity is also evident in acute exacerbations of COPD (AECOPD), which are brief and seemingly random periods of increased COPD symptoms requiring additional medical treatment and often hospitalization (Sethi et al., N Eng J Med 2008; 359:2355-65). Known exacerbation subtypes are defined by the nature of key triggering factors, including bacterial or viral infections and / or high eosinophil levels, and these events are often treated nonspecifically with a combination of antibiotics and steroids (Bafadhel et al., Am J Respir Crit Care Med 2011; 184:662). It is proposed to use protein D peptide from Haemophilus influenzae, together with PE-PilA fusion protein and UspA2 peptide from Moraxella catarrhalis, as a vaccine for the treatment or prevention of acute exacerbations of COPD (AECOPD), as described in WO2015125118A1.

[0006] There is a need for improved methods for preparing immunogenic compositions. In particular, there is a need for improved methods for preparing immunogenic compositions to help maintain the structure and function of protein antigens. These needs consider, but are not limited to, the chemical stability of the immunogenic composition (e.g., proteolysis or cleavage of proteins), the physical / thermal stability of the immunogenic composition (e.g., aggregation, precipitation, adsorption), the compatibility of the immunogenic composition with the container / sealing system, the interaction between the immunogenic composition and inactive components (e.g., buffers, salts, excipients, cryoprotectants), the method of preparation, the dosage form (e.g., lyophilized liquid), the environmental conditions encountered during transportation, storage, and handling (e.g., temperature, humidity, shear stress), and the duration between preparation and use.

[0007] A particular concern is the formation of visible particles in liquid compositions. The presence of particles depends on the preparation method and environment (design, identification, validation, and execution), as well as post-production handling, storage conditions, transportation, and end-user handling. This includes the selection and processing of the main packaging components, and the design and stability of the formulation, especially for biotechnology products. Regulatory monographs in Europe and the United States require parenteral medicines to be “virtually free” or “substantially free” of visible particles, respectively. (Serge Mathonet et al. PDA J Pharm Sci and Tech 2016, 70:392-408).

[0008] This invention addresses the need for an improved method for preparing liquid compositions of protein D peptides for use in preparing immunogenic compositions. According to the invention, the appearance of visible particles in liquid compositions of protein D peptides has been identified, and an improved method for improving stability and a liquid composition comprising a protein D peptide with improved stability are provided.

[0009] Invention Summary

[0010] According to the present invention, it has been found that protein D peptides readily form visible particles, particularly when held in a liquid composition. For example, the protein D peptide can be held in the liquid composition as an intermediate storage step (e.g., when measuring the amount of protein D peptide in the liquid composition) before mixing the liquid composition containing the protein D peptide with other antigens. It was previously unknown that protein D peptides readily aggregate, thus the observation of visible particles is surprising. The present invention provides a method for reducing the formation of visible particles from protein D peptides, thereby contributing to maintaining the structure and function of the protein antigen in an immunogenic composition. The method of the present invention comprises diluting the protein D peptide with a solution containing sucrose and poloxamer (e.g., poloxamer 188). According to the present invention, it has been found that adding sucrose and poloxamer to a liquid protein D peptide composition reduces particle formation while stabilizing the structure of the protein D peptide.

[0011] Therefore, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide (optionally the protein D polypeptide of SEQ ID NO: 2), wherein the method comprises mixing the protein D polypeptide with sucrose and poloxamer.

[0012] The present invention also provides a liquid composition comprising protein D polypeptide, sucrose and poloxamer.

[0013] The present invention also provides an immunogenic composition wherein the protein D polypeptide is prepared using the method of the present invention.

[0014] The present invention also provides immunogenic compositions of the present invention for treating or preventing acute exacerbations of COPD (AECOPD) in subjects (e.g., humans).

[0015] The present invention also provides the use of the immunogenic compositions of the present invention in the preparation of a medicament for treating or preventing acute exacerbations of COPD (AECOPD) in a subject (e.g., a human).

[0016] The present invention also provides a method for treating an acute exacerbation of COPD (AECOPD) in a subject (e.g., a human) at risk of developing an acute exacerbation of COPD (AECOPD), the method comprising administering an effective amount of the immunogenic composition of the present invention to the subject.

[0017] The present invention also provides a method for preventing acute exacerbation of COPD (AECOPD) in a subject (e.g., a human) at risk of developing an acute exacerbation of COPD (AECOPD), the method comprising administering an effective amount of the immunogenic composition of the present invention to the subject.

[0018] Detailed description

[0019] definition

[0020] As used herein, “adjuvant” refers to a compound or substance that, when administered to a subject together with a vaccine, immunotherapy agent or other composition containing an antigen or immunogen, increases or enhances the subject’s immune response to the administered antigen or immunogen (compared to the immune response obtained in the absence of an adjuvant).

[0021] As used herein, an "immunogenic fragment" is a smaller portion of an antigen than the whole that is capable of eliciting a fragment-specific humoral and / or cellular immune response in a host animal (e.g., a human). Therefore, a fragment, for example, of a genomic sequence does not include the genomic sequence itself, and a protein fragment does not include the full-length protein sequence itself. Protein fragments can be produced using techniques known in the art, such as recombination, proteolytic digestion, or chemical synthesis. Internal or terminal fragments of a polypeptide can be produced by removing one or more nucleotides from one end (for terminal fragments) or both ends (for internal fragments) of the nucleic acid encoding the polypeptide. The immunogenic fragments of the present invention can be derived from an amino acid sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a reference sequence (e.g., SEQ ID NO: 1 to 58 of the present invention), said reference sequence having been modified by deletion and / or addition and / or substitution of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids). Amino acid substitutions can be conserved or non-conserved. In one aspect, amino acid substitutions are conserved. Substitution, deletion, addition, or any combination thereof can be combined in a single variant, provided that the variant is an immunogenic polypeptide. For example, an immunogenic fragment can be derived by deleting a signal peptide.

[0022] As used herein, the term "conservative amino acid substitution" includes the substitution of a native amino acid residue with a non-native residue such that the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position has little or no effect, and does not result in a decrease in immunogenicity. Examples of such substitutions include: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications to a polypeptide sequence (and corresponding modifications to the encoding nucleotide) can produce polypeptides with similar functions and chemical characteristics to a reference polypeptide.

[0023] As used herein, "signal peptide" refers to a short polypeptide (less than 60 amino acids, e.g., 3-60 amino acids) present on a precursor protein (usually at the N-terminus) and is typically not present in the mature protein. Signal peptides (SPs) are usually rich in hydrophobic amino acids. Signal peptides direct the transport and / or secretion of translated proteins across the membrane. Signal peptides may also be referred to as targeting signals, transport peptides, localization signals, or signal sequences. For example, a signal sequence can be a co-translated or post-translational signal peptide.

[0024] As used herein, “subject” refers to mammals, including humans, non-human primates and non-primate mammals such as members of the genus Rodentia (including, but not limited to, mice and rats) and members of the order Lagomorpha (including, but not limited to, rabbits). In a particular embodiment, the subject is a human.

[0025] As further described below, an acute exacerbation of COPD (AECOPD) is an acute event characterized by a worsening of a patient's respiratory symptoms beyond the normal range of daily routine. AECOPD typically leads to changes in medications.

[0026] As used herein, the term “treatment of acute exacerbation of COPD (AECOPD)” refers to the improvement, stabilization, reduction, or elimination of increased symptoms that are characteristic of an acute exacerbation in a subject (e.g., a person).

[0027] As used in this article, “prevention of acute exacerbations of COPD (AECOPD)” means prevention of future acute exacerbations in subjects (e.g., humans), reducing their incidence or frequency, or reducing their severity (e.g., airflow obstruction, chronic bronchitis, bronchiolitis or small airway disease and emphysema).

[0028] As used herein, the term "treatment of disease caused by Haemophilus influenzae and / or Moraxella catarrhalis" refers to improving, stabilizing, reducing, or eliminating an increase in symptoms characteristic of bacterial infection caused by Haemophilus influenzae and / or Moraxella catarrhalis in a subject (e.g., a person).

[0029] As used in this article, the phrase “prevention of disease caused by Haemophilus influenzae and / or Moraxella catarrhalis” refers to the prevention of future bacterial infections caused by Haemophilus influenzae and / or Moraxella catarrhalis in subjects (e.g., humans), reducing their incidence or frequency, or reducing their severity.

[0030] As used in this article, "bacterial infection" refers to a bacterial infection with a total aerobic CFU count greater than or equal to 10 in routine cultures (Haemophilus influenzae or Moraxella catarrhalis). 7 A positive test for bacterial pathogens at a cell count. In a specific implementation, bacterial infection is associated with the following:

[0031] a) Haemophilus influenzae (e.g., untyped Haemophilus influenzae (NTHi));

[0032] b) Moraxella catarrhalis; or

[0033] c) Haemophilus influenzae (e.g., untyped Haemophilus influenzae (NTHi)) and Moraxella catarrhalis.

[0034] In the context of administering the immunogenic composition or vaccine of the present invention to a subject, as used herein, the term "effective amount" refers to the amount of the immunogenic composition or vaccine having preventive and / or therapeutic effects.

[0035] As used in this article, "w / v" refers to the weight / volume of the formulation.

[0036] The identity between peptides can be calculated using various algorithms. Typically, when calculating the percentage of identity, the two sequences to be compared are aligned to give the maximum correlation between them. This can include inserting “gap” in one or both sequences to enhance the degree of alignment. For example, the Needleman-Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48:443-453) for global alignment or the Smith-Waterman algorithm (Smith and Waterman 1981, J. Mol. Biol. 147:195-197) for local alignment can be used, for example, with default parameters (Smith-Waterman uses a BLOSUM 62 scoring matrix, with a gap opening penalty of 10 and a gap extension penalty of 1). Dufresne et al. described a preferred algorithm in Nature Biotechnology (Vol. 20, pp. 1269-71) in 2002, and this algorithm is used in GenePAST software (Genome Quest Life Sciences Inc., Boston, MA). The GenePAST "identity percentage" algorithm finds the best match between the query sequence and the subject sequence and expresses the alignment as a precise percentage. GenePAST does not adjust the alignment score based on considerations of biological relevance between the query and subject sequences. The identity between the two sequences is calculated over the entire length of both sequences and expressed as a percentage of a reference sequence (e.g., SEQ ID NO. 1 to 58 of this invention). For fragments, the reference sequence is the longest sequence.

[0037] As used herein, the term "particle" refers to both "visible particles" and "subvisible particles." In one embodiment, the particles have an average diameter of 35 to 70 μm.

[0038] As used herein, the term "visible particles" refers to insoluble or partially soluble solids in a liquid composition, such as an aqueous solution, that are visible to the human eye. In one embodiment, the visible particles have an average diameter of at least 50 μm. In another embodiment, the visible particles have an average diameter of 50-1000 μm. In another embodiment, the visible particles have an average diameter of 75-1000 μm. In yet another embodiment, the visible particles have an average diameter of 100-1000 μm. In one embodiment, the visible particles are visible when detected by the method described in Section 5.0, 2.9.20 of the European Pharmacopoeia. As used herein, "substantially free of visible particles" means a liquid composition free of visible particles according to the method described in Section 5.0, 2.9.20 of the European Pharmacopoeia.

[0039] As used in this article, "sub-visible particles" refers to particles that have passed the United States Pharmacopeia. <788> The Light Obscuration Particle Count Test described herein can detect particulate matter. In one embodiment, the subvisible particles have an average diameter of 2-175 μm. In another embodiment, the subvisible particles have an average diameter of 2-125 μm. In yet another embodiment, the subvisible particles have an average diameter of less than 50 μm. In yet another embodiment, the subvisible particles have an average diameter of 2-50 μm.

[0040] As used herein, "stable" means a composition that, when stored in a container or vial, does not show a significant increase in the number of visible particles over a specified period of time. In one embodiment, the composition also does not show a significant increase in the number of sub-visible particles over a specific period of time when stored in a container or vial. In some embodiments, the composition is stable for at least 1, 2, 3, 4, 5, 6, 7, or 14 days (i.e., the specified period of time is at least 1, 2, 3, 4, 5, 6, 7, or 14 days). Attached Figure Description

[0041] Figure 1 Visual inspection diagram; -, +, and ++ are depicted as 0, 5, and 10, respectively.

[0042] Figure 2 Day 1: The sum of visible particles from 35 to 70 micrometers: A significant interaction between sucrose and NaCl was observed.

[0043] Figure 3 Day 7, total of 35 to 70 micrometers: the significant effect of sucrose.

[0044] Figure 4 Day 7, total from 35 to 70 micrometers: the significant effect of NaCl.

[0045] Figure 5 On day 7, the average number of visible particles observed: a significant interaction between poloxamer 188 and pH was observed.

[0046] Figure 6 On day 7, the average value of visible particles observed: a significant effect of sucrose was observed.

[0047] Figure 7 Flowchart of the optimized method: Protein D dilution and filtration flowchart (in 150mM NaCl, 10% w / v sucrose, 1% w / v poloxamer 188, phosphate buffer 12.5mM PO4). 3 -KH2PO4 / K2HPO4, 1 mg / ml at pH 6.8).

[0048] Figure 8 :Flowchart of the reference method.

[0049] Figure 9 Occhio Particle Count: Represents the total number of particles ranging from 50 to 1000 μm detected by Occhio at three time points (1, 7, and 14 days) for the optimized liquid composition and the reference sample.

[0050] Figure 10 Example of a photograph of visible particles captured by Occhio for a protein D reference sample (1 mg / ml in 150 mM NaCl).

[0051] Figure 11 : Represents a multivariate analysis (PCA) that takes into account the entire range of light shading and Occhio measurements.

[0052] Figure 12 : Represents the average score given by observers from both black and white roles who conducted visual inspections on three different batches.

[0053] Figure 13 : Far UV circular dichroism.

[0054] Figure 14 : Far-UV circular dichroism differential spectrum.

[0055] Composition for diluting protein D peptide

[0056] This invention provides a method for preparing a liquid composition comprising a protein D polypeptide. The invention is based on the use of sucrose and / or poloxamer in diluting the protein D polypeptide to reduce protein D polypeptide particle formation. As described in the examples, it has been surprisingly found that adding sucrose and / or poloxamer to a liquid composition comprising a protein D polypeptide reduces the number of visible and subvisible particles formed in the liquid composition. The protein D polypeptide is mixed with a solution containing sucrose and / or poloxamer to form a liquid composition. Therefore, this invention provides an improved method for preparing a liquid composition of a protein D polypeptide with reduced particle formation. This invention also provides a liquid composition of a protein D polypeptide with improved stability. This invention provides a liquid composition of a protein D polypeptide that has improved stability compared to liquid compositions of a protein D polypeptide formulated without sucrose and poloxamer. Optionally, the method includes mixing the protein D polypeptide with sucrose and poloxamer. Therefore, a method for preparing a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2) includes mixing the protein D polypeptide with sucrose and poloxamer (e.g., poloxamer 188). In one embodiment, a method for preparing a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2) includes mixing the protein D polypeptide with sucrose, poloxamer (e.g., poloxamer 188), and a salt (e.g., NaCl). In another embodiment, a method for preparing a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2) includes mixing the protein D polypeptide with sucrose, poloxamer (e.g., poloxamer 188), a salt (e.g., NaCl), and a buffer (e.g., phosphate buffer). In another embodiment, the method includes mixing the protein D polypeptide with sucrose and poloxamer before mixing the protein D polypeptide with other antigens.

[0057] Protein D

[0058] As used herein, “protein D” and “PD” refer to protein D from Haemophilus influenzae. Protein D (PD) from Haemophilus influenzae is described in WO91 / 18926 and EP0594610. Protein D from Haemophilus influenzae can be EP0594610 (SEQ ID NO: 1). Figure 9 ( Figure 9The protein D sequence (a and 9b, a total of 364 amino acids). The protein D polypeptide can be the full-length protein D or an immunogenic fragment thereof (e.g., the protein D polypeptide is described in WO00 / 56360). For example, the protein D polypeptide may comprise (or consist of) the protein D fragment described in EP0594610, which begins with the sequence SSHSSNMANT(SerSerHisSerSerAsnMetAlaAsnThr) (SEQ ID NO: 3) and lacks the sequence from EP0594610. Figure 9 The 19 N-terminal amino acids in the protein D peptide are optionally supplemented with a tripeptide MDP from NS1 fused to the protein D fragment (348 amino acids) (i.e., SEQ ID NO: 2). Therefore, in one embodiment, the protein D peptide may comprise (or consist of) the amino acid sequence of SEQ ID NO: 2. In one embodiment, the protein D peptide is not conjugated to a polysaccharide (e.g., a polysaccharide from Streptococcus pneumoniae). In one embodiment, the protein D peptide is not conjugated to a polysaccharide from Streptococcus pneumoniae. In one embodiment, the protein D peptide is a free protein (e.g., unconjugated). In one embodiment, the protein D peptide is unlipidated.

[0059] SEQ ID NO 1: Protein D (364 amino acids)

[0060]

[0061] SEQ ID NO:2: Protein D fragment (348 amino acids) containing MDP tripeptide from NS1.

[0062]

[0063] Therefore, the protein D polypeptide sequence used in this invention can be modified, for example by truncating the N-terminal or C-terminal residues (e.g., deleting 19 amino acid residues from the N-terminus), by adding amino acid residues (e.g., adding a tripeptide MDP), or by substituting conserved amino acids. In one embodiment, the protein D polypeptide has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1. The immunogenic fragment of protein D may comprise an immunogenic fragment of at least 7, 10, 15, 20, 25, 30, or 50 consecutive amino acids of SEQ ID NO: 1. For example, the immunogenic fragment of protein D may comprise at least 7, 10, 15, 20, 25, 30, 50, 100, 200, or 300 consecutive amino acids of SEQ ID NO: 1, or at most 363 consecutive amino acids of SEQ ID NO: 1. The protein D polypeptide sequence (e.g., SEQ ID NO: 1) may be modified by deletion and / or addition and / or substitution of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids). The immunogenic fragment may trigger an antibody capable of binding to SEQ ID NO: 1. In another embodiment, the protein D polypeptide has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 2. The immunogenic fragment of protein D may contain at least 7, 10, 15, 20, 25, 30, or 50 consecutive amino acids of SEQ ID NO: 2. For example, the immunogenic fragment of protein D may contain at least 7, 10, 15, 20, 25, 30, 50, 100, 200, or 300 consecutive amino acids of SEQ ID NO: 2, or an immunogenic fragment of up to 347 consecutive amino acids of SEQ ID NO: 2. The immunogenic fragment of protein D may contain 100, 200, 300, 310, 320, 330, or 340 consecutive amino acids of SEQ ID NO: 2. The protein D polypeptide sequence (e.g., SEQ ID NO: 2) may be modified by deletion and / or addition and / or substitution of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids). The immunogenic fragment may trigger an antibody that binds to SEQ ID NO: 2.

[0064] In one embodiment, the method includes mixing the protein D peptide into a liquid composition at a concentration of 0.025 to 20 mg / ml, 0.5 to 10 mg / ml, or 0.5 to 1 mg / ml. Specifically, the concentration of the protein D peptide may be 0.5 mg / ml or 1 mg / ml. To achieve these target concentrations, the content of the protein D peptide can be analyzed using suitable techniques, such as RP-UPLC, and diluted accordingly.

[0065] sucrose

[0066] This invention is partly based on the use of sucrose in reducing particle formation in liquid formulations of protein D peptides. In one embodiment, the method includes mixing the protein D peptide with sucrose to a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v) sucrose. Specifically, the sucrose concentration can be 5%, 10%, 15%, or 20% (w / v). To achieve these target concentrations, a higher concentration of sucrose solution should be used during dilution. For example, to achieve a 10% (w / v) sucrose concentration, a 15.75% (w / v) sucrose solution can be mixed with the protein D peptide, but those skilled in the art will understand that variations are possible. In one embodiment, the invention provides a method for preparing a liquid composition comprising a protein D peptide (e.g., the protein D peptide of SEQ ID NO: 2), wherein the method includes mixing the protein D peptide with a solution containing sucrose. In another embodiment, the method includes mixing the protein D polypeptide with a solution containing sucrose to a concentration of, for example, 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v).

[0067] Polosham

[0068] This invention is partly based on the use of poloxamer in reducing particulate formation in liquid formulations of protein D peptides. Poloxamer is a nonionic triblock linear copolymer composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyethylene oxide (poly(ethylene oxide)). The length of the polymer can vary. The molecular weight of poloxamer can range from 7,500 to 15,000 or from 7,500 to 10,000. Suitably, poloxamer is selected from poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407. In one embodiment, the poloxamer is poloxamer 188 (PX188).

[0069]

[0070] Poloxamer 188 has a molecular weight of 7680 to 9510 Da. Khan et al. (European Journal of Pharmaceutics and Biopharmaceutics, 97(2015)60-67) generally describe the use of nonionic surfactants in therapeutic formulations.

[0071] In one embodiment, the method includes mixing the protein D peptide with poloxamer to a concentration of 0.1 to 1% (w / v) or 0.5 to 1% (w / v) poloxamer. Specifically, the concentration of poloxamer can be 0.5% or 1% (w / v). To achieve these target concentrations, a higher concentration of poloxamer solution should be used during the dilution process. For example, to achieve a concentration of 1% poloxamer (e.g., poloxamer 188), a 10% poloxamer solution (e.g., poloxamer 188) can be mixed with the protein D peptide, but those skilled in the art will understand that variations are possible.

[0072] In one embodiment, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), wherein the method comprises mixing the protein D polypeptide with a solution comprising poloxamer, for example to a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v). In another embodiment, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), wherein the method comprises mixing the protein D polypeptide with a solution comprising sucrose and poloxamer. In another embodiment, the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, for example to a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v), and (b) poloxamer (e.g., poloxamer 188), for example to a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v). In another embodiment, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide, wherein the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose at a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v), and (b) poloxamer (optionally poloxamer 188) at a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v).

[0073] Salt

[0074] As described in the examples, it has been found that adding salt to the liquid composition containing the protein D peptide also reduces the number of particles formed in the liquid composition (based on the sum of 35 to 70 micrometers). In one embodiment, a method for preparing a liquid composition containing a protein D peptide (e.g., the protein D peptide of SEQ ID NO: 2) includes mixing the protein D peptide with sucrose, poloxamer (e.g., poloxamer 188), and a salt (e.g., NaCl). Therefore, in one embodiment, the protein D peptide is mixed with sucrose, poloxamer (e.g., poloxamer 188), and a salt (e.g., NaCl). The salt may be, for example, sodium chloride, calcium chloride, or sodium phosphate. In one embodiment, the immunogenic composition of the present invention contains NaCl (sodium chloride).

[0075] A salt (e.g., NaCl) can be added to a concentration of 1 to 200 mM, suitably 10 to 200 mM, 50 to 200 mM, 100 to 200 mM, or 125 to 1755 mM. In particular, the concentration of the salt (e.g., NaCl) can be 150 mM. To achieve these target concentrations, a higher concentration of salt (e.g., NaCl) solution should be used during dilution. For example, to achieve a 150 mM salt (e.g., NaCl) concentration, a 1160 mM salt (e.g., NaCl) solution can be mixed with the protein D peptide, but those skilled in the art will understand that variations are possible.

[0076] In one embodiment, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide (optionally, the protein D polypeptide of SEQ ID NO: 2), wherein the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, (b) poloxamer (optionally, poloxamer 188), and (c) a salt (optionally, NaCl). In another embodiment, the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, for example, to a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v), and (b) poloxamer (e.g., poloxamer 188), for example, to a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v), and (c) a salt, such as NaCl.

[0077] buffer solution

[0078] In another embodiment, a method for preparing a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2) includes mixing the protein D polypeptide with sucrose, poloxamer (e.g., poloxamer 188), a salt (e.g., NaCl), and a buffer (e.g., phosphate buffer). In one embodiment, the buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is a phosphate, succinate, histidine, or citrate buffer. In some embodiments, the buffer is a phosphate buffer, suitably potassium phosphate (e.g., KH₂PO₄ / K₂HPO₄).

[0079] The buffer solution can be added to a concentration of 5 to 50 mM, suitably 10 to 40 mM, 10 to 30 mM, 10 to 20 mM, or 10 to 15 mM. Specifically, the buffer concentration can be 10.5 mM, 11.0 mM, 11.5 mM, 12.0 mM, 12.5 mM, 13.0 mM, 13.5 mM, 14.5 mM, or 15.0 mM. To achieve these target concentrations, a higher concentration of buffer solution (e.g., phosphate buffer) should be used during dilution. For example, to achieve a 12.5 mM buffer (e.g., phosphate buffer) concentration, a 100 mM buffer solution (e.g., phosphate buffer) can be mixed with the protein D peptide, but those skilled in the art will understand that variations are possible.

[0080] In one embodiment, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide (optionally, the protein D polypeptide of SEQ ID NO: 2), wherein the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, (b) poloxamer (optionally, poloxamer 188), (c) a salt (optionally, NaCl), and (d) a buffer (optionally, phosphate buffer). In another embodiment, the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, for example, at a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v), and (b) poloxamer (e.g., poloxamer 188), for example, at a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v), and (c) a salt, such as NaCl, and (d) a buffer (e.g., phosphate buffer).

[0081] pH

[0082] In one embodiment, the pH of the liquid composition can be adjusted to pH 5.5 to 8.5, pH 6.0 to 8.0, pH 6.4 to 7.7, pH 6.4 to 7.4, pH 6.4 to 6.9, pH 6.5 to 7.7, pH 6.5 to 7.4, pH 6.5 to 6.9, pH 6.8 to 7.7, pH 6.8 to 7.4, or pH 6.8 to 6.9. Specifically, the pH of the liquid composition of the present invention can be adjusted to pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, or pH 7.7. To achieve the target pH, a solution with a higher pH can be used during dilution. Adjusting the pH to achieve the target pH is within the scope of those skilled in the art. For example, to achieve a pH of 6.8, a solution with a pH of 6.9 can be mixed with a liquid composition containing the protein D polypeptide, but those skilled in the art will understand that variations are possible.

[0083] In one embodiment, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide (optionally, the protein D polypeptide of SEQ ID NO: 2), wherein the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, (b) poloxamer (optionally, poloxamer 188), (c) a salt (optionally, NaCl), and (d) a buffer (optionally, phosphate buffer) to achieve a pH of 6.4 to 7.7, for example, pH 6.8. In another embodiment, the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, for example, at a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v); and (b) poloxamer (e.g., poloxamer 188), for example, at a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v); and (c) a salt, for example, NaCl; and (d) a buffer (e.g., phosphate buffer) to achieve a pH of 6.4 to 7.7, for example, pH 6.8.

[0084] Thawing Protein D Peptide

[0085] Protein D peptides are typically stored frozen (e.g., at -45°C, pH 6.8) and must be thawed before formulation. Thawing is a change from a frozen state to a liquid or semi-liquid state. The method of the present invention suitably includes thawing protein D peptides. In one embodiment, the method includes the steps of: (i) thawing the protein D peptide, and (ii) mixing the protein D peptide with sucrose and poloxamer. This forms a liquid composition comprising the protein D peptide. In another embodiment, the method includes the steps of: (i) thawing the protein D peptide, and (ii) mixing the protein D peptide with sucrose, poloxamer, and salt. In yet another embodiment, the method includes the steps of: (i) thawing the protein D peptide, and (ii) mixing the protein D peptide with sucrose, poloxamer, salt, and buffer solution. In another embodiment, the method includes the steps of: (i) thawing the protein D polypeptide, and (ii) mixing the protein D polypeptide with: (a) sucrose, for example to a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v) and (b) poloxamer (e.g., poloxamer 188), for example to a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v). In another embodiment, step (ii) includes mixing the protein D polypeptide with: (a) sucrose, for example, at a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v), and (b) poloxamer (e.g., poloxamer 188), for example, at a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v), and (c) a salt, for example, NaCl. In another embodiment, step (ii) comprises mixing the protein D polypeptide with: (a) sucrose, for example, at a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v); and (b) poloxamer (e.g., poloxamer 188), for example, at a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v); and (c) a salt, such as NaCl; and (d) a buffer (e.g., phosphate buffer). In one embodiment, step (ii) is performed to achieve a pH of 6.4 to 7.7, suitably pH 6.8 (i.e., the pH of the mixed composition).

[0086] Steps (i) and (ii) can be performed simultaneously or sequentially. In one embodiment, steps (i) and (ii) occur simultaneously. In another embodiment, steps (i) and (ii) occur sequentially, with step (i) following step (ii). For example, step (i) can be performed by raising the temperature of the protein D polypeptide, for example, by raising the ambient temperature. Suitably, step (i) is performed statically. Suitably, step (i) is performed in an incubator. In one embodiment, step (i) is performed at 1 to 35°C. For example, step (i) can be performed at 2 to 35°C, 10 to 35°C, 20 to 35°C, 2 to 30°C, 10 to 30°C, 20 to 30°C, 2 to 25°C, or 23 to 27°C. In particular, step (i) can be performed at room temperature, for example, at 25°C. In one embodiment, step (i) is performed at 1 to 35°C, for example at 2 to 35°C, or 10 to 35°C, or 15 to 30°C, suitably at room temperature (e.g., 25°C). In one embodiment, step (i) is performed at 1 to 35°C, followed by step (ii).

[0087] Step (i) may also include homogenization of the protein D peptide. In one embodiment, step (i) includes thawing and homogenizing the protein D peptide. For example, the protein D peptide can be homogenized by stirring (e.g., with a magnetic rod) at 100 to 200 RPM, such as 150 RPM, for 5 to 10 minutes, such as 5 minutes.

[0088] Step (ii) comprises mixing the protein D peptide with: (a) sucrose, for example, to a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v), and (b) poloxamer (e.g., poloxamer 188), for example, to a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v). In one embodiment, step (ii) dilutes the protein D peptide in the liquid composition to a desired concentration (as determined by those skilled in the art). In one embodiment, step (ii) comprises stirring, optionally at 2 to 25°C. For example, the method comprises mixing the protein D peptide to a liquid composition at a concentration of 0.025 to 20 mg / ml, 0.5 to 10 mg / ml, or 0.5 to 1 mg / ml. In particular, the concentration of the protein D peptide may be 0.5 mg / ml or 1 mg / ml. Before mixing, a solution containing sucrose and poloxamer (and optional salts and buffers) can be added using a pipette or graduated glass cylinder. In one embodiment, a separate solution of sucrose and poloxamer (and optional salts and buffers) is added separately. In another embodiment, a separate solution of sucrose and poloxamer (and optional salts and buffers) is added simultaneously. In yet another embodiment, a single (combined) solution of sucrose and poloxamer (and optional salts and buffers) is added.

[0089] Therefore, as used herein, the term "solution" refers to a single solution or a single (combined) solution. For example, in a method for preparing a liquid composition comprising a protein D polypeptide, said method includes mixing the protein D polypeptide with a solution comprising (a) sucrose and (b) poloxamer, wherein a single solution of (a) sucrose and (b) poloxamer may be mixed with the protein D polypeptide, or a single (combined) solution of sucrose and poloxamer may be mixed with the protein D polypeptide. Suitably, a single (combined) solution of (a) sucrose and (b) poloxamer may be mixed with the protein D polypeptide. For example, in a method for preparing a liquid composition comprising a protein D polypeptide, said method includes mixing the protein D polypeptide with a solution comprising (a) sucrose, (b) poloxamer and (c) salt, wherein a single solution of (a) sucrose, (b) poloxamer and (c) salt may be mixed with the protein D polypeptide, or a single (combined) solution of sucrose, poloxamer and salt may be mixed with the protein D polypeptide. Suitablely, a single (combined) solution of (a) sucrose, (b) poloxamer, and (c) salt can be mixed with the protein D polypeptide. For example, in a method for preparing a liquid composition comprising the protein D polypeptide, said method includes mixing the protein D polypeptide with a solution comprising: (a) sucrose, (b) poloxamer, (c) salt, and (d) buffer, wherein a single solution of (a) sucrose, (b) poloxamer, (c) salt, and (d) buffer can be mixed with the protein D polypeptide, or a single (combined) solution of sucrose, poloxamer, salt, and buffer can be mixed with the protein D polypeptide. Suitablely, a single (combined) solution of (a) sucrose, (b) poloxamer, (c) salt, and (d) buffer can be mixed with the protein D polypeptide.

[0090] filter

[0091] In one embodiment, the method of the present invention includes filtering a liquid composition containing a protein D polypeptide. Therefore, the present invention provides a method for preparing a liquid composition comprising the protein D polypeptide as described above, followed by a filtration step, for example, using a 0.22 μm PVDF membrane. Suitably, filtration reduces or removes particles of the protein D polypeptide from the liquid composition. In one embodiment, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide, comprising steps (i) and (ii), followed by a filtration step (optionally using a 0.22 μm PVDF membrane) to obtain a liquid composition containing the protein D polypeptide in the filtrate. For example, the protein D polypeptide can be obtained by using... 47 filter (0.22μm) PVDF membrane 17.7cm 2 - Polypropylene column) and peristaltic pump (flow rate 0.7 ml / min / cm) 2Filtration. Other suitable membranes known to those skilled in the art, such as PES (polyethersulfone) and cellulose, may also be used. Therefore, the present invention provides a method for preparing a liquid composition comprising a protein D polypeptide, comprising a filtration step (optionally using a 0.22 μm PVDF membrane) after a step of mixing the protein D polypeptide with sucrose and poloxamer to obtain a liquid composition containing the protein D polypeptide in the filtrate. Therefore, the method of the present invention may comprise the following steps (in a sequential order): (i) thawing the protein D polypeptide, and (ii) mixing the protein D polypeptide with sucrose and poloxamer, followed by a filtration step.

[0092] store

[0093] This invention provides a method for preparing a liquid composition comprising a protein D peptide, said liquid composition reducing the formation of visible particles (and optionally subvisible particles) of the protein D peptide, particularly during storage (the period during which the protein D peptide remains in the liquid composition). Therefore, this invention provides a method for preparing a liquid composition comprising the protein D peptide as described above, subsequently including the step of storing the liquid composition comprising the protein D peptide. In one embodiment, this invention provides a method for preparing a liquid composition comprising a protein D peptide, comprising steps (i) and (ii) (optionally filtering), subsequently including the step of storing the liquid composition comprising the protein D peptide. Suitably, the liquid composition comprising the protein D peptide is stored for at least 1 day, at least 7 days, or at least 14 days. In some embodiments, the liquid composition comprising the protein D peptide is stored for at least 1, 2, 3, 4, 5, 6, 7, or 14 days. For example, the liquid composition comprising the protein D peptide may be stored for at least 1 day, suitably up to 7 days (e.g., between 1 and 7 days), or up to 14 days (e.g., between 1 and 14 days). The liquid compositions of this invention may be stored at +2 to +8°C. During storage as a liquid composition, the content of protein D peptide in the liquid composition can be measured. Therefore, the present invention provides a method for preparing a liquid composition containing protein D peptide, which includes a step of storing the liquid composition containing protein D peptide after a step of mixing the protein D peptide with sucrose and poloxamer (and optionally a filtration step). Thus, the method of the present invention may include the following steps (in a sequential order): (i) thawing the protein D peptide, (ii) mixing the protein D peptide with sucrose and poloxamer (and optionally a filtration step), and (iii) storing the liquid composition containing the protein D peptide.

[0094] Methods for reducing the formation of particulate protein D peptide particles

[0095] This invention provides a method for preparing a liquid composition containing a protein D peptide, which reduces the formation of protein D peptide particles in the liquid composition. Specifically, this invention provides a method for preparing a liquid composition containing a protein D peptide that reduces the formation of visible protein D peptide particles. In another embodiment, this invention provides a method for preparing a liquid composition containing a protein D peptide that reduces the formation of both visible and sub-visible protein D peptide particles. This invention also provides a method for reducing the formation of protein D peptide microparticles in a liquid composition, the method comprising the method of this invention. This invention also provides a method for preparing a stable liquid composition containing a protein D peptide. In one embodiment, the method reduces the formation of visible protein D peptide particles (and optionally, sub-visible particles) when the liquid composition is stored for at least 1 day. In another embodiment, the method reduces the formation of visible protein D peptide particles (and optionally, sub-visible particles) when the liquid composition is stored for at least 7 days. In yet another embodiment, the method reduces the formation of visible protein D peptide particles (and optionally, sub-visible particles) when the liquid composition is stored for at least 14 days.

[0096] The detection of visible particles in the composition can be determined by any technique deemed appropriate by a person skilled in the art. For example, visible particles can be detected by the method specified in Section 5.0, 2.9.20 of the European Pharmacopoeia. The detection of sub-visible particles in the composition can be determined by any technique deemed appropriate by a person skilled in the art. For example, visible particles can be detected by methods such as those specified in the United States Pharmacopeia. <788> The light-blocking particle count test described in the document is used for detection.

[0097] In one embodiment, the method of the present invention reduces the formation of visible particles (and optionally, subvisible particles) of the protein D peptide compared to methods that do not add sucrose and poloxamer to the protein D peptide composition. In one embodiment, the method of the present invention reduces the formation of visible particles (and optionally, subvisible particles) of the protein D peptide during subsequent storage of the liquid composition containing the protein D peptide for at least 1, 2, 3, 4, 5, 6, 7, or 14 days compared to methods that do not add sucrose and poloxamer to the liquid composition containing the protein D peptide. As used herein, “visible particles” refers to insoluble or partially soluble solids in a liquid composition (e.g., an aqueous solution) that are visible to the human eye. In one embodiment, the visible particles have an average diameter of at least 50 μm. In another embodiment, the visible particles have an average diameter of 50-1000 μm. In yet another embodiment, the visible particles have an average diameter of 75-1000 μm. In one embodiment, the visible particles have an average diameter of 100-1000 μm. In one implementation, visible particles are visible when detected by the method described in European Pharmacopoeia 5.0, 2.9.20. As used herein, "subvisible particles" refers to particles detected by the United States Pharmacopeia. <788> The light-shielding particle counting test described herein can detect particulate matter. In one embodiment, the subvisible particles have an average diameter of 2-175 μm. In another embodiment, the subvisible particles have an average diameter of 2-125 μm. In yet another embodiment, the subvisible particles have an average diameter of less than 50 μm. In yet another embodiment, the subvisible particles have an average diameter of 2-50 μm.

[0098] Mixing liquid compositions containing protein D peptides with other antigens

[0099] This invention provides a method for preparing a liquid composition comprising the protein D peptide as described above, and subsequently including a step of mixing the liquid composition comprising the protein D peptide with other antigens. In one embodiment, this invention provides a method for preparing a liquid composition comprising the protein D peptide, comprising steps (i), (ii), and (iii), and subsequently including the step: (iv) mixing the filtrate containing the protein D peptide with other antigens. In one embodiment, the other antigens include protein E or an immunogenic fragment thereof from Haemophilus influenzae, PilA or an immunogenic fragment thereof from Haemophilus influenzae, and the UspA2 peptide. In another embodiment, the other antigens comprise a PE-PilA fusion protein and the UspA2 peptide. This liquid composition can be used to prepare an immunogenic composition. Therefore, this invention provides a method for preparing a liquid composition comprising the protein D peptide, which, after the steps of mixing the protein D peptide with sucrose and poloxamer (and optionally, a filtration step) and storing the liquid composition comprising the protein D peptide, includes a step of mixing the liquid composition comprising the protein D peptide with other antigens. Therefore, the method of the present invention may include the following steps (in sequential order): (i) thawing the protein D polypeptide, (ii) mixing the protein D polypeptide with sucrose and poloxamer (and optionally, a filtration step), (iii) storing the liquid composition containing the protein D polypeptide, and (iv) mixing the liquid composition containing the protein D polypeptide with other antigens.

[0100] Protein E

[0101] Protein E (PE) is an outer membrane lipoprotein with adhesive properties. It plays a role in the adhesion / invasion of untyped Haemophilus influenzae (NTHi) to epithelial cells. (J. Immunology 183:2593-2601 (2009); The Journal of Infectious Diseases 199:522-531 (2009), Microbes and Infection 10:87-96 (2008)). It is highly conserved in both cysted and untyped Haemophilus influenzae and possesses a conserved epithelial-binding domain (The Journal of Infectious Diseases 201:414-419 (2010)). Thirteen different point mutations were described in different Haemophilus species when compared with Haemophilus influenzae Rd, which served as a reference strain. Its expression was observed in both logarithmic and stationary phase bacteria. (WO2007 / 084053). Protein E also participates in human complement resistance by binding to glassin (Immunology 183:2593-2601(2009)). PE binds to glassin, which is an important inhibitor of the terminal complement pathway (J.Immunology 183:2593-2601(2009)).

[0102] As used herein, “protein E”, “prot E”, and “PE” refer to protein E from Haemophilus influenzae. Protein E may contain (or consist of) the amino acid sequence of SEQ ID NO: 4 (corresponding to SEQ ID NO: 4 of WO2012 / 139225A1): (MKKIILTLSL GLLTACSAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDAVVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTLYNAAQIICAN YGEAFSVDKK).

[0103] In a particular embodiment, protein E or an immunogenic fragment thereof from Haemophilus influenzae suitably has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:4. In one embodiment, protein E from Haemophilus influenzae is an immunogenic fragment. In another embodiment, the immunogenic fragment of protein E from Haemophilus influenzae suitably has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4. For example, the immunogenic fragment of protein E may comprise at least 7, 10, 15, 20, 25, 30, or 50 consecutive amino acids of SEQ ID NO:4. For example, the immunogenic fragment of protein E may contain at least 7, 10, 15, 20, 25, 30, 50, 100, or 150 consecutive amino acids of SEQ ID NO: 4, and at most 159 consecutive amino acids of SEQ ID NO: 4. The immunogenic fragment may induce an antibody that binds to SEQ ID NO: 4.

[0104] In another embodiment, protein E or its immunogenic fragment from Haemophilus influenzae has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 5 (corresponding to SEQ ID NO: 125 of WO2012 / 139225A1).

[0105] SEQ ID NO:5: Amino acids 20-160 of protein E

[0106] I QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYVYPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICANYGEAFSVDKK.

[0107] In one embodiment, the immunogenic fragment of protein E from Haemophilus influenzae suitably has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 91%, 25%, 95%, 96%, 97%, 98%, or 100% identity with SEQ ID NO: 5 (corresponding to SEQ ID NO: 125 of WO2012 / 139225A1). In another embodiment, the immunogenic fragment of protein E from Haemophilus influenzae comprises (or consists of) the amino acid sequence of SEQ ID NO: 5 corresponding to (SEQ ID NO: 125 of WO2012 / 139225A1).

[0108] PilA

[0109] Pilin A (PilA) is likely a major fimbriae subunit of Haemophilus influenzae type IV Pilus (Tfp) involved in scurrying (Infection and Immunity, 73:1635-1643 (2005)). NTHi PilA is a conserved adhesin expressed in vivo. It has been shown to participate in NTHi adhesion, colonization, and biofilm formation (Molecular Microbiology 65:1288-1299 (2007)).

[0110] As used herein, “PilA” refers to fimbriae protein A from Haemophilus influenzae. PilA may contain (or consist of) the protein sequence of SEQ ID NO: 6 (corresponding to SEQ ID NO: 58 of WO2012 / 139225A1): (MKLTTQQTLKKGFTLIELMI VIAIIAILAT IAIPSYQNYT KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIAADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ).

[0111] In a particular embodiment, PilA or an immunogenic fragment thereof from Haemophilus influenzae suitably has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 96%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:6. In one embodiment, PilA from Haemophilus influenzae is an immunogenic fragment. In another embodiment, an immunogenic fragment of PilA from Haemophilus influenzae suitably has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 96%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:6. For example, the immunogenic fragment of PilA may comprise at least 7, 10, 15, 20, 25, 30, or 50 consecutive amino acids of SEQ ID NO:6. For example, the immunogenic fragment of PilA may contain at least 7, 10, 15, 20, 25, 30, 50, 100, or 150 consecutive amino acids of SEQ ID NO:6, and at most 148 consecutive amino acids of SEQ ID NO:6. The immunogenic fragment may induce an antibody that binds to SEQ ID NO:6.

[0112] In another embodiment, PilA or its immunogenic fragment from Haemophilus influenzae has at least 70%, 80%, 87%, 90%, 91%, 92%, 93%, 94%, 97%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 7 (corresponding to SEQ ID NO: 127 of WO2012 / 139225A1).

[0113] SEQ ID NO: 7 Amino acids 40-149 of PilA from Haemophilus influenzae strain 86-028NP

[0114] T KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIA ADITTAKGYV KSVTTSNGAITVKGDGTLAN MEYILQATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ.

[0115] In another embodiment, the immunogenic fragment of PilA suitably has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 7 (corresponding to SEQ ID NO: 127 of WO2012 / 139225A1). In another embodiment, the immunogenic fragment of PilA from Haemophilus influenzae comprises (or consists of) the amino acid sequence of SEQ ID NO: 7 (corresponding to SEQ ID NO: 127 of WO2012 / 139225A1).

[0116] PE-PilA fusion protein

[0117] Protein E or an immunogenic fragment thereof from Haemophilus influenzae and PilA or an immunogenic fragment thereof from Haemophilus influenzae can exist as a fusion protein. Therefore, protein E or an immunogenic fragment thereof from Haemophilus influenzae and PilA or an immunogenic fragment thereof from Haemophilus influenzae exist as a fusion protein. Suitably, the fusion protein may comprise protein E or an immunogenic fragment thereof from Haemophilus influenzae at the N-terminus and PilA or an immunogenic fragment thereof from Haemophilus influenzae at the C-terminus of the fusion protein (PE-PilA fusion protein). Specifically, the PE-PilA fusion protein may comprise the N-terminal immunogenic fragment of protein E from Haemophilus influenzae and the C-terminal immunogenic fragment of PilA from Haemophilus influenzae. In one embodiment, the PE-PilA fusion protein has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 8 (LVL-735, corresponding to SEQ ID NO: 194 of WO2012 / 139225A1).

[0118] SEQ ID NO: 8: LVL735 (protein): (pelB sp) (ProtE aa 20-160) (GG) (PilA aa40-149):

[0119]

[0120] In one embodiment, the PE-PilA fusion protein comprises (or consists of) the amino acid sequence of SEQ ID NO: 8 (LVL-735, corresponding to SEQ ID NO: 194 of WO2012 / 139225A1).

[0121] In another embodiment, the PE-PilA fusion protein has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 9 (LVL-735, wherein the signal peptide has been removed, corresponding to SEQ ID NO: 219 of WO2012 / 139225A1).

[0122] SEQ ID NO:9: PE-PilA fusion protein without signal peptide

[0123]

[0124] In one embodiment, the PE-PilA fusion protein comprises (or consists of) the amino acid sequence of SEQ ID NO: 9 (LVL-735, wherein the signal peptide has been removed, corresponding to SEQ ID NO: 219 of WO2012 / 139225A1).

[0125] The immunogenicity of the immunogenic fragments of protein E (PE) and fimbriae protein A (PilA) can be measured as described in WO2012 / 139225A1.

[0126] UspA2

[0127] UspA2 is a trimeric autotransporter that appears as a lollipop-like structure in electron micrographs (Hoiczyk et al., EMBO J.19:5989-5999 (2000)). It consists of an N-terminal head, followed by a stem terminated by an amphiphilic helix, and a C-terminal membrane domain (Hoiczyk et al. EMBO J.19:5989-5999 (2000)). UspA2 contains a highly conserved domain (Aebi et al., Infection & Immunity 65(11)4367-4377 (1997)) that is recognized by a monoclonal antibody that showed protective activity during passive transfer in a mouse Moraxella catarrhalis challenge model (Helminnen et al. J Infect Dis.170(4):867-72 (1994)). UspA2 has been shown to interact with host structural and extracellular matrix proteins such as fibronectin (Tan et al., J Infect Dis. 192(6): 1029-38(2005)) and laminin (Tan et al., J Infect Dis. 194(4): 493-7(2006)), suggesting that it may play a role in the early stages of Moraxella catarrhalis infection. UspA2 also appears to be involved in the ability of Moraxella catarrhalis to resist the bactericidal activity of normal human serum (Attia AS et al. Infect Immun 73(4): 2400-2410(2005)). It (i) binds to the complement inhibitor C4bp, enabling Moraxella catarrhalis to inhibit the classical complement system, (ii) prevents activation of the alternative complement pathway by absorbing C3 from serum, and (iii) interferes with the terminal phase of the complement system membrane attack complex (MAC) by binding to the complement regulatory protein polinecin. (de Vries et al., Microbiol Mol Biol Rev. 73(3):389-406 (2009)).

[0128] As used herein, “UspA2” refers to the ubiquitous surface protein A2 of *Moraxella catarrhalis*. UspA2 may contain (or consist of) the amino acid sequence of SEQ ID NO: 10 from ATCC25238 (corresponding to SEQ ID NO: 1 of WO2015 / 125118A1):

[0129] MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDIT

[0130] ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE

[0131] AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD

[0132] FGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSG

[0133] RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA

[0134] NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA

[0135] LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN

[0136] IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL

[0137] ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK

[0138] VNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAA

[0139] LGGYGSKSAVAIGAGYRV

[0140] NPNLAFKAGAAINTSGNKKGSYNIGVNYEF (SEQ ID NO:10). And sequences that are at least or exactly 63%, 66%, 70%, 72%, 74%, 75%, 77%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical in full length to SEQ ID NO:10.

[0141] The UspA2 polypeptide can be the full-length UspA2 or an immunogenic fragment thereof. In a particular embodiment, the UspA2 polypeptide has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 10. In another embodiment, the UspA2 polypeptide is an immunogenic fragment of UspA2 from Moraxella catarrhalis, which has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. For example, the immunogenic fragment of UspA2 may contain at least 7, 10, 15, 20, 25, 30, or 50 consecutive amino acids of SEQ ID NO: 10. For example, the immunogenic fragment of UspA2 may contain at least 7, 10, 15, 20, 25, 30, 50, 100, 200, 300, 400, 500, or 600 consecutive amino acids of SEQ ID NO: 10, or at most 629 consecutive amino acids of SEQ ID NO: 10. The immunogenic fragment may trigger an antibody that binds to SEQ ID NO: 10.

[0142] UspA2, as described in SEQ ID NO: 10, contains a signal peptide (e.g., amino acids 1 to 29 of SEQ ID NO: 10), a laminin-binding domain (e.g., amino acids 30 to 177 of SEQ ID NO: 10), a fibronectin-binding domain (e.g., amino acids 165 to 318 of SEQ ID NO: 10) (Tan et al., JID 192:1029-38 (2005)), a C3-binding domain (e.g., amino acids 30 to 539 of SEQ ID NO: 10 (WO2007 / 018463), or a fragment of amino acids 30 to 539 of SEQ ID NO: 10, such as amino acids 165 to 318 of SEQ ID NO: 1). T et al., J. Immunol. 186:3120-3129 (2011)), an amphiphilic helix (e.g., amino acids 519 to 564 of SEQ ID NO: 10 or amino acids 520-559 of SEQ ID NO: 10, identified using different prediction methods) and a C-terminal anchor domain (e.g., amino acids 576 to 630 of SEQ ID NO: 10) (Brooks et al., Infection & Immunity, 76(11), 5330-5340 (2008)). In one embodiment, the UspA2 polypeptide contains a laminin-binding domain and a fibronectin-binding domain. In another embodiment, the immunogenic fragment of UspA2 contains a laminin-binding domain, a fibronectin-binding domain, and a C3-binding domain. In a further embodiment, the UspA2 polypeptide contains a laminin-binding domain, a fibronectin-binding domain, a C3-binding domain, and an amphiphilic helix.

[0143] The amino acid differences of UspA2 for various Moraxella catarrhalis species have been described. See, for example, J Bacteriology 181(13):4026-34(1999), Infection and Immunity 76(11):5330-40(2008) and PLoS One 7(9):e45452(2012). The UspA2 polypeptide may contain (or consist of) an amino acid sequence selected from one or more of the following amino acids that differ from (or consist of) SEQ ID NO: 10: AA (amino acids) 30-298, AA 299-302, AA303-333, AA 334-339, AA 349, AA 352-354, AA 368-403, AA 441, AA 451-471, AA 472, AA474-483, AA 487, AA 490, AA 493, AA 529, AA 532, or AA 543. The UspA2 polypeptide may contain (or consist of) an amino acid sequence different from (or consist of) SEQ ID NO: 10, wherein it contains an amino acid insertion compared to SEQ ID NO: 10. UspA2 may contain (or consist of) an amino acid sequence from SEQ ID NO: 22 to SEQ ID NO: 58 that differs from (or consists of) SEQ ID NO: 10 at any one amino acid difference. For example, SEQ ID NO: 10 may contain K instead of Q at amino acid position 70, Q instead of G at amino acid position 135, and / or D instead of N at amino acid position 216.

[0144] UspA2 can be derived from the following UspA2 strain: Moraxella catarrhalis strain ATCC (US registered trademark) 25238TM , American 2933, American 2912, American 2908, Finnish 307, Finnish 353, Finnish358, Finnish 216, Dutch H2, Dutch F10, Norwegian 1, Norwegian 13, Norwegian 20, Norwegian 25, Norwegian 27, Norwegian 36, BC5SV, Norwegian 14. Norwegian 3, Finish414, Japanese Z7476, Belgian Z7530, German Z8063, American O12E, Greek MC317, American V1122, American P44, American V1171, American TTA24, American O35E, American SP12-6, American SP12-5, Swedish BC5, American 7169, Finnish FIN2344, American V1118, American V1145, or American V1156. UspA2 can be as shown in any of SEQ ID NO: 10 or SEQ ID NO: 22-SEQ ID NO: 38. UspA2 can be UspA2 from another source, corresponding to any of the UspA2 sequences in SEQ ID NO: 10 or SEQ ID NO: 22-SEQ ID NO: 58. The corresponding UspA2 sequence can be determined by those skilled in the art using various algorithms. For example, the GAP program or the Needle program can be used to determine the UspA2 sequence corresponding to any of SEQ ID NO: 10 or SEQ ID NO: 22-SEQ ID NO: 58.

[0145] UspA2 can be a sequence that has at least 95% identity in full length with any one of SEQ ID NO: 10 or SEQ ID NO: 22-SEQ ID NO: 58. In a particular embodiment, UspA2 may be a sequence selected from the amino acid sequences described below: SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58 or any subset of SEQ ID NO:1 or SEQ ID NO:22 to SEQ ID NO:58.

[0146] The immunogenic fragment of UspA2 comprises the following immunogenic fragments: at least 450 consecutive amino acids of SEQ ID NO:10, 490 consecutive amino acids of SEQ ID NO:10 (e.g., UspA2 fragments of MC-004 or MC-005), 511 consecutive amino acids of SEQ ID NO:10 (e.g., UspA2 fragments of constructs MC-001, MC-002, MC-003, or MC-004), 534 consecutive amino acids of SEQ ID NO:10 (e.g., UspA2 fragments of MC-009 or MC-011), or 535 consecutive amino acids of SEQ ID NO:10 (e.g., UspA2 fragments of MC-007, MC-008, or MC-010). The immunogenic fragment can elicit an antibody that binds to SEQ ID NO:10.

[0147] The immunogenic fragment of UspA2 may comprise an immunogenic fragment of at least 450, 490, 511, 534, or 535 consecutive amino acids of SEQ ID NO: 10. For example, the immunogenic fragment of UspA2 may comprise an immunogenic fragment of at least 450, 490, 511, 534, or 535 consecutive amino acids of SEQ ID NO: 10, or an immunogenic fragment of up to 629 amino acids of SEQ ID NO: 10. The immunogenic fragment of UspA2 may include an immunogenic fragment of UspA2, such as any one of the UspA2 constructs MC-001 (SEQ ID NO: 11), MC-002 (SEQ ID NO: 12), MC-003 (SEQ ID NO: 13), MC-004 (SEQ ID NO: 14), MC-005 (SEQ ID NO: 15), MC-006 (SEQ ID NO: 16), MC-007 (SEQ ID NO: 17), MC-008 (SEQ ID NO: 18), MC-009 (SEQ ID NO: 19), MC-010 (SEQ ID NO: 20), or MC-011 (SEQ ID NO: 21). The immunogenic fragment can induce an antibody that can bind to the full-length sequence derived therefrom.

[0148] In another embodiment, the UspA2 polypeptide has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identity with a polypeptide selected from the following: MC-001 (SEQ ID NO:11), MC-002 (SEQ ID NO:12), MC-003 (SEQ ID NO:13), MC-004 (SEQ ID NO:14), MC-005 (SEQ ID NO:15), MC-006 (SEQ ID NO:16), MC-007 (SEQ ID NO:17), MC-008 (SEQ ID NO:18), MC-009 (SEQ ID NO:19), MC-010 (SEQ ID NO:20), or MC-011 (SEQ ID NO:21). For example, the UspA2 polypeptide has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MC009 SEQ ID NO: 19 (corresponding to SEQ ID NO: 69 of WO2015 / 125118A1).

[0149] SEQ ID NO:19MC-009(protein)–(M)(UspA2 31-564)(HH)

[0150] .

[0151] In one embodiment, the UspA2 polypeptide comprises (or consists of) the amino acid sequence of SEQ ID NO: 19 (corresponding to SEQ ID NO: 69 of WO2015 / 125118A1).

[0152] The immunogenicity of the UspA2 peptide can be measured as described in WO2015 / 125118A1.

[0153] freeze-drying

[0154] The liquid composition of the protein D peptide prepared according to the method of the present invention can then be freeze-dried. Therefore, the present invention provides a method comprising preparing a liquid composition comprising the protein D peptide as described above, and subsequently freeze-drying the liquid composition comprising the protein D peptide. In one embodiment, the present invention provides a method for preparing a liquid composition comprising the protein D peptide, comprising steps (i), (ii), (iii), and (iv), and subsequently comprising the step of: (v) freeze-drying the liquid composition comprising the protein D peptide. "Freeze-drying" refers to a method of freezing a suspension and then removing water by sublimation. Sublimation is a change in the physical properties of a substance in which the solvent (e.g., water) in the substance changes directly from a solid (frozen) state to a gaseous state without becoming a liquid. Freeze-drying is a low-temperature dehydration method comprising freezing a formulation (e.g., an aqueous formulation) below its triple point (the lowest temperature at which the solid, liquid, and gaseous phases of a material can coexist), reducing the pressure and removing ice (solid solvent) by sublimation in a primary drying step, and removing the remaining water in a secondary drying step. Annealing may optionally be used prior to drying to increase the size of ice crystals by raising and lowering the temperature. Freeze-drying is commonly used in vaccine manufacturing. In one embodiment, the immunogenic composition is lyophilized. Lyophilization is a process of removing water from a product after it has been frozen and placed under a vacuum, allowing ice to be converted directly from a solid to vapor without passing through a liquid phase.

[0155] In one implementation, the freeze-drying process is performed using the following steps:

[0156] - Freezing step (below triple point)

[0157] -Optional, annealing step

[0158] - Initial drying steps

[0159] - Secondary drying step.

[0160] Freeze-drying increases the concentration of formulation components in a process known as low-temperature concentration.

[0161] Therefore, the present invention provides a method for preparing a liquid composition containing a protein D peptide, comprising, after the steps of mixing the protein D peptide with sucrose and poloxamer (and optionally, a filtration step), storing the liquid composition containing the protein D peptide, and mixing the liquid composition containing the protein D peptide with other antigens, freeze-drying the liquid composition containing the protein D peptide. Thus, the method of the present invention may include the following steps (in a sequential order): (i) thawing the protein D peptide, (ii) mixing the protein D peptide with sucrose and poloxamer (and optionally, a filtration step), (iii) storing the liquid composition containing the protein D peptide, (iv) mixing the liquid composition containing the protein D peptide with other antigens, and (v) freeze-drying the liquid composition containing the protein D peptide.

[0162] Liquid composition of protein D peptide

[0163] The present invention provides a liquid composition comprising a protein D polypeptide (optionally, the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (optionally or, poloxamer 188). In one embodiment, the present invention provides a liquid composition comprising, optionally, 0.025 to 20 mg / ml, 0.5 to 10 mg / ml, 0.5 to 1 mg / ml, or 1 mg / ml of a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2); optionally, 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v) of sucrose; and optionally, 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v) of a poloxamer (e.g., poloxamer 188). In another embodiment, the present invention provides a liquid composition comprising, optionally, 0.025 to 20 mg / ml, 0.5 to 10 mg / ml, 0.5 to 1 mg / ml or 1 mg / ml of a protein D polypeptide (optionally, the protein D polypeptide of SEQ ID NO: 2); optionally, 5 to 20% (w / v), 10 to 20% (w / v) or 10 to 15% (w / v) of sucrose; optionally, 0.1 to 1% (w / v), 0.5 to 1% (w / v) or 1% (w / v) of poloxamer (optionally, poloxamer 188); and a salt (optionally, NaCl). In another embodiment, the present invention provides a liquid composition comprising, optionally, 0.025 to 20 mg / ml, 0.5 to 10 mg / ml, 0.5 to 1 mg / ml or 1 mg / ml of a protein D polypeptide (optionally, the protein D polypeptide of SEQ ID NO: 2); optionally, 5 to 20% (w / v), 10 to 20% (w / v) or 10 to 15% (w / v) of sucrose; optionally, 0.1 to 1% (w / v), 0.5 to 1% (w / v) or 1% (w / v) of poloxamer (optionally, poloxamer 188); a buffer (optionally, phosphate buffer); and a salt (optionally, NaCl).

[0164] Protein D peptide, sucrose, and poloxamer can be combined in the above-mentioned dosage ranges. For example, the present invention provides a liquid composition comprising protein D peptide (e.g., protein D peptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.025-20 mg / ml of protein D peptide; 5 to 20% (w / v) of sucrose; and 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188). For example, the present invention provides a liquid composition comprising protein D peptide (e.g., protein D peptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5-10 mg / ml of protein D peptide; 5 to 20% (w / v) of sucrose; and 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.025 to 20 mg / ml of protein D polypeptide; 10 to 20% (w / v) of sucrose; and 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5 to 10 mg / ml of protein D polypeptide; 10 to 20% (w / v) of sucrose; and 0.5 to 1% (w / v) of poloxamer (e.g., poloxamer 188). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5 to 1 mg / ml of protein D polypeptide; 10 to 15% (w / v) of sucrose; and 0.5 to 1% (w / v) of poloxamer (e.g., poloxamer 188). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.025 to 20 mg / ml of protein D polypeptide; 5 to 20% (w / v) of sucrose; 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188); and a buffer (e.g., phosphate buffer).For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5 to 10 mg / ml of protein D polypeptide; 5 to 20% (w / v) of sucrose; 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188); and a buffer (e.g., phosphate buffer). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.025 to 20 mg / ml of protein D polypeptide; 10 to 20% (w / v) of sucrose; 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188); and a buffer (e.g., phosphate buffer). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5 to 10 mg / ml of protein D polypeptide; 10 to 20% (w / v) of sucrose; 0.5 to 1% (w / v) of poloxamer (e.g., poloxamer 188); and a buffer (e.g., phosphate buffer). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5 to 1 mg / ml of protein D polypeptide; 10 to 15% (w / v) of sucrose; 0.5 to 1% (w / v) of poloxamer (e.g., poloxamer 188); and a buffer (e.g., phosphate buffer). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.025 to 20 mg / ml of protein D polypeptide; 5 to 20% (w / v) of sucrose; 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188), a buffer (e.g., phosphate buffer), and a salt (e.g., NaCl). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5 to 10 mg / ml of protein D polypeptide; 5 to 20% (w / v) of sucrose; 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188), a buffer (e.g., phosphate buffer), and a salt (e.g., NaCl).For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.025 to 20 mg / ml of protein D polypeptide; 10 to 20% (w / v) of sucrose; 0.1 to 1% (w / v) of poloxamer (e.g., poloxamer 188), a buffer (e.g., phosphate buffer), and a salt (e.g., NaCl). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5 to 10 mg / ml of protein D polypeptide; 10 to 20% (w / v) of sucrose; 0.5 to 1% (w / v) of poloxamer (e.g., poloxamer 188), a buffer (e.g., phosphate buffer), and a salt (e.g., NaCl). For example, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (e.g., poloxamer 188), comprising: 0.5 to 1 mg / ml of protein D polypeptide; 10 to 15% (w / v) of sucrose; 0.5 to 1% (w / v) of poloxamer (e.g., poloxamer 188), a buffer (e.g., phosphate buffer), and a salt (e.g., NaCl).

[0165] In one embodiment, the present invention provides a liquid composition comprising a protein D polypeptide (e.g., the protein D polypeptide of SEQ ID NO: 2), poloxamer (e.g., poloxamer 188), and sucrose prepared by the method of the present invention. In one embodiment, the present invention provides a liquid composition comprising a stable protein D polypeptide. Suitably, the liquid composition comprising the protein D polypeptide is stable for at least 1 day, at least 7 days, or at least 14 days. In some embodiments, the liquid composition comprising the protein D polypeptide is stable for at least 1, 2, 3, 4, 5, 6, 7, or 14 days. For example, the liquid composition comprising the protein D polypeptide may be stable for at least 1 day, suitably up to 7 days (e.g., between 1 and 7 days), or up to 14 days (e.g., between 1 and 14 days). In one embodiment, the present invention provides a liquid composition comprising a protein D polypeptide, poloxamer, and sucrose, which, when stored as a liquid composition for at least 1, 2, 3, 4, 5, 6, 7, or 14 days, has fewer visible particles compared to a liquid composition comprising the protein D polypeptide but without poloxamer and without sucrose.

[0166] In one embodiment, the liquid composition containing the protein D peptide of the present invention is free of visible particles. In one embodiment, the liquid composition containing the protein D peptide of the present invention is free of visible particles when maintained as a liquid composition for at least 1 day. In one embodiment, the liquid composition containing the protein D peptide of the present invention is free of visible particles when maintained as a liquid composition for at least 7 days. In one embodiment, the liquid composition containing the protein D peptide of the present invention is free of visible particles when stored as a liquid composition for at least 14 days. For example, the liquid composition containing the protein D peptide is free of visible particles when maintained as a liquid composition for at least 1 day, suitably up to 7 days (e.g., between 1 and 7 days), or up to 14 days (e.g., between 1 and 14 days). In one embodiment, the liquid composition containing the protein D peptide of the present invention contains fewer than 100 particles in the size range of 50 to 1000 μm according to the Occhio particle count (as described herein). In one embodiment, the liquid composition containing the protein D peptide contains fewer than 100 particles in the size range of 50 to 1000 μm according to the Occhio particle count when maintained as a liquid composition for at least 1 day. In one embodiment, when maintained in a liquid composition for at least 7 days, the liquid composition containing the protein D peptide contains fewer than 100 particles in the size range of 50 to 1000 μm based on the Occhio particle count. In one embodiment, when maintained in a liquid composition for at least 14 days, the liquid composition containing the protein D peptide contains fewer than 100 particles in the size range of 50 to 1000 μm based on the Occhio particle count. In one embodiment, when maintained in a liquid composition for at least 1 day, suitably up to 7 days (e.g., 1-7 days) or up to 14 days (e.g., 1-14 days), the liquid composition containing the protein D peptide contains fewer than 100 particles in the size range of 50 to 1000 μm based on the Occhio particle count.

[0167] Uses, treatments, and preventions

[0168] The present invention also provides an immunogenic composition wherein the protein D polypeptide is prepared using the method of the present invention. The immunogenic composition may further comprise protein E or an immunogenic fragment thereof from Haemophilus influenzae, PilA or an immunogenic fragment thereof from Haemophilus influenzae, and UspA2 polypeptide from Moraxella catarrhalis. In another embodiment, the immunogenic composition may further comprise a PE-PilA fusion protein and the UspA2 polypeptide. The immunogenic composition may be used for the treatment or prevention of diseases caused by Haemophilus influenzae and / or Moraxella catarrhalis, or for the treatment or prevention of acute exacerbations of COPD (AECOPD) in a subject (e.g., a human).

[0169] The immunogenic compositions of the present invention may further comprise pharmaceutically acceptable adjuvants. Suitable adjuvants include aluminum salts, such as aluminum hydroxide gel or aluminum phosphate or alum, but may also be salts of calcium, magnesium, iron or zinc, or may be insoluble suspensions of acylated tyrosine or acylated sugars, cationic or anionic derived sugars or polyphosphazenes. In a particular embodiment, the protein antigen may be adsorbed onto aluminum phosphate. In another embodiment, the protein antigen may be adsorbed onto aluminum hydroxide. Suitable adjuvant systems for promoting major Th1 responses also include: lipid A, monophosphoryl lipid A (MPL) or derivatives thereof, particularly nontoxic derivatives of 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (see GB2220211A for its preparation); and monophosphoryl lipid A, such as 3-deoxy-acylated monophosphoryl lipid A, in mixtures with aluminum salts (e.g., aluminum phosphate or aluminum hydroxide) or oil-in-water emulsions. In these combinations, the antigen and 3D-MPL are contained in the same particulate structure, thereby allowing for more efficient delivery of antigen and immune stimulation signals. Studies have shown that 3D-MPL can further enhance the immunogenicity of alum-adsorbed antigens (Tholen et al., Vaccine (1998) 16:708-14; EP689454-B1). For example, a pharmaceutically acceptable adjuvant could be AS01. AS01 is an adjuvant system containing MPL (3-O-deacylated-4'-monophosphoryllipid A), QS21 ((Quillaja saponaria Molina, fraction 21) Antigenics, New York, NY, USA), and liposomes. AS01B is an adjuvant system containing MPL, QS21, and liposomes (50 μg MPL and 50 μg QS21). AS01E is an adjuvant system containing MPL, QS21, and liposomes (25 μg MPL and 25 μg QS21). The immunogenic compositions or vaccines of the present invention may contain AS01, such as AS01B or AS01E.

[0170] Therefore, the present invention provides an immunogenic composition for treating or preventing diseases caused by Haemophilus influenzae and / or Moraxella catarrhalis. The present invention also provides the use of the immunogenic composition of the present invention in the preparation of a medicament for treating or preventing diseases caused by Haemophilus influenzae and / or Moraxella catarrhalis. Additionally, the present invention provides a method for treating or preventing diseases caused by Haemophilus influenzae and / or Moraxella catarrhalis in a subject (e.g., a human) at risk, the method comprising administering an effective amount of the immunogenic composition of the present invention to the subject. Furthermore, the present invention provides a method for preventing diseases caused by Haemophilus influenzae and / or Moraxella catarrhalis in a subject (e.g., a human) at risk, the method comprising administering an effective amount of the immunogenic composition of the present invention to the subject. Finally, the present invention provides a method for treating diseases caused by Haemophilus influenzae and / or Moraxella catarrhalis in a subject (e.g., a human) at risk, the method comprising administering an effective amount of the immunogenic composition of the present invention to the subject. In addition, the present invention provides a method for inducing an immune response against Haemophilus influenzae and / or Moraxella catarrhalis in a subject (e.g., a human), the method comprising administering an effective amount of the immunogenic composition of the present invention to the subject.

[0171] This invention provides an immunogenic composition for treating or preventing acute exacerbations of COPD (AECOPD) in a subject (e.g., a human). This invention also provides the use of the immunogenic composition of this invention in the preparation of a medicament for treating or preventing acute exacerbations of COPD (AECOPD). Furthermore, this invention provides a method for treating or preventing acute exacerbations of COPD (AECOPD) in a subject (e.g., a human) at risk of developing AECOPD, the method comprising administering an effective amount of the immunogenic composition of this invention to the subject. Furthermore, this invention provides a method for preventing acute exacerbations of COPD (AECOPD) in a subject (e.g., a human) at risk of developing AECOPD, the method comprising administering an effective amount of the immunogenic composition of this invention to the subject. Furthermore, this invention provides a method for treating acute exacerbations of COPD (AECOPD) in a subject (e.g., a human) at risk of developing AECOPD, the method comprising administering an effective amount of the immunogenic composition of this invention to the subject.

[0172] Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by chronic obstruction of airflow in the lungs, which interferes with normal breathing and is not entirely reversible. COPD is diagnosed using a simple test called spirometry, which measures how deep a person can breathe and how quickly air can enter and leave the lungs. Such a diagnosis should be considered in any patient with symptoms of cough, sputum production, or dyspnea (difficulty or straining to breathe) and / or a history of exposure to risk factors for the disease. In cases where spirometry is unavailable, all available tools should be used for the diagnosis of COPD. Clinical symptoms and signs, such as abnormal shortness of breath and increased forced expiratory time, can aid in diagnosis. Low peak flow is consistent with COPD but may not be COPD-specific, as it can be caused by other lung diseases and by adverse events during testing. Chronic cough and sputum production often precede the development of airflow limitation for many years, although not all individuals with cough and sputum production continue to develop COPD.

[0173] An acute exacerbation of COPD (AECOPD) is an acute event characterized by a worsening of respiratory symptoms beyond the normal daily range. AECOPD typically leads to a change in medication. Acute exacerbations and comorbidities contribute to the overall disease severity in an individual COPD patient. AECOPD is an acute event characterized by a worsening of respiratory symptoms beyond the normal daily range and leading to a change in medication [Perez AC, Murphy TF. Potential impact of a Moraxella catarrhalis vaccine in COPD. Vaccine. 2017]. AECOPD increases morbidity and mortality, leading to a faster decline in lung function and a worse functional status [Sapey E, Stockley RA. COPD exacerbations. 2: aetiology. Thorax. 2006; 61(3): 250-8). Different types of bacteria are known to colonize the lungs [Erb-Downward JR, et al. PLoS One. 2011; 6(2):e16384 and Wilkinson TMA, et al. Thorax. 2017; 72(10):919-27]. In COPD patients, the collection of new bacterial strains is considered an important cause of AECOPD [Sethi S, et al. N Engl J Med. 2002; 347(7):465-71]. Despite the wide range of estimates, untyped Haemophilus influenzae (NTHi) appears to be the leading bacterial pathogen associated with AECOPD (11–38%), followed by Moraxella catarrhalis (3–25%) and Streptococcus pneumoniae (4–9%) [Alamoudi OS. et al. Respirology. 2007; 12(2):283–7, Bandi V, et al. FEMS Immunol Med Microbiol. 2003; 37(1):69–75, Beasley V, et al. Int J ChronObstruct Pulmon Dis. 2012; 7:555–69].

[0174] In one embodiment, the acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is associated with a bacterial infection in the subject, such as Haemophilus influenzae (e.g., untyped Haemophilus influenzae (NTHi)) and / or Moraxella catarrhalis infection. In another embodiment, the bacterial infection is present in the lungs of the subject (e.g., a person). In yet another embodiment, the subject (e.g., a person) is at risk of developing an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) caused by a bacterial infection.

[0175] Present

[0176] In some embodiments, the immunogenic composition is contained within a container tool, such as a vial or syringe, including a pre-filled syringe. In some embodiments, the container tool is siliconized. When the immunogenic composition of the present invention is presented in a vial, the vial is suitably made of glass or plastic material. The vial is preferably sterilized before the composition is added to it. The vial may comprise a single-dose vaccine, or it may comprise more than one dose (“multi-dose” vials), for example, 10 doses. When using multi-dose vials, each dose should be drawn under strict aseptic conditions with a sterile needle and syringe, taking care to avoid contaminating the vial contents. The vial may have a cap (e.g., a Luer lock) adapted to allow a pre-filled syringe to be inserted into the cap, the contents of the syringe to be extruded into the vial (e.g., for reconstitution of lyophilized material therein), and the contents of the vial to be transferred back into the syringe. After the syringe is removed from the vial, a needle can then be attached, and the composition can be administered to a patient. The cap is preferably located inside a seal or cap, such that the seal or cap must be removed before access to the cap is possible.

[0177] The immunogenic compositions of the present invention are suitable for administration via appropriate routes, such as intramuscular routes.

[0178] In another embodiment, the present invention provides a vaccine comprising the immunogenic composition of the present invention.

[0179] Embodiments of the present invention are further described in the following numbered paragraphs:

[0180] 1. A method for preparing a liquid composition comprising a protein D polypeptide (optionally the protein D polypeptide of SEQ ID NO: 2), wherein the method comprises mixing the protein D polypeptide with sucrose and poloxamer.

[0181] 2. The method of preparing a liquid composition comprising a protein D polypeptide according to paragraph 1, wherein the method comprises mixing the protein D polypeptide with sucrose and poloxamer before mixing the protein D polypeptide with other antigens.

[0182] 3. The method of preparing a liquid composition comprising a protein D polypeptide according to paragraph 1 or paragraph 2, wherein the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose at a concentration of 5 to 20% (w / v), 10 to 20% (w / v), or 10 to 15% (w / v), and (b) poloxamer (optionally poloxamer 188) at a concentration of 0.1 to 1% (w / v), 0.5 to 1% (w / v), or 1% (w / v).

[0183] 4. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 3, wherein the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, (b) poloxamer (optionally, poloxamer 188) and (c) a salt (optionally, NaCl).

[0184] 5. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 3, wherein the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, (b) poloxamer (optionally, poloxamer 188), (c) a salt (optionally, NaCl), and (d) a buffer (optionally, phosphate buffer).

[0185] 6. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 3, wherein the method comprises mixing the protein D polypeptide with a solution comprising: (a) sucrose, (b) poloxamer (optionally, poloxamer 188), (c) a salt (optionally, NaCl), and (d) a buffer (optionally, phosphate buffer) to achieve a pH of 6.4 to 7.7 (e.g., pH 6.8).

[0186] 7. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 6, wherein the method comprises the steps of: (i) thawing the protein D polypeptide, and (ii) mixing the protein D polypeptide with sucrose and poloxamer.

[0187] 8. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 7, wherein the method subsequently includes a filtration step (optionally using a 0.22 μm PVDF membrane) to obtain a liquid composition comprising the protein D polypeptide in the filtrate.

[0188] 9. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 8, followed by a step of storing the liquid composition comprising the protein D polypeptide.

[0189] 10. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 9, subsequently comprising the step of mixing the liquid composition comprising the protein D polypeptide with other antigens.

[0190] 11. The method of preparing a liquid composition comprising a protein D polypeptide according to paragraph 10, wherein the other antigen comprises a PE-PilA fusion protein and a UspA2 polypeptide.

[0191] 12. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 11, which reduces the formation of visible particles of the protein D polypeptide.

[0192] 13. A method comprising preparing a liquid composition comprising a protein D polypeptide according to any one of paragraphs 1 to 12, and subsequently freeze-drying the liquid composition comprising the protein D polypeptide.

[0193] 14. A liquid composition comprising a protein D polypeptide (optionally, the protein D polypeptide of SEQ ID NO: 2), sucrose, and poloxamer (optionally, poloxamer 188).

[0194] 15. The liquid composition according to paragraph 14, comprising optionally 0.025 to 20 mg / ml, 0.5 to 10 mg / ml, 0.5 to 1 mg / ml or 1 mg / ml of protein D polypeptide (optionally, protein D polypeptide of SEQ ID NO: 2); optionally 5 to 20% (w / v), 10 to 20% (w / v) or 10 to 15% (w / v) of sucrose; optionally 0.1 to 1% (w / v), 0.5 to 1% (w / v) or 1% (w / v) of poloxamer (optionally, poloxamer 188); a buffer (optionally, phosphate buffer); and a salt (optionally, NaCl).

[0195] To better understand the present invention, the following embodiments are provided. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. Example

[0196] Analytical techniques

[0197] Light shading

[0198] Light shielding is defined in the pharmacopoeia (Ph.Eur.2.9.19 and USP (United States Pharmacopeia)). <788> The pharmacopoeia-selected method for analyzing subvisible particles in parenteral products is listed in [reference needed]. The particle size detection range is between 2 and 175 μm. A volume of approximately 5 ml is required. To ensure that particles detected by light shielding do not originate from the culture medium, the culture medium was analyzed on day 1 at its maximum concentration by light shielding: 10% sucrose, 1% poloxamer 188, 1% NaCl, and 12.5 mM PO4 buffer. The equipment used was an APS-2000 (Automated Parenteral Sampling System).

[0199] The APSS-2000 hardware consists of two central components:

[0200] • Particle counter model E20P

[0201] • Syringe sampler model SLS-1000

[0202] The LiQuilaz-E20P particle counter uses extinction to measure and classify particles. When a particle passes through a light source (laser diode), it creates a momentary obscuration of light. This obscuration is converted into an electrical signal, which can be directly correlated with the size of the transient particle. A preset algorithm defines the particle distribution. A syringe sampler pulls the sample through the optical chamber at a predetermined and fixed flow rate.

[0203] The parameters used for analysis are:

[0204] • Analyze 1 ml of sample 4 times (total 4 ml) (discard the first measurement).

[0205] • Flow rate 10 ml / min

[0206] Occhio

[0207] Occhio is an emerging technology developed for the monitoring, measurement, and visualization of subvisible and visible particles. It integrates digital microscopy, microfluidics, and image processing into a single instrument for the automated analysis of particles or cells suspended in liquids. It operates by capturing images of the sample as it passes through the sensing zone of a flow cell. Each particle in each image is analyzed to generate a database of particle counts, sizes, transparency, and morphology (or shape). For immediate visual verification, the images are displayed in real-time on the system monitor. Particle sizes can be detected in the range of 0.4 to 1000 μm. Test volumes are approximately 2 ml. Occhio (IPAC2) was selected for analyzing fibrous aggregates with optimized hardware configurations using the following key parameters:

[0208] -400μm pool

[0209] -1ml syringe

[0210] -4x zoom

[0211] Protein D content was determined by RP UPLC.

[0212] The content of specific antigens was evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Zorbax 300SBC 34.6x50mm 3.5μm column with a guard column of 4.6x12.5mm coupled to a UV detector set at 215nm. Protein D was eluted at approximately 9 minutes.

[0213] Circular dichroism (CD) spectrum

[0214] FAR-UV CD: The ellipticity (mdeg) calculated based on the difference in absorption between left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL) is measured between 200 and 265 nm, corresponding to the absorption region of peptide linkages. Therefore, the obtained signal is related to the secondary structural composition of the antigen, such as α-helices and β-sheets. FAR-UV CD is used to detect modifications of secondary structures (α-helices, β-sheets, etc.).

[0215] Near-UV CD: This method is used to detect modifications of the tertiary structure of proteins that are related to environmental changes in aromatic amino acids.

[0216] ATR-FTIR

[0217] The ATR-FTIR method is based on reflectivity. IR radiation is directed into a crystal with a high refractive index in contact with the sample. The beam is reflected within the crystal before being guided to the detector. When the beam strikes the reflecting surface, it is partially absorbed, and the incident beam is recorded.

[0218] The infrared spectrum of proteins contains contributions from peptide amide groups, termed amide I, II, etc., and relatively weaker contributions from amino acid side chains. The amide II band (1550-1450 cm⁻¹) is also significant. -1 The majority of the structure is δN-H, which is primarily associated with peptide bonds. This is observed at 1700-1600 cm⁻¹. -1 In the amide I region, the υC=O band, which belongs to the peptide bond, is the most sensitive to protein secondary structure to date. Because the strength of hydrogen bonds present in each secondary structure is different, each secondary structure absorbs at different wavelengths in the amide I region. Frequency limits for each secondary structure have been specified based on theoretical and experimental data (Goormaghtigh et al, 2006, Evaluation of the Information Content in Infrared Spectra for Protein Secondary Structure Determination; Biophysical Journal, 90(8)2946–2957): α-helix 1662–1645 cm -1 β-fold 1689-1682cm -1 irregular 1644-1637cm -1 And β-turn angle 1682-1662.

[0219] Intrinsic fluorescence

[0220] The fluorescence emission (AU) of a protein is related to its aromatic amino acid content, primarily by the contributions of tryptophan and tyrosine residues. The obtained signals are correlated with the larger or smaller polar environments of these chromophores, thus relating them to their location within the protein. The shape of the fluorescence spectrum, with its maximum value, is then correlated with the protein's tertiary structure.

[0221] Example 1: Screening of excipients and their effect on particle formation during liquid storage (Part 1)

[0222] The research objective is to identify excipients and / or parameters that positively influence the colloidal stability of liquid protein D at 2–8 °C. A comprehensive factorial screening study will be conducted to determine parameters that have a positive or negative impact on the appearance of visible particles. The parameters to be studied are:

[0223] - Protein D concentration (2 levels)

[0224] o 0.5mg / ml

[0225] o 1mg / ml

[0226] -pH (2 levels)

[0227] o 6.8

[0228] o 7.7

[0229] - The presence of sucrose (2 levels)

[0230] o 0% m / v sucrose

[0231] o 10% m / v sucrose

[0232] - The existence of Polosham 188 (2 levels)

[0233] o 0%

[0234] o 0.5%

[0235] -The presence of NaCl (2 levels)

[0236] o 0mM

[0237] o 150mM

[0238] Frozen protein D was thawed in an incubator at 25°C for 1 hour and 30 minutes. After thawing, protein D was diluted to 20 mg / ml in 150 mM NaCl, and then... (The sentence is incomplete and requires more context to translate accurately.) TM Filter using a sterile syringe filter (SLGV033RS). Then, use... Thirty-seven conditions were established. These conditions correspond to the whole factorial studies (32 samples, see Table 1), with an additional 3 centerpoints (0.75 mg / ml Protein D, 75 mM NaCl, 5% sucrose, 0.25% poloxamer 188, and pH 7.4) and 2 actual processes (1 mg / ml PD in 150 mM NaCl). Preparations were made in PEN glass containers (unsiliconized) (2 × 10 ml each time). Two PEN containers were combined into a single Duran Schott container (unsiliconized) (20 ml) and stored at 2–8 °C for different time points (1 day, 7 days, 14 days, and 21 days).

[0239] For time points 7 and 14, a control without visible particles was added to the light-shielding measurements. This control served as a reference for filtering during the day (actual procedure: 1 mg / ml protein D in 150 mM NaCl). After 21 days, the control without visible particles was not analyzed because sufficient data were generated at time points 7 and 14.

[0240] Visual inspection

[0241] All visual inspections were performed by the same person at each time point (days 1, 7, 14, and 21) (see Table 1). The visual inspections were conducted in a laboratory setting, not at a black-and-white visual inspection station. The aim was to define the conditions that would allow for a reduction or elimination of the presence of visible particles (±50 μm).

[0242] surface 1. Visual inspection was conducted at four time points (T1 day, T7 day, T14 day, and T21 day). - indicates no particles, + indicates almost no particles, and ++ indicates a large number of particles (this classification is determined by the person conducting the visual inspection).

[0243]

[0244]

[0245] As shown in Table 1 above, visible particles were present in some samples after 24 hours of storage at 2 / 8°C. After 7 days of storage at 2 / 8°C, only 20% of the samples were free of visible particles. At time points of 14 and 21 days, visual inspection revealed visible particles in 100% of the samples. For all samples, an increase in the number of fibers was observed over time.

[0246] Statistical analysis was performed, and visual inspections were graded (=0, +=5, and ++=10). Based on this grading, the visual inspection process was described (see...). Figure 1 This statistical analysis was performed to confirm the visual observations. (As follows...) Figure 1As can be seen, the values ​​on the left side of the graph (samples without poloxamer 188) are always greater than the values ​​on the right side (samples containing 0.5% poloxamer 188). Based on the current visual inspection results, poloxamer 188 appears to have an effect. There is no clear evidence of the effect of sucrose or NaCl in the visual inspection.

[0247] Light shading

[0248] Light shading measurements were performed at each time point (T1, 7, 14, and 21 days). After data generation, two decisions were made for statistical analysis. The first was to consider only particles larger than 35 μm (50 μm particles visible to the naked eye). The second was to sum all visible particles. This decision helped standardize the data.

[0249] On day 1, significant effects were observed between sucrose alone and NaCl alone (see Figure 2 Fewer visible particles were observed with or without sucrose (with or without NaCl) compared to the absence of NaCl and the absence of sucrose. The presence of NaCl, other than sucrose, did not have a greater impact on the reduction of visible particles.

[0250] On day 7, significant effects of sucrose and NaCl were observed (see [link]). Figure 3 and Figure 4 For both, fewer visible particles were observed in their presence.

[0251] in conclusion:

[0252] This evaluation proves that:

[0253] • Based on the light-blocking results of visible particles (based on the sum of 35-70 micrometers), the addition of NaCl is advantageous.

[0254] • Based on the light-blocking results of visible particles (based on the sum of 35-70 micrometers), the addition of 10% m / v sucrose is advantageous.

[0255] • No effect was observed for protein D concentrations between 0.5 and 1 mg / ml.

[0256] For poloxamer 188 in the range of 0% m / v to 0.5% m / v, an effect on subvisible particles (less than 25 micrometers) was observed from the light-shielding results, but no effect on visible particles. However, poloxamer 188 may have an effect from visual inspection.

[0257] • Due to the difference between theoretical and measured pH, there is no experience to learn regarding pH.

[0258] Example 2: Screening of excipients and their effect on particle formation during liquid storage (Part 2)

[0259] In this study, the following parameters were investigated in two batches of protein D:

[0260] -pH (2 levels)

[0261] o 6,4

[0262] o 7,4

[0263] -Sucrose (2 levels)

[0264] o 10% m / v sucrose

[0265] o 20% m / v sucrose

[0266] -Polosham 188 (2 levels)

[0267] o 0%

[0268] o 1%

[0269] -NaCl (1 level)

[0270] o 150mM

[0271] - Protein D concentration (1 level)

[0272] o 1mg / ml

[0273] Two batches of frozen protein D were thawed in an incubator at 25°C (air) for 1 hour and 30 minutes. After thawing, protein D was diluted to 20 mg / ml in 150 mM NaCl, and then... (The sentence is incomplete and requires more context to translate accurately.) TM Filtration was performed using a sterile syringe filter (SLGV033RS). Then, the filter was moved by a Tecan robot. 28 conditions were set.

[0274] Table 2: Excluding the 6 facet center points and 2 current processes, the total factorial analysis of DoE (at 150mM) 1 mg / ml NaCl PD)

[0275]

[0276]

[0277] Visual inspection

[0278] All visual inspections were conducted at a black-and-white visual inspection station (using only a black background) by five people at 1-day time points, and by seven people at 7-day and 14-day time points. All samples were classified using a five-level scale (0, -, +, ++ & +++), representing no particles, few particles, some particles, many particles, and a large number of particles, respectively. Statistical analysis was performed, and visual inspection results were graded (0 = 0, - = 1, + = 2, ++ = 3, and +++ = 4).

[0279] As in Example 1, visible particles were present in some samples after storage at 2 / 8°C for 24 hours. For all samples, the number of visible particles was observed to increase over time. Statistical analysis was performed, and visual inspection was graded (0 = 0, - = 1, + = 2, ++ = 3, and +++ = 4). Based on this grading, the visual inspection was described. This citation was then processed in the statistical analysis to confirm the visual observations. The results were sorted, first at time points 1, 7, and 14 days, according to poloxamer 188, sucrose, or pH.

[0280] Considering the average scores of all observers on days 1, 7, and 14, poloxamer 188 was observed to have a significant effect on lower (i.e., visible particle reduction) scores in the presence of poloxamer 188.

[0281] Considering the average scores from all observers, a trend towards lower scores (i.e., visible particle reduction) was observed as pH increased. However, a significant effect was only observed on day 7 (p-value = 0.0129), with lower scores at pH 6.9. A significant interaction between poloxamer 188 and pH was also observed on day 7 (see [link to poloxamer 188]). Figure 5 In fact, pH has a significant effect when poloxamer 188 is absent. As pH increases, the number of visible particles decreases.

[0282] Considering the average scores of all observers on day 1, no significant effect was observed. On day 7, a slight but significant effect of sucrose was observed (p = 0.0179) (see [link to relevant documentation]). Figure 6 The presence of 20% sucrose resulted in a lower score (i.e., visible particle reduction). On day 14, the effect of sucrose was observed (p = 0.08), with a lower score (i.e., visible particle reduction) in the presence of 20% sucrose.

[0283] Considering three time points (days 1, 7, and 14), the presence of poloxamer 188 was beneficial in reducing the number of visible particles. This reduction may have been slightly improved at the highest sucrose levels (20% m / v) (see [link to study]). Figure 5 However, although a statistically relevant effect was observed for sucrose, the actual correlation is considered to be limited.

[0284] Light shading

[0285] Light shading measurements were performed at each time point (T1, 7, and 14 days). Only particles larger than or equal to 35 μm were considered (particles of 50 μm are visible to the naked eye). Statistical analysis was performed based on the sum of particles between 35 μm and 70 μm.

[0286] Sample 18COP02003 (poloxamer-free, pH 6.4; sucrose 20% m / v) was detected as atypical across the entire particle size range (2 to 125 μm). No explanation was found for this atypical result.

[0287] Statistical analysis was performed. Results were analyzed at time points 1, 7, and 14, sorted according to poloxamer 188, sucrose, or pH. Analysis was based on the average of the measurements. Light shading measurements were obtained by analyzing four 1 ml samples. The first value obtained in the first ml was discarded and used only for rinsing the equipment.

[0288] Considering the average of three measurements of the total number of particles ranging from 35 to 70 micrometers on days 1, 7, and 14, the number of particles was lower in the presence of poloxamer for each configuration tested. This was also the case when atypical results were removed (configuration: no poloxamer, pH 6.4, 20% m / v sucrose).

[0289] in conclusion:

[0290] This evaluation proves that:

[0291] • Whether measured by light shielding, visual inspection, or Occhio, the addition of poloxamer 188 showed a significant effect in reducing visible particles. This is consistent with the results observed in Example 1, which showed reductions of up to 0.5% m / v. The observed reductions were almost identical at 1% m / v and 0.5% m / v.

[0292] Increasing sucrose from 10% m / v to 20% m / v had no practically significant effect on the reduction of visible particles. The increase in sucrose raised the melting temperature and the temperature at which aggregation began. From a visual inspection point of view, this increase slightly improved the reduction of visible particles, but this observation was not correlated with the fact that 10% m / v sucrose was beneficial for light shading.

[0293] Example 3: Optimized methods for thawing, dilution, and filtration of protein D

[0294] For the first step, statically thaw protein D (4.5 ml Nunc container) in an incubator at 25°C. Once thawed, homogenize protein D by stirring with a magnetic stir bar. Then, follow the flowchart below ( Figure 1Protein D was placed in a Duran Schott glass container in 150 mM NaCl, 10% w / v sucrose, 1% w / v poloxamer 188, and 12.5 mM PO4. 3- Dilute to 1 mg / mL in KH₂PO₄ / K₂HPO₄ phosphate buffer, pH 6.8. Add via pipette or graduated glass. To achieve these target concentrations, use 15.75% w / v sucrose solution, 100 mM KH₂PO₄ / K₂HPO₄ 1160 mM NaCl pH 6.9 buffer, and 10% w / v poloxamer 188 solution. Protein D dilution is based on protein D content determined by RP-UPLC, which was pre-obtained on other aliquots from the same three batches of pharmaceutical material. Once diluted, use... 47 filter (0.22μm) PVDF membrane 17.7cm 2 - Polypropylene column) and peristaltic pump (flow rate 0.7 ml / min / cm) 2 Filter protein D.

[0295] Example 4: Comparison of protein D dilution processes

[0296]

[0297] Optimization methods:

[0298] According to Example 3 and Figure 7 The flowchart provides a method (optimization method) for diluting protein D.

[0299] Reference method:

[0300] according to Figure 8 The flowchart provides a method (reference method) for diluting protein D. Thaw frozen PD drug material (stored at -45°C, pH 6.8) as follows:

[0301] -2-4g aliquots: incubate at 2-8℃ for at least 7h-72h, or at 25±1℃ for at least 1h-2h (water bath).

[0302] -18g aliquots: incubate at 2-8℃ for at least 24h-72h, or at 25±1℃ for at least 2h-3h (water bath).

[0303] Once thawed, dilute PD to approximately 1 mg / ml with 150 mM NaCl and filter through a 0.22 μm filter. Filter characteristics: Millex (0.45-) 0.22 μm PVDF, optimal protein loading to area ratio: 90 mg prot / cm². 2(For example, 20ml PD, 20mg / ml, at Millex GV33 mm 0.22μm 4.5cm) 2 (Upper filter).

[0304] Three different batches of protein D drug material (APDOAPA024, APDOBPA027 & APDOBPA029) were evaluated. For each batch, eight dilutions of 1 mg / ml protein D were performed: four with optimized formulations (10% m / v sucrose, 1% m / v poloxamer 188, 150 mM NaCl, 1 mg / ml protein D, 12.5 mM phosphate buffer K2HPO4 / KH2PO4, pH 6.8) and four with the current method as a reference (150 mM NaCl, pH 6.8). The target protein D concentration of 1 mg / ml was based on Lowry values.

[0305] The optimized and reference protein D dilution methods were compared using the following analytical techniques (as described above):

[0306] • Particle detection using light masking, Occhio (Flow Cam), and visual inspection.

[0307] Secondary and tertiary structures determined by intrinsic fluorescence, FTIR, and far-UV circular dichroism.

[0308] • Protein D content determined by RP-UPLC.

[0309] At each time point, all visual inspections were conducted by eleven people in a black-and-white visual inspection station (using only a black background), not by all observers. All samples were categorized, resulting in scores from 0 (no particles) to 6 (fully visible particles). A graph showing only the average scores from all observers at the three time points (days 1, 7, and 14) is shown. Figure 12 middle.

[0310] Multivariate analysis (principal component analysis) was performed using the PCA method. Multivariate analysis aims to synthesize information from several variables into a two-dimensional representation to better interpret the data.

[0311] result:

[0312] · Figure 9 This represents the total number of particles ranging from 50 to 1000 μm detected by Occhio at three time points (1, 7 & 14 days) for the optimized liquid composition and the reference sample. A significant evolution in particle number was observed for the reference method, while the number remained more stable for the optimized composition.

[0313] · Figure 10Examples of photographs of visible particles captured by Occhio in a protein D reference sample (1 mg / ml in 150 mM NaCl) are provided.

[0314] · Figure 11 Multivariate analysis (PCA) was performed, taking into account the entire range of light shading and Occhio measurements. A significant difference was observed between the optimized and reference samples. The optimized sample was more homogeneous than the reference sample. The horizontal axis summarizes the particle number across the entire range: more particles were measured for the reference sample across the entire range of light shading and Occhio measurements. The vertical axis is more discriminative for the reference sample (no expansion was observed on the vertical axis for the optimized sample). The top sample was characterized by a higher number of visible particles and a lower number of invisible particles. It can be inferred that the optimized method has better reproducibility.

[0315] · Figure 12 The score represents the average score from observers who visually inspected three different batches at the black and white workstations. The optimized sample scored lower regardless of the day or the protein D batch.

[0316] · Figure 13 and 14 The far-UV CD spectrum represents the differential spectrum and shows subtle differences in the 208 nm and 222 nm regions. This reflects a slight change in secondary structure (an increase in α-helical content).

[0317] in conclusion:

[0318] All evaluations of protein D confirmed that:

[0319] When added to liquid protein D, a significant reduction in the number of particles was observed:

[0320] 1% w / v Poloxamer 188

[0321] 150mM NaCl

[0322] 10% w / v sucrose

[0323] 12.5mM K2HPO4 / KH2PO4 buffer, pH 6.8

[0324] It has no effect on the distribution, size, and molar mass of protein D.

[0325] It has no significant impact on PD levels and antigenicity.

[0326] There are slight differences between the secondary and tertiary structures (protein D folds slightly more in this optimized composition).

[0327] Furthermore, filtration of protein D after dilution with the new composition showed no loss of content.

[0328] sequence:

[0329] SEQ ID NO 1: Protein D (364 amino acids)

[0330]

[0331]

[0332] SEQ ID NO:3:SerSerHisSerSerAsnMetAlaAsnThr

[0333] SEQ ID NO:4: Protein E from Haemophilus influenzae

[0334]

[0335] SEQ ID NO:5: Amino acids 20-160 of protein E

[0336]

[0337]

[0338] SEQ ID NO:6 PilA from Haemophilus influenzae

[0339]

[0340] SEQ ID NO:7 Amino acids 40-149 of PilA from Haemophilus influenzae strain 86-028NP

[0341]

[0342] SEQ ID NO: 8: LVL735 (protein): (pelB sp) (ProtE aa 20-160) (GG) (PilA aa40-149)

[0343]

[0344] SEQ ID NO:9: PE-PilA fusion protein without signal peptide

[0345]

[0346]

[0347] SEQ ID NO:10: UspA2 from ATCC 25238

[0348] MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDIT

[0349] ALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGE

[0350] AIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYD

[0351] FGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSG

[0352] RLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQA

[0353] NIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDA

[0354] LNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINN

[0355] IYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKL

[0356] ITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK

[0357] VNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0358] SEQ ID NO:11:MC-001 (Protein)–(M)(UspA2 amino acids 30 - 540)(ASHHHHHH)

[0359] MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKASHHHHHH

[0360] SEQ ID NO:12 MC-002 (Protein)–(M)(UspA2 Amino Acids 30 - 540)

[0361] MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRIDQKADIDNNIINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIADLAAYNELQDAYAKQQQQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTK

[0362] SEQ ID NO:13 MC-003 (Protein)–(M)(Amino acids 30 - 540 of UspA2)(H)MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKH

[0363] SEQ ID NO:14 MC-004 (Protein)–(M)(Amino acids 30 - 540 of UspA2)(HH)

[0364] MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKHH

[0365] SEQ ID NO:15 MC-005 (Protein)–(M)(UspA2 amino acids 30 - 519)(ASHHHHHH)

[0366] MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSASHHHHHH

[0367] SEQ ID NO:16 MC-006 (Protein)–(M)(UspA2 amino acids 30 - 519)

[0368] MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKS

[0369] SEQ ID NO:17 MC-007 (Protein)–(M)(UspA2 amino acids 30 - 564)(ASHHHHHH)

[0370] MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAASHHHHHH

[0371] SEQ ID NO:18 MC-008 (Protein)–(M)(UspA2 30-564)(HH)

[0372] MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAHH

[0373] SEQ ID NO:19 MC-009(Protein)–(M)(UspA2 31-564)(HH)

[0374] MAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAAHH

[0375] SEQ ID NO:20 MC-010 (Protein)–(M)(UspA2 amino acids 30 - 564)

[0376] MQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAA

[0377] SEQ ID NO:21 MC-011 (Protein)–(M)(UspA2 amino acids 31 - 540)(ASHHHHHH)

[0378] MAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADINNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHL IDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNNAITKNAKSITDLGTKVDGFDSRVTALDTKASHHHHH

[0379] SEQ ID NO:22 UspA2 American 2933(613 aa)

[0380] MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDINTLKQDQQKMNKYLLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLIEKDEEHDRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAERIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0381] SEQ ID NO:23 UspA2 American 2912(644 aa)

[0382] MKTMKLLPLKIAVTSALIIGLGAASTANAQQQLQTETFLPNFLSNDNYDLTDPFYHNMILGDTALLDKQDGSQPQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKKGDTIIPLDKDGKPVYQVDYKLDGKGKKQKRRQVYSVTTKTATDDDVNSAYSRGILGKVDDLDDEMNFLNHDITSLYDVTANQQDAIKDLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0383] SEQ ID NO:24 UspA2 American 2908(591 aa)

[0384] MKTMKLLPLKIAVTSALIVGLGAASTANAQLVERFFPNIFLDKPLAKQHYHNVVVGDTSIVSDLQSNSDQLKFYSDDEGLVPDSLLFNKMLHEQLLNGFKEGDTIIPLDENGKPVYKVDYKLDGKEPRKVYSVTTKIATAEDVATSSYANGIQKDIDDLYDFDHQVTERLTQHGKTIYRNGERILANEESVQYLNKEVQNNIEHIYELAQQQDQHSSDIKTLESNVEKGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLIDQKADLTKDIKTLESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0385] SEQ ID NO:25 UspA2 Finnish 307(687 aa)

[0386] MKTMKLLPLKIAVTSAMIIGLGAASTANAQQQQQQQQQQQSRTEIFFPNIFFNENHDELDDAYHNIILGDTALLDKQDGSQPQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKKGDTIIPLDKDGKPVYQVDYKLDGKGKKQKRRQVYSVTTKTATDDDVNSAYSRGILGKVDDLDDEMNFLNHDITSLYDVTANQQDAIKGLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0387] SEQ ID NO:26 UspA2 Finnish 353 (683 amino acids)

[0388] MKTMKLLPLKIAVTSAMIVGLGMASTANAQQQKSPKTETFLPNIFFNEYADDLDTLYHNMILGDTAITHDDQYKFYADDATEVPDSLFFNKILHDQLLYGFKEGDKIIPLDENGKPVYKLDKRLENGVQKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0389] SEQ ID NO:27 UspA2 Finnish 358(684 amino acids)

[0390] MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGNTALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGGTIIPLDENGKPVYKLDSIVEQGKTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0391] SEQ ID NO:28 UspA2 Finnish 216(684 amino acids)

[0392] MKTMKLLPLKIAVTSAMIIGLGAASTANAQQQQKTKTEVFLPNLFDNDYYDLTDPLYHSMILGDTATLFDQQDNSKSQLKFYSNDKDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTQDTRTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVGKDLLDLSGRLIAQKEDIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0393] SEQ ID NO:29 UspA2 Dutch H2 (684 amino acids)

[0394] MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGNTALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKKGDTIIPLDENGKPVYKLDSIVEQGKTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIYELVQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0395] SEQ ID NO:30 UspA2 Dutch F10 (574 amino acids)

[0396] MKTMKLLPLKIAVTSAMIIGLGAASTANAQLAEQFFPNIFSNHAPVKQHYHNVVVGDTSIVENLQDSDDTQLKFYSNDEYSVPDSLLFNKMLHEQQLNGFKKGDTIIPLDENGKPVYKVDYKLDGQEPRRVYSVTTKIATQDDVDNSPYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0397] SEQ ID NO:31 UspA2 Norwegian 1 (678 amino acids)

[0398] MKTMKLLPLKIAVTSALIVGLGAASTANAQQQPQTETFFPNIFFNENHDALDDVYHNMILGDTAITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGVKKSVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0399] SEQ ID NO:32 UspA2 Norwegian 13 (678 amino acids)

[0400] MKTMKLLPLKIAVTSAMIVGLGAASTANAQQQQQPRTETFFPNIFFNENHDALDDVYHNMILGDTAITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGVKKSVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0401] SEQ ID NO:33 UspA2 Norwegian 33 (587 amino acids)

[0402] MKTMKLLPLKIAVTSALIVGLGAASTANAQLVERFFPNIFLDKPLAKQHYHNVVVGDTSIVSDLQSNSDQLKFYSDDEGLVPDSLLFNKMLHEQLLNGFKEGDTIIPLDENGKPVYKVDYKLDGKEPRKVYSVTTKIATAEDVATSSYANGIQKDIDDLYDFDHQVTERLTQHGKTIYRNGERILANEESVQYLNKEVQNNIEHIYELAQQQDQHSSDIKTLESNVEKGLLELSGHLIDQKADLTKDIKTLENNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0403] SEQ ID NO:34 UspA2 Norwegian 25(678 amino acids)

[0404] MKTMKLLPLKIAVTSAMIVGLGAASTANAQQQQQPRTETFFPNIFFNENHDALDDVYHNMILGDTAITQDNQYKFYADAISEVPDSLLFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEKVENGVKKSVYSVTTKTATRADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDTNKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0405] SEQ ID NO:35 UspA2 Norwegian 27(616 amino acids)

[0406] MKTMKLLPLKIAVTSALIVGLGAASTANAQVRDKSLEDIEALLGKIDISKLEKEKKQQTELQKYLLLSQYANVLTMEELNKNVEKNTNSIEALGYEIGWLENDIADLEEGVEELTKNQNTLIEKDEEHDRLIAQNQADIKTLENNVVEELFNLSDRLIDQEADIAKNNASIEELYDFDNEVAERIGEIHAYTEEVNKTLEKLITNSVKNTDNIDKNKADIQALENNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0407] SEQ ID NO:36 UspA2 Norwegian 36 (676 amino acids)

[0408] MKTMKLLPLKIAVTSALIVGLGAASTANAQATETFLPNLFDNDYTETTDPLYHGMILGNTAITQDTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENGVKRKVYSVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0409] SEQ ID NO:37 UspA2 BC5SV (629 amino acids)

[0410] MKTMKLLPLKIAVTSALIVGLGAASTANAQNGTSTKLKNLKEYAQYLDNYAQYLDDDIDDLDKEVGELSQNIAKNQANIKDLNKKLSRDIDSLREDVYDNQYEIVNNQADIEKNQDDIKELENNVGKELLNLSGRLLDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0411] SEQ ID NO:38 UspA2 Norwegian 14(683 amino acids)

[0412] MKTMKLLPLKIAVTSAMIVGLGMASTANAQQQRSPKTETFLPNIFFNEYADDLDTLYHNMILGDTAITHDDQYKFYADDATEVPDSLFFNKILHDQLLYGFKEGDKIIPLDENGKPVYKLDKRLDNGVQKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNRIKALENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0413] SEQ ID NO:39 UspA2 Norwegian 3 (700 amino acids)

[0414] MKTMKLLPLKIAVTSAMIVGLGAASTANAQAQSNRSLDQVQALLRGIDETKIKKEIQQSQQPELNKYLTFNQLANALNIEELNNNVQKNTQRLDSAATLYGDLSKTVPKSIKENKESIKENKESIKENKESIKENKESIKENKESIKENKESITTLTRKSFQNQVDIVRNNASIEDLYAYGQEVAKSIGEIHAYTEEVNKTLENLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTVIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0415] SEQ ID NO:40 UspA2 Finnish 414(676 amino acids)

[0416] MKTMKLLPLKIAVTSALIVGLGAASTANAQATETFLPNLFDNDYIETTDPLYHGMILGNTAITQDTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENGVKRKVYSVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKADLTKDIKTLENNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0417] SEQ ID NO:41 UspA2 Japanese Z7476 (678 amino acids)

[0418] MKTMKLLPLKIAVTSAMIIGLGAASTANAQLAEQFFPNIFSNHAPVKQHYHNVVVGDTSIVENLQDSDDTQLKFYSNDEYSVPDSLLFNKMLHEQQLNGFKKGDTIIPLDENGKPVYKVDYKLDGQEPRRVYSVTTKIATQDDVDNSPYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIYELAQQQDQHSSDIKTLKKNVEEGLLELSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0419] SEQ ID NO:42 UspA2 Belgian Z7530 (613 amino acids)

[0420] MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDINTLKQDQQKMNKYLLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLIEKDEEHDRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAERIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0421] SEQ ID NO:43 German Z8063 (589 amino acids)

[0422] MKTMKLLPLKIAVTSALIVGLGAASTANAQATNKDITLEDVLKSIEEIDPYELRDYIEYPTAIERFLLLSQYGNTLTLEEFDNDIELLDQDVEDLEESVTELAKNQNSLIEQGEAIKEDLQGLADFVERQEDKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAKSIGEIHAHNEAQNETLKDLITNSVKNTDNITKNKADIQALESNVEKGLLELSGHLIDQKADIDNNINNIHELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0423] SEQ ID NO:44 UspA2 American O12E (684 amino acids)

[0424] MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFD

[0425] NDNTELTDPLYHNMILGNTALLTQENQYKFYADDGNGVPDSLLFNKI

[0426] LHDQLLHGFKEGDTIIPLDENGKPVYKLDSIVEQGKTKTVYSVTTKT

[0427] ATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQ

[0428] YLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIA

[0429] QKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNI

[0430] AKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLL

[0431] DLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNK

[0432] ASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0433] SEQ ID NO:45 UspA2 Greek MC317 (650 amino acids)

[0434] MKTMKLLPLKIAVTSALIVGLGAASTANAQQQQKTKTEVFLPNLFYNDYIEETDLLYHNMILGDTAALVDRQNYSNSQLKFYSNDEESVPDSLLFSKMLNNQQLNGFKAGDIIIPVDANGQVIYQKDTRVEGGKTRTVLSVTTKIATQQDVDSAYSRGIQGKVNDLDDEMNFLNHDITSLYDVTANQQDDIKGLKKGVKDLKKGVKGLNKELKELDKEVGVLSRDIGSLNDDVAQNNESIEDLYDFSQEVADSIGEIHAHNKAQNETLQDLITNSVENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADLTKDIKTLESNVEEGLLELSGHLIDQKADIAKNQADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0435] SEQ ID NO:46 UspA2 American V1122 (616 amino acids)

[0436] MKTMKLLPLKIAVTSALIVGLGAVSTTNAQAQSRSLDQIQTKLADLAGKIAAGKNGGGQNNQNNQNDINKYLFLSQYANILTMEELNNNVVKNSSSIETLETDFGWLENDVADLEDGVEELTKNQNTLIEKDEEHDRLIAQNQADIQTLENNVVEELFNLSDRLIDQKADIAKNQADIAQNNESIEELYDFDNEVAEKIGEIHAYTEEVNKTLQDLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLENNVEEGLLDLSGRLIDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0437] SEQ ID NO:47 UspA2 American P44 (668 amino acids)

[0438] MKTMKLLPLKIAVTSALIVGLGTASTANAQVASPANQKIQQKIKKVRKELRQDIKSLRNDIDSNTADIGSLNDDVADNQDDILDNQADIAKNQDDIEKNQADIKELDKEVGVLSREIGSLNDDIADNYTDIIDNYTDIIDNQANIAKNQDDIEKNQADIKELDKEVGVLSREIGSLNDDVADNQDDIAKNQADIQTLENNVEEGLLELSGHLLDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQEQTEAIDALNKASSENTQNIAKNSNRIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKVSADTKFAATADAITKNGNAITKNAKSITDLGTKVDAFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0439] SEQ ID NO:48 UspA2 American V1171 (674 amino acids)

[0440] MKTMKLLPLKIAVTSAMIVGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTAIVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDTRTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0441] SEQ ID NO:49 UspA2 American TTA24 (613 amino acids)

[0442] MKTMKLLPLKIAVTSAMIIGLGAASTANAQSRDRSLEDIQDSISKLVQDDIDTLKQDQQKMNKYLLLNQLANTLITDELNNNVIKNTNSIEALGDEIGWLENDIADLEEGVEELTKNQNTLIEKDEEHDRLIAQNQADIQTLENNVVEELFNLSGRLIDQEADIAKNNASIEELYDFDNEVAERIGEIHAYTEEVNKTLENLITNSVKNTDNIDKNKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTNRIATAELGIAENKKDAQIAKAQANANKTAIDENKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0443] SEQ ID NO:50 UspA2 American O35E (576 amino acids)

[0444] MKTMKLLPLKIAVTSAMIVGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTAIVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDTRTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0445] SEQ ID NO:51 UspA2 American SP12-6 (684 amino acids)

[0446] MKTMKLLPLKIAVTSAMMVGLGMASTANAQQQKSPKTEIFLPNLFDNDNTELTDPLYHNMILGNTALLTQENQYKFYADDGNGVPDSLLFNKILHDQLLHGFKEGDTIIPLDENGKPVYKLDSIVEQGKTKTVYSVTTKTATADDVNSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0447] SEQ ID NO:52 UspA2 American SP12-5 (686 amino acids)

[0448] MKTMKLLPLKIAVTSAMIIGLGAASTANAQATETFLPNLFDNDYTETTDPLYHGMILGNTAITQDTQYKFYAENGNEVPDSLFFNKILHDQQLNGFKEGDTIIPLDENGKPVYKLDEITENGVKRKVYSVTTKTATREDVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNKEVQNNIENIHELAQQQDQHSSDIKTLKKNVEEGLLELSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAKNQADIAQNQTDIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0449] SEQ ID NO:53 UspA2 Swedish BC5 (630 amino acids)

[0450] MKTMKLLPLKIAVTSAMIIGLGAASTANAQAKNDITLEDLPYLIKKIDQNELEADIGDITALEKYLALSQYGNILALEELNKALEELDEDVGWNQNDIANLEDDVETLTKNQNALAEQGEAIKEDLQGLADFVEGQEGKILQNETSIKKNTQRNLVNGFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAHNEAQNETLKGLITNSIENTNNITKNKADIQALENNVVEELFNLSGRLIDQKADIDNNINNIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKTDIAQNQANIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0451] SEQ ID NO:54 UspA2 American 7169(616 amino acids)

[0452] MKTMKLLPLKIAVTSALIVGLGAASTANAQAQDRSLEQIQDKLANLVEKIEQAKSQNGQSQKDINQYLLLSQYANVLTMEELNNNVVKNSSSIETLDNDIAWLNDDLIDLDKEVGVLSRDIGSLHDDVAQNQADIKTLKNNVVEELFNLSDRLIDQEADIAQNNESIEDLYDFGREVAESIGEIHAHNEAQNETLKDLITNSVKNTDNITKNKADIQALENDVGKELLNLSGRLIDQKADIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLLDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0453] SEQ ID NO:55 UspA2 Finnish FIN2344 (614 amino acids)

[0454] MKTMKLLPLKIAVTSAMIIGLGATSTVNAQVVEQFFPNIFFNENHDELDDAYHNMILGDTAIVSNSQDNSTQLKFYSNDEDSVPDSLLFSKLLHEQQLNGFKAGDTIIPLDKDGKPVYTKDTRTKDGKVETVYSVTTKIATQDDVEQSAYSRGIQGDIDDLYDINREVNEYLKATHDYNERQTEAIDALNKASSANTDRIDTAEERIDKNEYDIKALESNVGKDLLDLSGRLIAQKEDIDNNINHIYELAQQQDQHSSDIKTLKNNVEEGLLELSGHLIDQKADLTKDIKTLESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKVSAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0455] SEQ ID NO:56 UspA2 American V1118 (679 amino acids)

[0456] MKTMKLPPLKIAVTSAMIIGLGAASTANAQTTETFLPNLFDNDYTETTDPLYHGMILGDTAITQDTQYKFYAENGNEVPDSLFFNKILHDQLLNGFKAGDTIIPLDENGKPVYKLDERTENGVKRKVYSVTTKTATQADVEQSAYSRGIQGDIDDLYEANKENVNRLIEHGDKIFANEESVQYLNREVQNNIENIHELAQQQDQHSSDIKTLKKNVEKDLLDLSGRLIAQKEDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIAKNSNHIKTLENNIEECLLELSGHLIDQKADLTKDIKALESNVEEGLLDLSGRLIDQKADIAQNQANIQDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDGRVTALDTKVNALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0457] SEQ ID NO:57 UspA2 American V1145 (724 amino acids)

[0458] MKTMKLLPLKIAVTSALIVGLGAASTANAQETLEEVLESIKQINEQDLQDDIGYNSALDRYLVLSQYGNLLIAKELNENVEKNSNSIAKNSNSIADLEADVGYLAENQNTLIEQNETINQELEGITHELESFIAYAHAQDQKNLVNEFEIEKNKDAIAKNNESIEDLYDFGHEVAESIGEIHAYTEEVNKTLENLITNSVKNTDNITKNKADIQALESNVEKELLNLSGRLIDQKADIDNNINHIYELAQQQDQHSSDIKTLKKNVEEGLLELSGHLIDQKSDIAQNQTDIQDLATYNELQDQYAQKQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIEDLAAYNELQDAYAKQQTEAIDALNKASSENTQNIAKNQADIANNINNIYELAQQQDQHSSDIKTLAKASAANTDRIAKNKADADASFETLTKNQNTLIEKDKEHDKLITANKTAIDANKASADTKFAATADAITKNGNAITKNAKSITDLGTKVDGFDSRVTALDTKVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYRVNPNLAFKAGAAINTSGNKKGSYNIGVNYEF

[0459] SEQ ID NO:58 UspA2 American V1156 (611 amino acids)

[0460] 。

Claims

1. A method for preparing a liquid composition comprising a protein D polypeptide with the amino acid sequence shown in SEQ ID NO: 2, wherein the method comprises mixing the protein D polypeptide with a solution of (a) sucrose, (b) poloxamer 188, (c) NaCl, and (d) a buffer solution to achieve a pH of 6.4 to 7.4, and wherein the concentration of the protein D polypeptide is 0.5 to 1 mg / ml, the concentration of sucrose is 5 to 20% w / v, and the concentration of poloxamer 188 is 0.1 to 1% w / v.

2. The method for preparing a liquid composition comprising a protein D polypeptide according to claim 1, wherein the buffer is a phosphate buffer.

3. The method for preparing a liquid composition comprising a protein D polypeptide according to claim 1, comprising mixing the protein D polypeptide with the solution to achieve a pH of 6.

8.

4. A method for preparing a liquid composition comprising a protein D polypeptide according to any one of claims 1 to 3, wherein the method comprises the following steps: (i) Thaw the protein D polypeptide, and (ii) mix the protein D polypeptide with the solution.

5. The method for preparing a liquid composition comprising a protein D polypeptide according to any one of claims 1 to 3, further comprising a filtration step to obtain a liquid composition comprising a protein D polypeptide in the filtrate.

6. The method for preparing a liquid composition comprising a protein D polypeptide according to claim 5, wherein the filtration step uses a 0.22 µm PVDF membrane.

7. The method for preparing a liquid composition comprising a protein D polypeptide according to any one of claims 1 to 3, further comprising the step of storing the liquid composition comprising the protein D polypeptide.

8. The method for preparing a liquid composition comprising a protein D polypeptide according to any one of claims 1 to 3, further comprising the step of mixing the liquid composition comprising the protein D polypeptide with other antigens.

9. The method of claim 8 for preparing a liquid composition comprising a protein D polypeptide, wherein the other antigen comprises a PE-PilA fusion protein and a UspA2 polypeptide.

10. A method comprising preparing a liquid composition comprising a protein D polypeptide according to any one of claims 1 to 9, and subsequently freeze-drying the liquid composition comprising the protein D polypeptide.

11. A liquid composition comprising a protein D polypeptide with the amino acid sequence shown in SEQ ID NO: 2, sucrose, poloxamer 188, a buffer solution, and NaCl, said composition having a pH of 6.4 to 7.4, and wherein the concentration of the protein D polypeptide is 0.5 to 1 mg / ml, the concentration of the sucrose is 5 to 20% w / v, and the concentration of the poloxamer 188 is 0.1 to 1% w / v.

12. The liquid composition of claim 11, wherein the buffer solution is a phosphate buffer solution.

13. The liquid composition according to claim 11, having a pH of 6.8.

Images