Recombinant human alpha-1 antitrypsin glycoproteins for use in treatment of lung non-viral inflammatory diseases

Abstract

The present invention relates to a human recombinant AAT (rhAAT) protein or fragment thereof expressed in a genetically modified yeast for use in the treatment and / or prevention of lung non-viral diseases associated with inflammation and / or pathological immune responses. In embodiments, the rhAAT or fragment has one or more N-linked glycosylation, including at least one HexNAC1 glycosylation. In other embodiments, the rhAAT glycoprotein or a fragment thereof comprises at least one of the glycosylation of Hex9 HexNac2, Hex10 HexNac2, Hex11 HexNac2, Hex12 HexNac2, Hex13 HexNac2, Hex14 HexNac2, Hex15 HexNac2, and / or Hex16 HexNac2. The invention further relates to the use of the rhAAT glycoprotein or the fragment thereof. In embodiments, the invention also relates to a protein formulation comprising a recombinant human alpha 1-antitrypsin (rhAAT) glycoprotein or a fragment thereof having a heterologous N-linked glycosylation. The invention also relates to a pharmaceutical composition comprising said rhAAT protein or fragment thereof or a protein preparation for use in the treatment of lung non-viral diseases associated with inflammation and / or pathological immune responses.

Description

[0001] manual

[0002] This invention belongs to the field of recombinant glycoprotein production, including methods for yeast fermentation, recombinant protein expression and purification, and the medical uses of said recombinant glycoprotein. Specifically, this invention relates to the use of said recombinant glycoprotein in the treatment and / or prevention of non-viral inflammatory diseases of the lungs.

[0003] Therefore, the present invention relates to a human recombinant AAT (rhAAT) protein or a fragment thereof, expressed in genetically modified yeast, for use in the treatment and / or prevention of nonviral lung diseases associated with inflammation and / or pathological immune responses.

[0004] In embodiments, the various glycoprotein structures disclosed herein can be used to define the AAT protein of the present invention. In embodiments, rhAAT or fragments have one or more N-linked glycosylations, including at least one HexNAc1 glycosylation. In other embodiments, the rhAAT glycoprotein or fragments thereof include at least one Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2 glycosylation. In embodiments, the present invention also relates to a protein formulation comprising a recombinant human α1-antitrypsin (rhAAT) glycoprotein or fragment thereof having heterologous N-linked glycosylations.

[0005] The present invention also relates to a pharmaceutical composition comprising the rhAAT protein or a fragment or protein formulation thereof for use in the treatment of nonviral lung diseases associated with inflammation and / or pathological immune responses. Background Technology

[0006] α-1 antitrypsin (also known as A1AT, AAT, PI, SERPINA1, or AAT protein) is a glycoprotein of approximately 52 kDa and is one of the most abundant endogenous serine protease inhibitors (SERPIN superfamily). AAT is considered an acute-phase protein, therefore its concentration can increase several times during acute inflammation.

[0007] Although AAT is primarily known for its antiprotease and anti-inflammatory activities, studies over the past decade have cumulatively demonstrated that it is also an immunomodulator and cytoprotective molecule. Consequently, AAT-rich microenvironments have been shown to contain lower levels of pro-inflammatory cytokines (such as IL-1, IL-6, and TNF-α) and higher levels of anti-inflammatory mediators (such as IL-1 receptor antagonists and IL-10). This phenomenon has also been confirmed in in vitro studies of human PBMCs and in samples obtained from cystic fibrosis patients receiving inhaled AAT. Meanwhile, AAT has been shown to directly bind to IL-8 and risk-associated molecular pattern molecules (DAMPs), such as extracellular HSP70 and gp96, which otherwise act as adjuvants to the relevant immune responses (Lior et al., Expert Opinion on Therapeutic Patents, 2016).

[0008] AAT also exerts a significant effect on neutrophils through the IL-8 / CXCR1 signaling pathway (Curley et al., Clinical Implications. Chest. 2016). It binds to IL-8, thereby preventing CXCR1 activation. Simultaneously, it reduces soluble immune complexes by binding to ADAM-17, which induce neutrophil chemotaxis (Bergin et al., J Clin Invest., 2010). The reduction of TNF-α, free radicals, and neutrophil elastase in tissues, along with the regulation of the immune response, alleviates lung periphery damage. AAT further reduces monocyte IL-1β secretion by downregulating the receptor P2X7 and increases cAMP levels. Simultaneously, it stimulates macrophages to secrete anti-inflammatory IL-1R (Janciauskiene et al., J Biol Chem. 2007). The immunomodulatory effects of AAT are not limited to macrophages and neutrophils. It has been shown to reduce the effects of TNF-α on pulmonary microvascular endothelial cells. This delays the disruption of the alveolar fluid barrier and prevents the formation of secondary pulmonary edema (Lockett et al., Am J Respir Cell Mol Biol. 2013).

[0009] In vitro, neutrophil extracellular traps (NETs) have been shown to disrupt the cell membrane integrity of lung epithelial cells. This leads to cell death via apoptosis. Simultaneously, cell junctions between epithelial cells are disrupted. This not only causes lung tissue damage but also increases the risk of microthrombus formation. AAT counteracts this mechanism by forming a complex with NETs and neutrophil elastase (Hudock et al., Front Immunol. 2023).

[0010] Surprisingly, the anti-inflammatory properties of AAT still allow for: innate immune cells to respond to real threats; macrophages readily engulf bacteria, neutrophils cleanse infection sites, and antigen-laden dendritic cells migrate to draining lymph nodes. On the other hand, T lymphocytes indirectly respond to an AAT-rich environment, pending stimulation by innate immune cells. For example, AAT has been shown to induce the proliferation of semi-mature antigen-presenting cells, which favor the expansion of protective regulatory T cells. B lymphocytes, belonging to both the innate and adaptive immune systems, appear to exert a modified response in the presence of AAT, manifested as reduced allotype switching, leading to enhanced protective IgM production. Studies have shown that during AAT treatment, as observed in patients with CF, the reduction in bacterial load may be associated with an enhanced antipathogen immune response; simultaneously, local tissues are protected from inappropriate excessive damage, which may promote harmful adaptive responses by increasing local DAMP levels (Lior et al., Expert Opinion on Therapeutic Patents, 2016).

[0011] Inflammatory lung diseases and diseases associated with adverse pathological immune responses represent a significant cause of health problems and death worldwide, and represent a medical condition requiring improved inflammation regulation for effective treatment. Nonviral inflammatory diseases of the lungs can severely impact patient health and encompass a variety of pathological conditions affecting lung tissue.

[0012] Asthma is a chronic inflammatory disease of the lungs characterized by cough, wheezing, chest tightness, and shortness of breath, with reversible airway obstruction and bronchospasm being the most common symptoms (Huang et al., Cytokine 2021). Worldwide, asthma affects more than 300 million people, including children and adults. Symptomatic treatment for asthma includes several medications, such as beta-2 agonists and corticosteroids. However, because the pathology of asthma is not fully understood, there is currently no curative treatment available. Overall, oxygen free radicals and autophagy have been reported to be involved in the pathophysiology of asthma.

[0013] Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by tissue edema resulting from impaired pulmonary capillary permeability and an inflammatory response of neutrophil elastase, leading to severe disturbances in gas exchange. The severity of the disease depends on the degree of excessive inflammation. Especially in the early stages of the disease, the strong immune response associated with a rapid and significant increase in pro-inflammatory cytokines and the infiltration of immune cells into lung tissue necessitates early intervention in immunomodulation. Therefore, a rapid increase in pro-inflammatory cytokines is associated with poor patient prognosis (Pugin et al., Am J Respir CritCare Med. 1996). If patients survive the early stages of the disease, they may progress along this pathway to persistent and severe pulmonary dysfunction.

[0014] Bronchiolitis obliterans (BO), also known as bronchiolitis obliterans syndrome (BOS), is a rare chronic lung disease characterized by inflammation and fibrotic thickening of the bronchioles, leading to narrowing of the lumen and restricting air circulation (Jerkic et al., Can Respir J. 2020). BO can occur secondary to lung infections (post-infectious bronchiolitis obliterans (PiBO)) or as a comorbidity of related conditions (such as lung transplantation or allogeneic hematopoietic stem cell transplantation (chronic graft-versus-host disease)), in which case it is referred to as bronchiolitis obliterans syndrome (BOS) (Walther et al., Pediatr Pulmonol. 2020). The pathogenesis of BO is not fully understood. However, its pathological mechanisms may involve the infiltration of leukocytes into the airway submucosa. A cascade of inflammatory cytokines and mediators leads to the accumulation of inflammatory cells. Studies by Rosewich et al. have shown that a neutrophil-dominated inflammatory response exists in the bronchioles, accompanied by elevated concentrations of inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α (Rosewich et al., Cytokine 2015). In summary, bronchogenic embolism (BO) develops as a complex response of the immune system, involving cytokines, growth factors, chemokines, and cells of alloimmune reactivity, humoral immunity, autoimmunity, and innate immunity.

[0015] Observations of induced sputum in PiBO patients have shown that neutrophilic inflammation persists over time and is not self-limiting (Eckrich et al., Lung 2016). Recent studies have also shown upregulation of IL-33 in patient sputum, indicating that alveolar epithelial type II cells (AEC-II) are involved in the inflammatory process (Kriszeleit, Johann Wolfgang). Frankfurt am Main, Department of Medicine, PhD dissertation, 2023. In the occurrence and progression of inflammation-induced lung injury, AEC-II cells are damaged by a variety of cellular and humoral inflammatory mediators. Conversely, AEC-II itself can amplify lung inflammation by producing inflammatory cytokines and chemokines, leading to the activation and recruitment of phagocytes and neutrophils (Standiford et al., J Clin Invest 1990).

[0016] Body stagnation (BOS) after lung transplantation is a leading cause of death more than one year after transplantation, with 48% and 76% of patients developing BOS within 5 and 10 years, respectively (Weigt et al., Semin Respir Crit Care Med 2013). In addition to impacting long-term survival, BOS leads to significant morbidity, impairs quality of life, and increases healthcare costs.

[0017] Given the uncontrolled inflammation in BO lung disease, eliminating the chronic inflammatory response is crucial (Jerkic et al., Clin Transl Immunology. 2022). To date, the most established treatment for BO is azithromycin; however, it is accompanied by several side effects (such as headache, diarrhea, and abdominal pain) and has also been shown to have serious interactions with several drugs, including antacids, digoxin, zidovudine, ergotamine, statins, warfarin, cyclosporine, terfenadine, cisapride, astemizole, alfentanil, and substances that prolong the QT interval.

[0018] AAT alleviates airway inflammation in other immune-mediated lung diseases, such as cystic fibrosis, by reducing neutrophil elastase activity (Griese et al., European Respiratory Journal 2007). However, obtaining AAT has so far relied primarily on purification from human plasma. For example, available methods include protein precipitation by adding ammonium sulfate. The logical evolution of AAT purification involves combining ammonium sulfate fractionation with other procedures that utilize the physicochemical properties of AAT. To facilitate the separation of large protein libraries (often the result of ammonium sulfate precipitation) into several smaller libraries, enriching one (or more) libraries with AAT, the first parameter researchers consider is protein charge. Recently, with the emergence of various complex materials in ion exchange and affinity chromatography, improved purification levels have been observed. For example, in Morihara et al.'s study, human plasma was saturated with ammonium sulfate (80%), the precipitate was dissolved in phosphate buffer at pH 8.0, dialyzed, and loaded onto an Affi-GEL Blue column. Subsequent two steps of Zn chelation column and DE ion exchange chromatography yielded homogeneous AAT.

[0019] Kwon et al. also disclosed the purification of recombinant AAT from yeast, which was secreted into the culture medium in a glycosylated form. Their developed procedure involved precipitating the protein with ammonium sulfate (60%–75% saturation), followed by a series of subsequent chromatographic steps consisting of anion exchange (DEAE and mono Q columns) and affinity (A-Gel Blue column) chromatography. Although the yeast-produced AAT was fully functional as a protease inhibitor (compared to its plasma form), its molecular weight decreased to that of recombinant AAT produced by *Escherichia coli* upon treatment with endoglycosidase H, unlike plasma-produced AAT. This indicates that the N-linked glycosylation in this form is of the high-mannose type. However, Saccharomyces diastaticus is generally not ideal for the secretion of recombinant proteins (Purification and characterization of alpha1-antitrypsin secreted by recombinant yeast Saccharomyces diastaticus. J. Biotechnol. 1995, 42, 191–195.).

[0020] Arjmand et al. disclosed the expression and purification of recombinant human AAT in the methyltrophic yeast *Pichia pastoris* in 2011 (Avicenna J Med Biotechnol. 2011, 3(3): 127–134). Human AAT was expressed in a secretory manner under the control of the inducible alcohol oxidase 1 (AOX1) promoter. The amount of AAT protein in the culture medium was measured to be 60 mg / L 72 hours after induction with methanol. Arjmand et al. disclosed the use of *Pichia pastoris* as a host for the efficient production and secretion of recombinant AAT in 2013 (Electronic Journal of Biotechnology, 2013, Vol. 16, No. 1, 1-14). The results revealed that optimizing the codon usage of the AAT gene in *Pichia pastoris* under the control of the inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters had a positive impact on the level of secreted AAT.

[0021] US7914771 discloses methods for treating and preventing chronic obstructive pulmonary disease (COPD) or emphysema partially caused by smoking, and compositions comprising recombinant human AAT protein. Although the document teaches the production of AAT in yeast, it emphasizes production in Saccharomyces cerevisiae, and the invention focuses on the use of non-glycosylated AAT.

[0022] WO2020 / 092448 discloses a recombinant engineered AAT serine protease inhibitor domain fused with a human serum albumin binding domain or a serum albumin domain.

[0023] US2022 / 204646 discloses the generation and utilization of AAT constructs, as well as methods for purifying and scaling up recombinant AAT for therapeutic applications. These recombinant AAT constructs can be used to treat a variety of conditions, including AAT deficiency, inflammatory diseases, and immune dysregulations.

[0024] WO2903 / 096458 discloses compositions containing AAT, wherein AAT is recombinantly produced in mammary epithelial cells of nonhuman mammals.

[0025] Several drawbacks remain to be addressed in isolating AAT from human plasma. For example, the high abundance of albumin in human plasma presents a challenge, and the yield of AAT from human plasma is limited. Therefore, optimized methods for producing secretory recombinant proteins are needed. Furthermore, recombinant AAT production in yeast is currently limited by relatively low yields, poor glycosylation characterization, and a lack of clinical applications.

[0026] Despite progress in purifying AAT from human plasma and in the recombinant production of AAT, to the best of the inventors' knowledge, no convincing solution has yet been found to overcome the shortcomings of the prior art. Methods and means to reduce or avoid the problems associated with isolating AAT from human plasma are needed, as are novel means and uses for recombinant AAT. Means to obtain the required amounts and purity levels of AAT suitable for therapeutic use remain necessary. Another problem that needs to be addressed, given the prevalence and severity of nonviral lung diseases associated with inflammation and / or pathological immune responses, is to provide effective means of preventing and / or treating these medical conditions. Summary of the Invention

[0027] According to the prior art, a potential technical problem of the present invention is to provide improved or alternative means for treating and / or preventing non-viral medical conditions of the lungs associated with inflammation and / or pathological immune responses.

[0028] Another potential problem with the present invention is to provide disease-modifying treatment for nonviral medical conditions of the lungs associated with inflammatory and / or pathological immune responses in subjects.

[0029] According to the prior art, another potential technical problem of the present invention is to provide an improved or alternative means for producing recombinant human AAT.

[0030] Another potential problem of the present invention is to provide a method for producing recombinant human AAT that provides higher yields than previously established methods, while reducing costs and minimizing limitations on production capacity.

[0031] These problems are addressed by the features in the independent claims. The dependent claims provide preferred embodiments of the invention.

[0032] Therefore, in one aspect, the present invention relates to a human recombinant AAT (rhAAT) protein or a fragment thereof, expressed in genetically modified yeast, for use in the treatment and / or prevention of nonviral lung diseases associated with inflammation and / or pathological immune responses.

[0033] Although the production of AAT in yeast has been previously described, to the inventors' knowledge, the efficacy of rhAAT glycoprotein from yeast in treating nonviral lung diseases associated with inflammation and / or pathological immune responses has not previously been evaluated. Therefore, this invention represents a novel and unexpectedly effective rhAAT glycoprotein formulation using yeast as an expression system. Accordingly, in embodiments, the isolated rhAAT glycoprotein itself, the genetically modified yeast used to produce recombinant AAT, and the method for preparing and using AAT produced in yeast can be defined by the findings of this invention regarding its efficacy in treating nonviral lung diseases associated with inflammation and / or pathological immune responses, such as asthma, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), bronchiectasis, or bronchiolitis obliterans syndrome (BOS).

[0034] In one implementation, the human recombinant AAT (rhAAT) protein is a glycoprotein.

[0035] In one implementation, the human recombinant AAT (rhAAT) protein is the human recombinant α-1 antitrypsin (rhAAT) protein.

[0036] In one embodiment, the human recombinant AAT (rhAAT) glycoprotein is the human recombinant α-1 antitrypsin (rhAAT) glycoprotein.

[0037] In one implementation, lung disease associated with inflammation and / or pathological immune response is associated with the following: elevated levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ and / or TNF-α, elevated levels of one or more transcription factors such as NF-κB, and / or increased immune cell infiltration in the lung tissue of the subject compared to healthy subjects.

[0038] In one implementation, lung disease associated with inflammation and / or pathological immune response is associated with the following: elevated levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ and / or TNF-α and / or elevated levels of one or more transcription factors such as NF-κB in the subject's whole blood, serum and / or plasma compared to healthy subjects.

[0039] In one implementation, lung disease associated with inflammation and / or pathological immune response is associated with the following: elevated levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ and / or TNF-α and / or elevated levels of one or more transcription factors such as NF-κB in the bronchoalveolar lavage fluid (BALB) of the subject compared to healthy subjects.

[0040] In one embodiment, compared to healthy subjects, the levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ, and / or TNF-α in the lung tissue of the subjects are increased by at least 1.2 times, preferably at least 2 times, more preferably at least 10 times. In one embodiment, compared to healthy subjects, the levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ, and / or TNF-α in the lung tissue of the subjects are increased by 1.2, 1.5, 1.6, 2, 2.5, 4, 5, 10, 12, 14, 15, or 20 times.

[0041] In one embodiment, compared to healthy subjects, the levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ, and / or TNF-α in the subjects' whole blood, serum, and / or plasma are increased by at least 1.2 times, preferably at least 2 times, and more preferably at least 10 times. In one embodiment, compared to healthy subjects, the levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ, and / or TNF-α in the subjects' whole blood, serum, and / or plasma are increased by 1.2, 1.5, 1.6, 2, 2.5, 4, 5, 10, 12, 14, 15, or 20 times.

[0042] In one embodiment, compared to healthy subjects, the levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ, and / or TNF-α in the bronchoalveolar lavage fluid (BALB) of the subjects are increased by at least 1.2 times, preferably at least 2 times, and more preferably at least 10 times. In one embodiment, compared to healthy subjects, the levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ, and / or TNF-α in the bronchoalveolar lavage fluid (BALB) of the subjects are increased by 1.2, 1.5, 1.6, 2, 2.5, 4, 5, 10, 12, 14, 15, or 20 times.

[0043] In one embodiment, compared to healthy subjects, the levels of one or more transcription factors, such as NF-κB, are increased by at least 1.2 times, preferably at least 2 times, and more preferably at least 10 times in the lung tissue of the subject. In one embodiment, compared to healthy subjects, the levels of one or more transcription factors, such as NF-κB-α, are increased by 1.2, 1.5, 1.6, 2, 2.5, 4, 5, 10, 12, 14, 15, or 20 times in the lung tissue of the subject.

[0044] In one embodiment, compared to healthy subjects, the levels of one or more transcription factors, such as NF-κB, are increased by at least 1.2 times, preferably at least 2 times, and more preferably at least 10 times in the subject's whole blood, serum, and / or plasma. In one embodiment, compared to healthy subjects, the levels of one or more transcription factors, such as NF-κB, are increased by 1.2, 1.5, 1.6, 2, 2.5, 4, 5, 10, 12, 14, 15, or 20 times in the subject's whole blood, serum, and / or plasma.

[0045] In one embodiment, compared to healthy subjects, the levels of one or more transcription factors, such as NF-κB, are increased by at least 1.2 times, preferably at least 2 times, and more preferably at least 10 times in the bronchoalveolar lavage fluid (BALB) of the subjects. In one embodiment, compared to healthy subjects, the levels of one or more transcription factors, such as NF-κB, are increased by 1.2, 1.5, 1.6, 2, 2.5, 4, 5, 10, 12, 14, 15, or 20 times in the bronchoalveolar lavage fluid (BALB) of the subjects.

[0046] In one implementation, lung disease associated with inflammation and / or pathological immune response is associated with the following: increased infiltration levels of macrophages, monocytes, and / or neutrophils in the lung tissue of the subject compared to healthy subjects.

[0047] In one implementation plan, the lung disease is asthma.

[0048] In one implementation, the lung disease is acute respiratory distress syndrome (ARDS).

[0049] In one implementation, the lung disease is bronchiolitis obliterans (BO).

[0050] In one implementation, the lung disease is bronchiolitis obliterans syndrome (BOS).

[0051] In one implementation, obliterative bronchiolitis syndrome (BO) is associated with lung transplantation and / or allogeneic hematopoietic stem cell transplantation (chronic graft-versus-host disease).

[0052] In one implementation, the lung disease is bronchiolitis obliterans syndrome (BOS) associated with lung transplantation and / or allogeneic hematopoietic stem cell transplantation (chronic graft-versus-host disease).

[0053] In the implementation scheme, the recombinant AAT as described herein is used for the prevention and / or treatment of chronic respiratory diseases, preferably obstructive diseases, restrictive diseases and / or vascular diseases.

[0054] In the implementation plan, recombinant AAT as described herein is used for the prevention and / or treatment of inflammatory lung conditions.

[0055] In the implementation plan, the recombinant AAT as described herein is used for the prevention and / or treatment of bronchiectasis, chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans syndrome (BOS), pulmonary fibrosis, interstitial lung disease (ILD), pneumonia, and / or pulmonary hypertension.

[0056] In one embodiment, the rhAAT protein or a fragment thereof comprises a sequence according to SEQ ID NO 4, or encoded by SEQ ID NO 1, 2 or 3, or a sequence having at least 80% identity with them.

[0057] In one embodiment, the rhAAT protein or a fragment thereof has a serum half-life, pulmonary half-life, and / or activity not less than that of AAT purified from human plasma.

[0058] In one embodiment, the intrabronchial half-life and / or activity of the rhAAT protein or a fragment thereof is not less than that of AAT purified from human plasma.

[0059] In one embodiment, the alveolar half-life and / or activity of the rhAAT protein or a fragment thereof is not less than that of AAT purified from human plasma.

[0060] In one embodiment, the rhAAT protein or a fragment thereof is expressed in yeast of the family Saccharomycetaceae, preferably Pichia.

[0061] In one implementation, the rhAAT protein or a fragment thereof contains post-translational modifications.

[0062] In one embodiment, the post-translational modification is N-glycosylation, O-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation or any combination thereof, preferably comprising one or more N-linked glycosylations, including at least one HexNAc1 glycosylation.

[0063] In one implementation, the rhAAT protein or a fragment thereof is administered by inhalation, nasal administration, and / or injection.

[0064] In one embodiment, the rhAAT protein or a fragment thereof is administered by inhalation, preferably as a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation.

[0065] In one implementation, the rhAAT protein or a fragment thereof is administered via inhalation of an aerosol solution.

[0066] In one embodiment, the rhAAT protein or a fragment thereof is administered by injection, preferably as a solution suitable for injection. In one embodiment, the rhAAT protein or a fragment thereof is administered by intravenous injection, preferably as a solution suitable for intravenous injection. In one embodiment, the rhAAT protein or a fragment thereof is administered by subcutaneous injection, preferably as a solution suitable for subcutaneous injection.

[0067] In embodiments of the invention, recombinant AAT is administered for multiple consecutive days. In these embodiments, patients may receive inhalation therapy multiple times a day, multiple times a week, or even for several weeks.

[0068] In the implementation plan, patients also receive standard medical treatment for nonviral lung diseases associated with inflammation and / or pathological immune responses. For example, standard medical treatment includes oxygen support, noninvasive ventilation, high-flow oxygen therapy, mechanical ventilation, and extracorporeal membrane oxygenation; or administration of azithromycin, cyclophosphamide, cyclosporine, azathioprine, anti-CD3 antibodies, tacrolimus, mycophenolate mofetil, or statins for the treatment of bronchiolitis obliterans (BO); or administration of β-2 agonists (such as salbutamol, bambuterol, and indacaterol), glucocorticoids (such as beclomethasone, budesonide, and fluticasone), theophylline, leukotriene antagonists (such as montelukast), cromoglycine, nedocromil, and loxamethasone for the treatment of asthma.

[0069] In embodiments of the invention, recombinant AAT is administered as a preventative treatment, for example, by directly administering rhAAT after lung transplantation and / or allogeneic hematopoietic stem cell transplantation to prevent bronchiolitis obliterans syndrome (BOS) associated with lung transplantation and / or allogeneic hematopoietic stem cell transplantation.

[0070] In embodiments of the invention, the treatment results in at least one of the following outcomes: reduced hospitalization rates, reduced oxygen dependence, reduced need for intensive care or mechanical ventilation, reduced use or burden of medical resources, reduced absences or absenteeism, reduced need for drugs (such as β-2 agonists) and / or steroids, reduced relapse frequency and / or reduced morbidity or risk of disease.

[0071] In one embodiment, rhAAT glycoprotein is administered to treat nonviral lung diseases associated with inflammation and / or pathological immune responses, wherein the rhAAT protein or a fragment thereof is expressed by Pichia pastoris, and the rhAAT protein or a fragment thereof is administered by inhalation of an aerosol solution.

[0072] In one embodiment, rhAAT glycoprotein is administered to treat nonviral lung diseases associated with inflammation and / or pathological immune responses, wherein the rhAAT protein or a fragment thereof is expressed by Pichia pastoris and isolated using chromatographic methods (such as affinity, size exclusion, ion exchange and / or hydrophobic interaction chromatography), and the rhAAT protein or a fragment thereof is administered by inhalation of a nebulized solution.

[0073] In one aspect, the present invention relates to a pharmaceutical composition comprising an rhAAT protein or a fragment thereof according to any one of the preceding claims and a pharmaceutically acceptable carrier for use in the treatment of nonviral lung diseases associated with inflammation and / or pathological immune responses.

[0074] In one aspect, the present invention relates to a pharmaceutical composition comprising an rhAAT protein or a fragment thereof or a protein formulation according to any one of the preceding claims, and a pharmaceutically acceptable carrier, for use in the treatment of nonviral lung diseases associated with inflammation and / or pathological immune responses.

[0075] In one embodiment, the pharmaceutical composition is a solution, a soluble powder, or a dry powder. In another embodiment, the composition is suitable for inhalation, nasal administration, and / or injection.

[0076] In one embodiment, the composition is a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation.

[0077] In one embodiment, the rhAAT protein of the present invention does not show or shows a negligible immune response in mammalian subjects, preferably humans.

[0078] In one implementation, the rhAAT protein is non-immunogenic.

[0079] The rhAAT protein of the present invention is produced to be non-immunogenic in mammalian systems. Advantageously and unexpectedly, when administered to mammalian subjects, the rhAAT protein produced in yeast as described herein leads to an acceptable immune response (or no adverse immune response).

[0080] In the implementation plan, recombinant AAT is used for the treatment of bacterial infections and / or bacterial respiratory infections.

[0081] In the implementation scheme, the recombinant AAT as described herein is used for the prevention and / or treatment of chronic respiratory diseases, preferably obstructive diseases, restrictive diseases and / or vascular diseases.

[0082] In the implementation plan, recombinant AAT as described herein is used for the prevention and / or treatment of inflammatory lung conditions.

[0083] In the implementation plan, the recombinant AAT as described herein is used for the prevention and / or treatment of bronchiectasis, chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans syndrome (BOS), pulmonary fibrosis, interstitial lung disease (ILD), pneumonia, and / or pulmonary hypertension.

[0084] In the implementation scheme, recombinant AAT as described herein exhibits effective interaction with and blockade of proteases, such as those in pathological organisms that play a role in viral, bacterial, or inflammatory (non-infectious) diseases, particularly lung diseases.

[0085] Surprisingly, experimental work presented in the examples below demonstrates that yeast-derived rhAAT glycoproteins not only interact with proteases such as the V8 protease from Staphylococcus aureus, but also exhibit improvements compared to existing therapeutic formulations of purified AAT derived from human plasma. For example, yeast-derived rhAAT glycoproteins show relatively superior properties in binding to and / or inhibiting V8 compared to Prolastin AAT formulations.

[0086] As also shown below, and as demonstrated by gel migration assays as described in Examples 29–31, it is particularly surprising that the rhAAT protein is more stable than Prolastin. For example, all the proteases tested were able to cleave and degrade Prolastin, but not rhAAT. This was especially evident in protease V8, which almost completely cleaves and degrades Prolastin, which was not observed in rhAAT.

[0087] Therefore, this invention represents a novel and unexpectedly effective rhAAT glycoprotein formulation using yeast as an expression system. Accordingly, in embodiments, the isolated rhAAT glycoprotein itself, the genetically modified yeast used to produce recombinant AAT, and the methods for preparing and using AAT produced in yeast can be defined by inventive findings of efficacy in treating viral respiratory diseases, bacterial diseases, and / or other inflammatory lung diseases, or by other inventive findings of improving nature regarding the physiological and biological properties of the rhAAT glycoprotein of this invention, as shown herein.

[0088] An implementation scheme involving human α1-antitrypsin (rhAAT) glycoprotein:

[0089] In one embodiment, the present invention relates to a recombinant human α1-antitrypsin (rhAAT) glycoprotein or a fragment thereof having one or more N-linked glycosylations, including at least one HexNAc1 glycosylation. This rhAAT is suitable for the medical treatments disclosed herein.

[0090] As described in more detail below, the present invention provides a novel method for producing rhAAT from Pichia pastoris, resulting in the production of an rhAAT glycoprotein with a unique glycosylation pattern that, to the inventors' knowledge, has not been previously described. Furthermore, this novel glycoprotein and protein formulations containing it represent therapeutic agents that exhibit unexpected advantages in preventing respiratory viruses from entering target cells.

[0091] Recombinant protein expression in *Pichia pastoris* is a well-established technique that results in protein glycosylation that typically has shorter chain lengths than those found in *Saccharomyces cerevisiae*. Expression in yeasts, including *Pichia pastoris*, generally leads to N-linked oligosaccharides derived from an oligosaccharide (Glc3Man9GlcNAc2) assembled in the endoplasmic reticulum (ER). In most yeasts studied, three glucose residues and a specific α-1,2-linked mannose residue are removed by a specific glycosidase in the ER, resulting in an N-linked Man8GlcNAc2 core structure for further processing in the Golgi complex. This can involve the addition of additional mannose or other sugar residues, either linearly or branched, resulting in a complex extended polysaccharide that binds to the glycosylated protein via a GlcNAc2 disaccharide unit (as a proximal unit of the protein). In other words, *Pichia pastoris* can typically N-glycosylate proteins via mannose oligosaccharides linked to asparagine via two N-acetylglucosamines.

[0092] This invention provides an rhAAT glycoprotein with a unique glycosylation that has not been previously observed in AAT or in AAT proteins expressed by Pichia pastoris. As outlined in the examples below, the rhAAT glycoprotein of this invention exhibits at least one HexNAc1 glycosylation. This structure preferably occurs at the amide nitrogen of an asparagine residue in AAT, found in the consensual sequence Asn-Xaa-Thr / Ser, thereby providing N-linked glycosylation of HexNAc1.

[0093] In the implementation scheme, the rhAAT glycoprotein or fragment thereof contains at least two or at least three HexNAc1 glycosylations.

[0094] As illustrated in the examples below, the three N-linked glycosylation sites of AAT can be fully or partially occupied by HexNAc1, a finding that is unexpected given typical glycosylation patterns or recombinant proteins (especially those expressed in yeast).

[0095] In the implementation, the rhAAT glycoprotein or fragment thereof comprises at least one Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2 glycosylation.

[0096] As illustrated in the examples below, any one or more of the N-linked glycosylation sites of AAT may be completely or partially occupied by Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2, Hex16HexNAc2, Hex17HexNAc2 and / or Hex18HexNAc2.

[0097] In the implementations, these glycosylations appear to be prevalent, but to a lesser extent than HexNAc1. In other implementations, longer chain structures, such as Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2, Hex16HexNAc2, Hex17HexNAc2, and / or Hex18HexNAc2, are more prevalent than HexNAc1.

[0098] As used herein, the term "Hex" refers to a hexose. In embodiments, Hex may be mannose, for example, Hex9HexNAc2 may refer to Man9HexNAc2. In embodiments, Hex may be glucose, for example, Hex9HexNAc2 may refer to Hex9GlcNAc2. In a preferred embodiment, the HexNAc2 nomenclature refers to two N-acetylglucosamines, in other words, it refers to GlcNAc2.

[0099] In a preferred embodiment, the polysaccharides HexNAc1, Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2, Hex17HexNAc2 and / or Hex18HexNAc2 can be represented as GlcNAc1, Man9GlcNAc2, Man10GlcNAc2, Man11GlcNAc2, Man12GlcNAc2, Man13GlcNAc2, Man14GlcNAc2, Man15GlcNAc2, Man16GlcNAc2, Man17GlcNAc2 and / or Man18GlcNAc2, respectively.

[0100] This nomenclature of "Man(n)GlcNAc2" (where n is the number of mannoses) is applicable to any given embodiment of the nomenclature "Hex(n)HexNAc2". In embodiments, the nomenclature can be applied to polysaccharides as Hex(n)HexNAc2 or Man(n)GlcNAc2, where n is 0-20, more preferably 1-19, 2-18, 5-18, and more preferably 9-16. In embodiments, the nomenclature can be applied to polysaccharides as Hex(n)HexNAc2 or Man(n)GlcNAc2, where n is 0-25, more preferably 1-22, 2-20, 5-20, and more preferably 9-18.

[0101] Given that recombinant proteins expressed in yeast typically possess complex and long polysaccharides, the glycans detected in rhAAT as presented in this paper represent an unexpected glycosylation pattern. Compared to the expected results obtained from yeast expression, the presence of HexNAc1 as the major glycan and other glycans with relatively short hexose (mannose) chains is surprising. Unbound by theory, this glycosylation pattern may show advantages over other rhAAT protein formulations in terms of protein secretion, protein yield, glycoprotein stability, enzymatic efficacy, and / or therapeutic effects.

[0102] It has been shown that N-glycosylation and N-glycan structure can influence the biophysical and pharmacokinetic properties of therapeutic glycoproteins. Furthermore, N-glycans at different sites can play a role in the secretion and final yield of therapeutic recombinant proteins. The report presented below demonstrates high protein yield and putatively good protein stability based on N-linked glycosylation modifications, which appear to be optimal for the biotechnological production of therapeutic rhAAT products.

[0103] In summary, this invention is based on the development of a novel method for producing rhAAT in yeast (particularly Pichia pastoris), resulting in a stable and good yield of rhAAT glycoprotein that exhibits a unique glycosylation pattern and shows potential for improved therapeutic efficacy.

[0104] Implementation scheme involving the amount of each glycosylation:

[0105] In one embodiment, HexNAc1 glycosylation accounts for 50%-100% of the total N-glycans, preferably 60%-90%, more preferably 70%-80%.

[0106] As illustrated in the examples below, HexNAc1 glycosylation at any one or more of the three N-linked glycosylation sites represents an unexpected modification following the production of rhAAT in yeast (Pichia pastoris). Unbound by theory, this highly generalized modification also appears to convey properties of improved secretion efficiency and / or enzymatic efficacy and / or therapeutic effect compared to other AAT formulations (such as Prolastin). Particularly unexpectedly, as demonstrated by the gel migration assays described in Examples 32-34, the rhAAT protein is more stable than Prolastin. For example, all the proteases tested were able to cleave and degrade Prolastin but not rhAAT. This was particularly evident in protease V8, which almost completely cleaves and degrades Prolastin, which was not observed in rhAAT.

[0107] In one embodiment, the glycosylation of Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2 and Hex14HexNAc2 accounts for 5%-50% of the total N-glycans, preferably 10%-40%, more preferably 15%-35%.

[0108] In one embodiment, the glycosylation of Hex12HexNAc2, Hex13HexNAc2 and Hex14HexNAc2 accounts for 5%-30% of the total N-glycans, preferably 10%-25%, more preferably 12%-20%.

[0109] In one embodiment, the glycosylation of Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2 and Hex15HexNAc2 accounts for 5%-50% of the total N-glycans, preferably 10%-40%, more preferably 15%-35%.

[0110] In one embodiment, Hex9HexNAc2 glycosylation accounts for 0.05%-5% of the total N-glycans, preferably 0.1%-2%, more preferably 0.2%-1%.

[0111] In one embodiment, Hex10HexNAc2 glycosylation accounts for 0.1%-10% of the total N-glycans, preferably 0.5%-5%, more preferably 1%-3%.

[0112] In one embodiment, Hex11HexNAc2 glycosylation accounts for 0.1%-10% of the total N-glycans, preferably 0.5%-5%, more preferably 1%-4%.

[0113] In one embodiment, Hex12HexNAc2 glycosylation accounts for 1%-20% of the total N-glycans, preferably 2%-10%, more preferably 3%-8%.

[0114] In one embodiment, Hex13HexNAc2 glycosylation accounts for 1%-20% of the total N-glycans, preferably 2%-10%, more preferably 3%-8%.

[0115] In one embodiment, Hex14HexNAc2 glycosylation accounts for 1%-20% of the total N-glycans, preferably 2%-10%, more preferably 3%-8%.

[0116] In one embodiment, Hex15HexNAc2 glycosylation accounts for 0.1%-10% of the total N-glycans, preferably 0.5%-7%, more preferably 1%-5%.

[0117] In one embodiment, Hex16HexNAc2 glycosylation accounts for 0.1%-10% of the total N-glycans, preferably 0.5%-7%, more preferably 1%-5%.

[0118] Implementation schemes involving AAT sequences and N-linked glycosylation sites:

[0119] The rhAAT glycoprotein according to any one of the preceding claims comprises the amino acid sequence or a fragment thereof according to SEQ ID NO 4. SEQ ID NO 4 relates to the primary amino acid sequence of human AAT commonly known from protein databases, etc.

[0120] In the implementation, rhAAT or a fragment thereof comprises an amino acid sequence according to SEQ ID NO 4 and at least one N-linked glycosylation at N46.

[0121] In the implementation, rhAAT or a fragment thereof comprises an amino acid sequence according to SEQ ID NO 4 and at least two N-linked glycosylations at N46 and N247.

[0122] In the implementation, rhAAT or a fragment thereof comprises an amino acid sequence according to SEQ ID NO 4 and at least three N-linked glycosylations at N46, N83 and N247.

[0123] In the embodiments, rhAAT or a fragment thereof comprises an amino acid sequence according to SEQ ID NO 4 or a fragment thereof and at least one N-linked glycosylation at N46, N83 or N247, at least two N-linked glycosylations at N46 and N83, N46 and N247, or N83 and N247, or three N-linked glycosylations at N46, N83 and N247.

[0124] In the implementation, rhAAT or a fragment thereof comprises an amino acid sequence according to SEQ ID NO 4 and at least one N-linked glycosylation at N46, including HexNAc1.

[0125] In the embodiments, rhAAT or fragments thereof comprise an amino acid sequence according to SEQ ID NO 4 and at least one N-linked glycosylation at N83, including Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2.

[0126] In the embodiments, rhAAT or fragments thereof comprise an amino acid sequence according to SEQ ID NO 4 and at least one N-linked glycosylation at N247, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2.

[0127] Implementation schemes involving the AAT sequence, the location and type of N-linked glycosylation:

[0128] In the embodiments, rhAAT or a fragment thereof comprises an amino acid sequence according to SEQ ID NO 4 or a fragment thereof and at least one N-linked glycosylation at N46, N83 or N247, including HexNAc1, at least two N-linked glycosylations at N46 and N83, N46 and N247 or N83 and N247, including HexNAc1, or at least three N-linked glycosylations at N46, N83 and N247, including HexNAc1.

[0129] In the embodiments, rhAAT or a fragment thereof comprises the amino acid sequence according to SEQ ID NO 4 or a fragment thereof, and comprises at least one N-linked glycosylation at N83, including Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 or Hex16HexNAc2, and / or

[0130] In the embodiments, rhAAT or a fragment thereof comprises an amino acid sequence or a fragment thereof according to SEQ ID NO 4, and comprises at least one N-linked glycosylation at N247, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 or Hex16HexNAc2.

[0131] Implementation schemes involving AAT sequence, position, and N-linked glycosylation type, with modifications at each glycan site. Indications of (total) occupancy and frequency of decorations:

[0132] The present invention also relates to a protein formulation comprising a recombinant human α1-antitrypsin (rhAAT) glycoprotein or a fragment thereof having heterologous N-linked glycosylation, wherein the N-linked glycosylation includes at least one HexNAc1 glycosylation.

[0133] As described in the examples below, the expression, secretion, and isolation of rhAAT result in protein formulations with various glycan structures. These can be homogeneous or heterogeneous. In a preferred embodiment, the formulation comprises an rhAAT glycoprotein or fragment thereof with heterologous N-linked glycosylation. The amount (relative or absolute) of each glycosylation can be used to define the protein formulation. Quantitative (or semi-quantitative) peptide mapping analysis has identified rhAAT protein sequences with varying degrees of glycosylation at different sites on the rhAAT protein. These glycosylation patterns can be used to define the rhAAT protein or protein formulation and appear to be beneficial for secretion, yield, enzymatic activity, and / or therapeutic activity.

[0134] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N46, including HexNAc1, wherein the HexNAc1 at N46 is present in more than 50%, preferably more than 80%, more preferably more than 90% of the glycoprotein in the formulation.

[0135] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N83, including HexNAc1, wherein the HexNAc1 at N83 is present in 20%-60%, preferably 30%-50%, more preferably 35%-45% of the glycoprotein in the formulation.

[0136] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N247, including HexNAc1, wherein the HexNAc1 at N247 is present in more than 60%, preferably 75%-95%, more preferably 80%-90% of the glycoprotein in the formulation.

[0137] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N83, including Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2, wherein any one or more of said glycosylations are present alone or in combination in 0.5%-30%, preferably 1%-20%, more preferably 2%-18% of the glycoprotein in the formulation.

[0138] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N247, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2, wherein any one or more of said glycosylations are present, alone or in combination, in 0.1%-15%, preferably 0.5%-10%, more preferably 1%-5% of the glycoprotein in the formulation.

[0139] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N46, wherein the N-linked glycosylation is present in more than 80%, preferably more than 90%, of the glycoprotein in the formulation.

[0140] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N83, the N-linked glycosylation being present in more than 80%, preferably more than 90%, of the glycoprotein.

[0141] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N247, wherein the N-linked glycosylation is present in more than 50% of the glycoprotein in the formulation, preferably 60%-80% of the glycoprotein.

[0142] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N46, including HexNAc1, wherein the HexNAc1 at N46 is present in more than 50%, preferably more than 80%, more preferably more than 90% of the glycoprotein in the formulation.

[0143] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N83, including HexNAc1, wherein the HexNAc1 at N83 is present in 20%-60%, preferably 30%-50%, more preferably 35%-45% of the glycoprotein in the formulation.

[0144] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N247, including HexNAc1, wherein the HexNAc1 at N247 is present in more than 60%, preferably 75%-95%, more preferably 80%-90% of the glycoprotein in the formulation.

[0145] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N83, including Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2, wherein one or more of said glycosylations are present alone or in combination in 0.5%-30%, preferably 1%-20%, more preferably 2%-18% of the glycoprotein in the formulation.

[0146] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N247, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2 and / or Hex16HexNAc2, wherein one or more of said glycosylations are present, alone or in combination, in 0.1%-15%, preferably 0.5%-10%, more preferably 1%-5% of the glycoprotein in the formulation.

[0147] Implementation schemes involving the amounts of each glycosylation (for clones 6E2 and 6B2):

[0148] The aspects and embodiments relating to the amounts of each glycosylation of clones 6E2 and 6B2 are unified, benefited from, based on and / or associated with the above-mentioned aspects and embodiments relating to the amounts of each glycosylation, and optionally also with the obvious beneficial properties in terms of expression, secretion, yield, isolation, enzyme activity and / or therapeutic activity in these aspects of the invention.

[0149] As illustrated in the examples below, glycosylation at any one or more of the three N-linked glycosylation sites in clones 6E2 and 6B2 represents an unexpected modification following the production of rhAAT in yeast (Pichia pastoris). Unbound by theory, these highly generalized modifications also appear to convey properties of improved secretion efficiency and / or enzymatic efficacy and / or therapeutic effect compared to other AAT formulations (such as Prolastin).

[0150] As described in the examples below, the expression, secretion, and isolation of rhAAT result in protein formulations having various glycan structures. These can be homogeneous or heterogeneous. In a preferred embodiment, the formulation comprises a fragment of an rhAAT glycoprotein or thereof with heterologous N-linked glycosylation.

[0151] The amount (relative or absolute) of each glycosylation can be used to define protein formulations from clones 6E2 and 6B2. Quantitative (or semi-quantitative) peptide mapping analysis has identified rhAAT protein sequences with varying degrees of glycosylation at different sites in the rhAAT protein. These glycosylation patterns can be used to define rhAAT proteins or protein formulations and appear to be beneficial for secretion, yield, enzymatic activity, and / or therapeutic activity.

[0152] In one embodiment, the glycosylation of Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2 and Hex14HexNAc2 accounts for 5%-50% of the total N-glycans, preferably 6%-40%, more preferably 7%-35%.

[0153] In one embodiment, the glycosylation of Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2 and Hex13HexNAc2 accounts for 5%-50% of the total N-glycans, preferably 6%-40%, more preferably 7%-35%.

[0154] In one embodiment, Hex9HexNAc2 glycosylation accounts for 0.05%-20% of the total N-glycans, preferably 1%-17%, more preferably 2%-15%.

[0155] In one embodiment, Hex10HexNAc2 glycosylation accounts for 0.1% to 45% of the total N-glycans, preferably 1% to 40%, more preferably 2% to 35%.

[0156] In one embodiment, Hex11HexNAc2 glycosylation accounts for 0.1%-40% of the total N-glycans, preferably 0.31%-35%, more preferably 0.5%-30%.

[0157] In one embodiment, Hex12HexNAc2 glycosylation accounts for 0.1%-35% of the total N-glycans, preferably 0.1%-30%, more preferably 0.1%-25%.

[0158] In one embodiment, Hex13HexNAc2 glycosylation accounts for 0.1%-35% of the total N-glycans, preferably 0.1%-20%, more preferably 0.1%-15%.

[0159] In one embodiment, Hex14HexNAc2 glycosylation accounts for 0.1%-20% of the total N-glycans, preferably 0.1%-10%, more preferably 0.1%-8%.

[0160] In one embodiment, Hex15HexNAc2 glycosylation accounts for 0.1%-10% of the total N-glycans, preferably 0.1%-7%, more preferably 1%-5%.

[0161] In one embodiment, Hex16HexNAc2 glycosylation accounts for 0.1%-10% of the total N-glycans, preferably 0.1%-7%, more preferably 0.1%-5%.

[0162] In one embodiment, Hex17HexNAc2 glycosylation comprises 0.1%-7% of total N-glycans, preferably 0.3%-5%, more preferably 0.5%-3%.

[0163] In one embodiment, Hex18HexNAc2 glycosylation accounts for 0.1%-3% of the total N-glycans, preferably 0.1%-2%, more preferably 0.1%-1%.

[0164] Implementation schemes involving AAT sequences and N-linked glycosylation sites (for clones 6E2 and 6B2):

[0165] The various aspects and embodiments relating to the AAT sequence and N-linked glycosylation position of clones 6E2 and 6B2 are unified, benefited from, based on and / or associated with the above-described aspects and embodiments relating to the AAT sequence and N-linked glycosylation position, and optionally also through the obvious beneficial properties in terms of expression, secretion, yield, isolation, enzyme activity and / or therapeutic activity of these aspects of the invention.

[0166] In the embodiments, rhAAT or a fragment thereof comprises an amino acid sequence according to SEQ ID NO 4 and at least one N-linked glycosylation at N46, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2 and / or Hex14HexNAc2.

[0167] In the embodiments, rhAAT or fragments thereof comprise an amino acid sequence according to SEQ ID NO 4 and at least one N-linked glycosylation at N83, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2 and / or Hex14HexNAc2.

[0168] In the embodiments, rhAAT or fragments thereof comprise an amino acid sequence according to SEQ ID NO 4 and at least one N-linked glycosylation at N247, including Hex9HexNAc2 and / or Hex10HexNAc2.

[0169] Implementation schemes involving the AAT sequence, the location and type of N-linked glycosylation (for clones 6E2 and 6B2):

[0170] The various aspects and embodiments related to the type of AAT sequence and N-linked glycosylation of clones 6E2 and 6B2 are unified, benefited from, based on and / or associated with the above-mentioned aspects and embodiments related to the type of AAT sequence and N-linked glycosylation, and optionally also through the obvious beneficial properties in terms of expression, secretion, yield, isolation, enzyme activity and / or therapeutic activity of these aspects of the invention.

[0171] In the embodiments, rhAAT or a fragment thereof comprises the amino acid sequence according to SEQ ID NO 4 or a fragment thereof, and comprises at least one N-linked glycosylation at N46, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2 and / or Hex14HexNAc2, and / or

[0172] In the embodiments, rhAAT or a fragment thereof comprises the amino acid sequence according to SEQ ID NO 4 or a fragment thereof, and comprises at least one N-linked glycosylation at N83, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2 and / or Hex14HexNAc2, and / or

[0173] In the embodiments, rhAAT or a fragment thereof comprises an amino acid sequence or a fragment thereof according to SEQ ID NO 4, and comprises at least one N-linked glycosylation at N247, including Hex9HexNAc2 and / or Hex10HexNAc2.

[0174] Implementation schemes involving AAT sequences, the location and type of N-linked glycosylation, and information about each glycan site. Indication of (total) occupancy and frequency for each modification (for clones 6E2 and 6B2):

[0175] The aspects and embodiments relating to the occupancy and frequency of each modification at each glycan site of clones 6E2 and 6B2 are unified, benefited from, based on and / or associated with the above-mentioned aspects and embodiments relating to the occupancy and frequency of each modification at each glycan site, and optionally also through the obvious beneficial properties in terms of expression, secretion, yield, separation, enzyme activity and / or therapeutic activity of these aspects of the invention.

[0176] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N46, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2 and / or Hex15HexNAc2, wherein any one or more of said glycosylations are present, alone or in combination, in 0.1%-100%, preferably 1%-85%, preferably 1%-50%, more preferably 2%-35% of the glycoprotein in the formulation.

[0177] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N83, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2 and / or Hex15HexNAc2, wherein any or more of said glycosylations are present alone or in combination at 0.1%-90%, preferably 0.1%-45%, more preferably 1%-40%.

[0178] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N247, including Hex9HexNAc2 and / or Hex10HexNAc2, wherein any or more of said glycosylations are present alone or in combination at 0.1%-30%, preferably 1%-25%, more preferably 2%-12%.

[0179] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N46, wherein the N-linked glycosylation is present in more than 80%, preferably more than 90%, of the glycoprotein in the formulation.

[0180] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N83, the N-linked glycosylation being present in more than 70%, preferably more than 80% of the glycoprotein.

[0181] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 or a fragment thereof and at least one N-linked glycosylation at N247, wherein the N-linked glycosylation is present in more than 1% of the glycoprotein in the formulation, preferably 5%-30% of the glycoprotein.

[0182] In an embodiment, the protein formulation comprises an rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N46, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2 and / or Hex15HexNAc2, wherein the Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2 and / or Hex15HexNAc2 at N46 are present in more than 50%, preferably more than 80%, more preferably more than 90% of the glycoprotein in the formulation.

[0183] In an embodiment, the protein formulation comprises an rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N83, including Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2 and / or Hex15HexNAc2, wherein the Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2 and / or Hex15HexNAc2 at N83 are present in more than 60%, preferably more than 70%, more preferably more than 80% of the glycoprotein in the formulation.

[0184] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N247, including Hex9HexNAc2 and / or Hex10HexNAc2, wherein the Hex9HexNAc2 and / or Hex10HexNAc2 at N247 are present in more than 1%, preferably 1%-25%, more preferably 5%-20% of the glycoprotein in the formulation.

[0185] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N46, wherein the most abundant N-linked glycosylation at N46 is one or more of Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2 and Hex12HexNAc2, preferably wherein each glycosylation is present in more than 5%, preferably more than 10%, of the glycoprotein in the formulation.

[0186] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N83, wherein the most abundant N-linked glycosylation at N83 is one or more of Hex10HexNAc2 and Hex11HexNAc2, preferably wherein each glycosylation is present in more than 10%, preferably more than 15%, of the glycoprotein in the formulation.

[0187] In an embodiment, the protein formulation comprises rhAAT glycoprotein having an amino acid sequence according to SEQ ID NO4 and at least one N-linked glycosylation at N247, wherein the most abundant N-linked glycosylation at N247 is one or more of Hex9HexNAc2 and Hex10HexNAc2, preferably wherein each glycosylation is present in more than 1%, preferably more than 2%, of the glycoprotein in the formulation.

[0188] Other aspects and implementation plans:

[0189] In the implementation scheme, the rhAAT protein or a fragment thereof is expressed in *Pichia pastoris*. As detailed below, a novel production method has been developed that results in the efficient expression, secretion, and isolation of the desired protein or protein formulation. Therefore, this production method can be used to characterize the rhAAT glycoprotein itself or the protein formulation. Without being bound by theory, any production method using *Pichia pastoris* as defined by any one or more of the characteristics disclosed herein can convey the glycosylation pattern and / or associated beneficial properties (e.g., in terms of expression, secretion, yield, isolation, enzymatic activity, and / or therapeutic activity) of the glycoprotein to the implementation scheme.

[0190] In this embodiment, peptide mapping analysis is used to measure, determine, and / or analyze one or more N-linked glycosylations. Peptide mapping analysis is a well-established mass spectrometry-based technique used to identify proteins and their modifications based on mass measurements. In this embodiment, peptide mapping analysis involves protease degradation of a sample containing the glycoprotein or a fragment thereof or a protein formulation, followed by mass spectrometry analysis of the sample. In this embodiment, liquid chromatography-mass spectrometry (LC-MS) may be used.

[0191] Other aspects and implementation schemes related to the manufacturing method:

[0192] The present invention also relates to a method for producing recombinant human α1-antitrypsin (rhAAT) glycoprotein or fragments thereof in Pichia pastoris, the method comprising:

[0193] i. Provide genetically modified Pichia pastoris mut s The strain contains an exogenous hAAT-encoding nucleotide sequence controlled by the AOX1 promoter (PAOX1).

[0194] ii. The strain is cultured in a yeast growth medium, the culture comprising at least:

[0195] a) Batch stage, which includes incubation in the presence of glycerol, preferably for 10-24 hours.

[0196] b) A transition phase, which includes incubation in the presence of glycerol and methanol, preferably for 10-24 hours, and

[0197] c) The induction phase, which includes culturing in the presence of methanol, preferably for 40-100 hours, more preferably 60-90 hours, and

[0198] iii. Obtain the secreted rhAAT protein or a fragment thereof from the culture supernatant, and optionally isolate the rhAAT or the fragment thereof from the supernatant.

[0199] In embodiments, the method is used to produce the rhAAT glycoprotein or fragments thereof or protein formulations described in the foregoing aspects and embodiments. Unexpectedly, unique rhAAT glycoproteins with beneficial properties have been obtained by developing the novel methods described herein. Therefore, in embodiments, the method can be defined by and / or used to produce the inventive rhAAT described herein. The methods for manufacturing rhAAT as described herein result in the efficient expression, secretion, and / or isolation of the desired protein or protein formulation. Therefore, the production methods can be used to characterize the rhAAT glycoprotein itself or the protein formulation, and vice versa. Without being bound by theory, the production methods using Pichia pastoris, defined by any one or more of the features disclosed herein, can convey the unique glycosylation pattern and / or associated beneficial properties (e.g., in terms of expression, secretion, yield, isolation, enzymatic activity, and / or therapeutic activity) of the glycoprotein in embodiments.

[0200] The method described herein is further characterized by high levels of rhAAT expression, rhAAT secretion, and final rhAAT yield from the supernatant obtained after fermentation using the methods disclosed herein. This method can achieve high yields, for example, rhAAT levels in the supernatant exceeding 100 mg / L or 200 mg / L. Using a 10 L fermentation scale, Table 18 below shows, for example, rhAAT concentrations exceeding 200 mg / L in the supernatant after 60 or 90 hours of induction with methanol.

[0201] In an embodiment of the method, the culture is carried out in a bioreactor containing 500 mL to 25,000 L of yeast growth medium, and preferably the yeast growth medium is basal salt medium (BSM).

[0202] Technicians are able to select appropriate container sizes or production conditions based on the intended use and / or the required production scale. To the knowledge of the inventors, this large-scale production of rhAAT has not previously been established in Pichia pastoris and represents a beneficial and efficient method for producing large quantities of rhAAT for clinical use.

[0203] Therefore, cultivation can be carried out in any given container, bioreactor, or other suitable vessel with dimensions of 500 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 10 L, 15 L, 20 L, 50 L, or 100 L. Large-scale bioreactors for industrial manufacturing can also be used with vessels and / or bioreactors of 500 L, 1000 L, 2000 L, 3000 L, 5000 L, or even up to 10,000 L, 20,000 L, or 25,000 L. The dimensions of the container, vessel, and / or bioreactor can be any or more of the values ​​disclosed herein, or the dimensions of the container, vessel, and / or bioreactor can be defined by a range using any or more of the values ​​disclosed herein.

[0204] In an embodiment of the method, the supernatant containing the secreted rhAAT (optionally treated by centrifugation and filtration) is then treated with affinity chromatography, anion exchange chromatography and / or size exclusion chromatography to obtain isolated rhAAT glycoprotein or protein formulations.

[0205] Any suitable chromatographic or purification protocol may be used, such as those established in the art and known to those skilled in the art. Preferred methods, such as affinity chromatography, ion exchange, or size exclusion chromatography, represent feasible approaches to obtaining the desired rhAAT glycoprotein or its fragments or formulations described herein.

[0206] In an embodiment of the method, the rhAAT protein comprises the sequence according to SEQ ID NO 4, or encoded by SEQ ID NO 1, 2 or 3, preferably encoded by SEQ ID NO 3.

[0207] In an implementation of this method, the strain contains an exogenous hAAT-encoded nucleotide sequence integrated into the Pichia pastoris genome.

[0208] In an implementation of this method, the cultivation includes:

[0209] a. Batch stage, which includes culturing for 10-24 hours in the presence of glycerol.

[0210] b. The transition phase, which includes incubation for 12-24 hours in the presence of glycerol and methanol, and subsequently...

[0211] c. Induction phase, which includes culturing in the presence of methanol for 40-100 hours, preferably 60-90 hours.

[0212] In an embodiment of the method, in step a), a glycerol feeding phase is initiated 8-16 hours after inoculation into BSM medium, including glycerol feeding for a period of 6-10 hours after inoculation, wherein the glycerol feeding phase includes feeding the medium with glycerol at a rate of 5-20 g glycerol per liter of medium per hour (g / h / L).

[0213] In the implementation of this method, during the period of 6-10 hours after inoculation, the glycerol supplementation is gradually increased from 5g / h / L-8g / h / L to 12g / h / L-20g / h / L.

[0214] In an embodiment of the method, in step b), a transition phase is initiated 16-24 hours after inoculation into BSM medium, including glycerol and methanol feeding for a period of 12-24 hours after inoculation. The transition phase includes feeding the medium with glycerol at a rate of 2 to 15 g / L and feeding the medium with methanol at a rate of 1 to 6 g / L.

[0215] In the implementation of this method, during the period of 12-24 hours after inoculation, the glycerol supplementation is gradually increased from 2g / h / L-8g / h / L to 8g / h / L-15g / h / L, and the methanol supplementation is gradually increased from 1g / h / L-3g / h / L to 4g / h / L-6g / h / L.

[0216] In an embodiment of the method, in step c), an induction phase is initiated 24-48 hours after inoculation into BSM medium, including methanol feeding for a period of 40-100 hours after inoculation, wherein the induction phase includes feeding the medium with methanol at a rate of 2 to 15 grams of methanol per liter of medium per hour (g / h / L).

[0217] In the implementation of this method, during the period of 40-100 hours after inoculation, the methanol feed is gradually increased from 2g / h / L-6g / h / L to 6g / h / L-15g / h / L.

[0218] In an embodiment of this method, the strain contains a nucleotide sequence encoding a secretion guide sequence for an α-mating factor precursor-propeptide from *Saccharomyces cerevisiae*. Unbound by theory, this factor appears in the embodiment to enhance the secretion of rhAAT glycoprotein into the supernatant.

[0219] In an embodiment of this method, the yeast growth medium is a basal salt medium (BSM). This medium is preferred, but not required, for the production of rhAAT of the present invention.

[0220] In an embodiment of the method, for one or more of steps a)-c), preferably for two or more of steps a)-c), more preferably for the entire method, the oxygen saturation of the culture medium is 20%-40%, preferably 25%-35%, more preferably about 30%.

[0221] In an embodiment of this method, the supernatant containing the secreted rhAAT is subsequently processed by centrifugation and filtration, followed by purification and / or separation using the chromatographic methods described herein. Other options for protein separation are also described in detail below.

[0222] Further aspects and implementation schemes relating to manufacturing methods with shortened induction time:

[0223] The present invention also relates to a method for producing recombinant human α1-antitrypsin (rhAAT) glycoprotein or fragments thereof in Pichia pastoris, wherein the induction time includes culturing in the presence of methanol, preferably for 10-70 hours, more preferably for 12-60 hours, and most preferably for 15-50 hours (referred to herein as "shortened induction time").

[0224] The various aspects and embodiments related to the manufacturing method with shortened induction time are unified, benefited from, based on and / or associated with the above-described manufacturing method, and optionally also by the obvious beneficial properties in terms of expression, secretion, yield, separation, enzyme activity and / or therapeutic activity in these aspects of the invention.

[0225] The present invention also relates to a method for producing recombinant human α1-antitrypsin (rhAAT) glycoprotein or fragments thereof in Pichia pastoris, the method comprising:

[0226] i. Provide genetically modified Pichia pastoris mut s The strain contains an exogenous hAAT-encoding nucleotide sequence controlled by the AOX1 promoter (PAOX1).

[0227] ii. The strain is cultured in a yeast growth medium, the culture comprising at least:

[0228] a) Batch stage, which includes incubation in the presence of glycerol, preferably for 10-24 hours.

[0229] b) A transition phase, which includes incubation in the presence of glycerol and methanol, preferably for 10-24 hours, and

[0230] c) The induction phase, which includes culturing in the presence of methanol, preferably for 10-70 hours, more preferably 12-60 hours, and most preferably 15-50 hours.

[0231] iii. Obtain the secreted rhAAT protein or a fragment thereof from the culture supernatant, and optionally isolate the rhAAT or the fragment thereof from the supernatant.

[0232] Surprisingly, even with the shortened induction time as described herein, this method resulted in highly efficient expression, secretion, and / or isolation of the desired protein or protein formulation. Furthermore, the shortened induction time led to an increase in the full-length protein and a corresponding decrease in the relative amounts of cleaved variants or protease-digested fragments. Those skilled in the art would not have anticipated that a shortened induction time would result in a larger relative amount of full-length rhAAT.

[0233] Therefore, the shortened induction time reduces the total production time while maintaining high output, thereby improving efficiency and reducing production costs.

[0234] In an embodiment of the method, in step c), an induction phase is initiated 24-48 hours after inoculation into BSM medium, including methanol feeding for a period of 10-80 hours, more preferably 15-70 hours, and most preferably 20-60 hours after inoculation, wherein the induction phase includes methanol feeding into the medium, preferably at a rate of 2 to 15 grams of methanol per liter of medium (g / h / L).

[0235] In an implementation of this method, the shortened induction time in step c) results in an increase in the full-length protein.

[0236] In an implementation of this method, the cultivation includes:

[0237] a. Batch stage, which includes culturing for 10-24 hours in the presence of glycerol.

[0238] b. The transition phase, which includes incubation for 12-24 hours in the presence of glycerol and methanol, and subsequently...

[0239] c. An induction phase, comprising culturing in the presence of methanol for 10-70 hours, preferably 15-50 hours. In other embodiments, a protease inhibitor is added to the culture program to reduce the amount of cleaved / cut rhAAT variants. In embodiments, the protease inhibitor may be added to the culture medium during the batching phase, transition phase, and / or induction phase, or subsequently during rhAAT purification. In embodiments, the protease inhibitor is used at a concentration sufficient to reduce rhAAT cleavage to levels lower than those without the protease inhibitor.

[0240] In one embodiment, the protease inhibitor is EDTA, and it is preferably used at a concentration of 1 mM-100 mM, more preferably 1 mM-10 mM. In another embodiment, the protease inhibitor is PMSF, and it is preferably used at a concentration of 1 mM-100 mM, more preferably 1 mM-10 mM. In yet another embodiment, the protease inhibitor is pepsin A, and it is preferably used at a concentration of 0.1 μM-10 μM, more preferably 0.5 μM-5 μM.

[0241] The embodiments and features of the invention described with respect to methods, glycoproteins, protein formulations or other aspects are considered to be disclosed with respect to each other aspect of this disclosure, such that the features of the characterization methods can be used to characterize glycoproteins or protein formulations, and vice versa.

[0242] The various aspects of the invention are unified, benefited from, based on, and / or associated with the common and unexpected discovery of the glycosylation of rhAAT described herein, and optionally also with the evidently beneficial properties in terms of expression, secretion, yield, isolation, enzymatic activity, and / or therapeutic activity in these aspects of the invention.

[0243] An overview of the sequences disclosed in the specification:

[0244]

[0245]

[0246]

[0247]

[0248] Trypsin: Arginine (R) or Lysine (K); GluC: Aspartic acid (D) or Glutamic acid (E) Detailed Implementation

[0249] All cited patent documents and non-patent literature are incorporated herein by reference in their entirety.

[0250] α-1 antitrypsin (AAT):

[0251] α-1 antitrypsin (also known as A1AT, AAT, PI, SERPINA1) is a glycoprotein of approximately 52 kDa and is one of the most abundant endogenous serine protease inhibitors (SERPIN superfamily). AAT is considered an acute-phase protein, therefore its concentration can increase several times during acute inflammation.

[0252] The AAT coding region is preferably any nucleic acid encoding a naturally occurring or synthetic AAT protein sequence that expresses AAT function, having reduced, identical, similar, or increased activity compared to human AAT, or being functionally similar to human AAT. The amino acid sequence of AAT is available from the NCBI database under accession number 1313184B. The corresponding nucleic acid sequence encoding AAT is available to those skilled in the art of molecular biology or genetics. This invention covers the use of AAT sequence variants that are functionally similar or analogous to the unmodified form of human AAT.

[0253] An AAT coding sequence (CDS) is disclosed at http: / / www.ncbi.nlm.nih.gov / nuccore / NM_000295.4 and is a preferred embodiment. The sequence contains bases 262 to 1518 of the complete sequence. SEQ ID NO 1 represents an exemplary AAT coding sequence.

[0254] In some embodiments of the present invention, the CDS is codon-optimized to increase protein yield. The codon-optimized coding sequence is preferably read as SEQ ID NO 2.

[0255] The human AAT protein is encoded by the nucleotide sequence of SEQ ID NO 1 and / or 2 and / or 3, and the amino acid sequence of SEQ ID NO 4.

[0256] In the implementation scheme, the human AAT protein according to SEQ ID NO 4 is used, which corresponds to the human AAT sequence as known in the art.

[0257] As used herein, recombinant human AAT (rhAAT) glycoprotein is any protein containing a segment or fragment of the human rhAAT sequence that exhibits substantially equivalent or similar activity to the rhAAT evaluated in the examples below. Therefore, the fragment of SEQ ID NO4 is also considered human AAT, and sequence variants exhibiting equivalent or substantially similar function to rhAAT as described herein are also considered human AAT.

[0258] Therefore, this invention covers the use of genetically modified yeast expressing recombinant AAT as described herein, preferably Pichia pastoris, wherein the yeast comprises nucleic acid molecules selected from the group consisting of:

[0259] a) A nucleic acid molecule containing a nucleotide sequence encoding an AAT protein (such as according to SEQ ID NO 4), preferably encoded by a nucleotide sequence according to SEQ ID NO 1 or 2 or 3, more preferably SEQ ID NO 3.

[0260] b) Nucleic acid molecules complementary to the nucleotide sequence according to a);

[0261] c) A nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to the nucleotide sequence according to a) or b), preferably comprising having at least 70%, 75%, 80%, 85%, preferably 90%, more preferably 95% sequence identity to the nucleotide sequence according to a) or b.

[0262] d) Nucleic acid molecules that are derived from the nucleotide sequences of a) to c) due to the degeneracy of the genetic code; and / or

[0263] e) Nucleic acid molecules based on the nucleotide sequences of a) to d), which are modified by deletion, addition, substitution, translocation, inversion and / or insertion, and are functionally similar to / equivalent to the nucleotide sequences based on a) to d).

[0264] Therefore, the present invention covers the use of the AAT protein according to SEQ ID NO 4 or a variant thereof, or the use of genetically modified yeast, preferably Pichia pastoris, as described herein, which contains a nucleotide sequence encoding the amino acid sequence according to SEQ ID NO 4 5.

[0265] This invention covers other sequence variants of SEQ ID NO 3, particularly those having at least 70% sequence identity with SEQ ID NO 4, preferably at least 75%, 80%, 85%, 90%, or at least 95% sequence identity with SEQ ID NO 4. Such sequence variants are preferably functionally similar to or equivalent to the human AAT disclosed herein.

[0266] While maintaining functional equivalence or similarity to human AAT of SEQ ID NO 4, the present invention also covers variations in protein length. Truncation or extension of protein length, for example, up to 50, 40, 30, 20, or 10 amino acids, can maintain AAT activity and is therefore included in the present invention.

[0267] The term rhAAT glycoprotein fragment also refers to, for example, a truncated form (or fragment) of SEQ ID NO 4, which substantially maintains the activity observed in the rhAAT glycoprotein of the present invention. Sufficient details regarding the enzyme and / or therapeutic activity are disclosed in the examples below, which can be used by those skilled in the art to determine that the rhAAT fragments exhibit substantially the same, similar, or analogous activity.

[0268] Therefore, the rhAAT glycoprotein fragment preferably contains all three rhAAT glycosylation sites described herein and maintains the activity described herein. Any fragment lacking up to 20 amino acids from the N-terminal and / or C-terminal sequence of SEQ ID NO 4 can be considered a functional fragment.

[0269] In the implementation scheme, AAT may include 0 to 10 amino acid additions or deletions at the N-terminus and / or C-terminus of the relevant sequence. This means that the polypeptide may have: a) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids added to its N-terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted at its C-terminus, or b) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids added to its C-terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted at its N-terminus. A nucleotide, or c) having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids added to its N-terminus and having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids added to its N-terminus, or d) having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted from its N-terminus and having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted from its C-terminus.

[0270] Functionally similar sequences are those that can provide a functional AAT gene product and achieve the same or similar functional effects as human AAT. AAT function can be determined by its ability to inhibit various proteases (such as trypsin) in vitro or by its ability to inhibit neutrophil elastase (as described below). Appropriate assays for determining protease activity or for determining neutrophil elastase activity are known to those skilled in the art.

[0271] Protein modifications to AAT proteins (which can occur through substitutions in the amino acid sequence and the nucleic acid sequence encoding such molecules) are also included within the scope of this invention. A “substitution” as defined herein refers to a modification of the amino acid sequence of a protein in which one or more amino acids are replaced by the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from the original protein. In some embodiments, such modification will not significantly alter the function of the protein. Similar to addition, substitution can be natural or artificial. It is known in the art that amino acid substitutions can be performed without significantly altering protein function. This is especially true when the modification involves a “conserved” amino acid substitution, in which one amino acid is replaced by another amino acid having similar properties. Such “conserved” amino acids can be natural or synthetic amino acids that can be substituted for in size, charge, polarity, and conformation without significantly affecting the structure and function of the protein. Typically, multiple amino acids can be substituted with conserved amino acids without adversely affecting the function of the protein. Generally, the nonpolar amino acids Gly, Ala, Val, Ile, and Leu; the nonpolar aromatic amino acids Phe, Trp, and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn, and Met; the positively charged amino acids Lys, Arg, and His; and the negatively charged amino acids Asp and Glu represent a group of conserved amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and sometimes Cys can be interchanged with each other, even if they belong to different groups.

[0272] The term "conserved amino acid substitution" is well known in the art and refers to the substitution of a specific amino acid with an amino acid having similar properties (e.g., similar charge or hydrophobicity, similar side chain volume). Examples include the substitution of glutamic acid with aspartic acid or the substitution of leucine with isoleucine. Conserved substitution variants will: 1) have only conserved amino acid substitutions relative to the parental sequence; 2) have at least 90% sequence identity relative to the parental sequence, preferably at least 95%, 96%, 97%, 98%, or 99% or higher; and 3) retain neuroprotective or neuroreparative activity. In this respect, any conserved substitution variant of the above-described polypeptide sequence is considered according to the present invention. Such variants are considered "AAT proteins".

[0273] As used herein, the “percentage of sequence identity (%)” relative to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in the candidate sequence that are identical to those in the reference polypeptide or nucleic acid sequence after alignment of the candidate and reference sequences, with vacancies introduced where necessary to achieve the maximum percentage of sequence identity, and any conserved substitutions are not considered part of the sequence identity. Alignment to determine the percentage of amino acid or nucleic acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software programs. Software such as BLAST or Clustal is capable of performing such sequence alignments and calculating the percentage of identity. As used herein, the percentage of homology between two sequences is equivalent to the percentage of identity between the two sequences. The determination of the percentage of identity or homology between sequences can be performed, for example, using the GAP program (Genetics Computer Group software; now available through Accelrys, http: / / www.accelrys.com), and alignment can be performed using, for example, the ClustalW algorithm (VNTI software, InforMax Inc.). Sequence databases can be searched using the target nucleic acid sequence. Algorithms used for database searching are typically based on BLAST software (Altschul et al., 1990). In some implementations, the percentage of homology or identity can be determined along the full length of the nucleic acid.

[0274] In addition to the peptides described herein, peptide analogs are also considered. In the pharmaceutical industry, peptide analogs are commonly used as non-peptide drugs that possess properties similar to the template peptide. These types of non-peptide compounds are referred to as “peptide mimics” or “peptide simulants” (Fauchere (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS ​​p. 392; and Evans et al. (1987) J. Med. Chem. 30:1229) and are typically developed using computerized molecular modeling. Peptide mimics with structures similar to therapeutic peptides can be used to produce equivalent therapeutic or preventative effects. In some embodiments, peptide mimics are preferred to prolong the stability of the peptide when administered to a subject. For this purpose, peptide mimics of peptides that are not cleaved by the human proteasome are preferred.

[0275] The nucleic acid molecule encoding the AAT of this invention can be codon-optimized according to standard methods in the art for expression in cells containing the target DNA. For example, if the intended target nucleic acid is in yeast cells, a yeast codon-optimized polynucleotide encoding AAT is considered for use in the construct described herein.

[0276] In other embodiments, the α-1 antitrypsin (AAT) protein may be replaced by other serine protease inhibitors. Therefore, any features of AAT disclosed herein may also relate to other serine protease inhibitors. In one embodiment, the serine protease inhibitor is a trypsin-like serine protease inhibitor.

[0277] Trypsin-like serine protease inhibitors inhibit serine proteases with trypsin-like activity. Serine acts as a nucleophilic amino acid at the enzyme's active site of serine proteases. Numerous trypsin-like serine proteases have become a research hotspot for potential therapeutic targets. Viral entry via endocytosis depends on target-cell proteases, such as serine proteases. If a virus requires target-cell serine proteases for entry and growth, viral infection is target-cell serine protease dependent.

[0278] Target-cell serine proteases activate viral spike proteins, which are required for viral-target cell membrane fusion and the release of the viral genome into the target cell's cytosol. Targeting host-cell serine proteases can prevent the growth of endocytosis-dependent viruses; therefore, inhibiting target-cell serine proteases is an effective method for treating subjects infected with endocytosis-dependent viruses. Serine protease inhibitors, preferably trypsin-like serine protease inhibitors, inhibit viral infection and viral growth. As non-limiting examples, trypsin-like serine protease inhibitors include carmostat, aprotinin, benzalkonium chloride, gabexate, leucopeptidase, naftomostat, pepsin A, ribavirin, spiculstat, ulinastatin, and palmostat.

[0279] molecular:

[0280] As used herein, "nucleic acid" or "nucleic acid molecule" refers to a molecule composed of chains of monomeric nucleotides, such as DNA molecules (e.g., cDNA or genomic DNA). Nucleic acids may encode, for example, promoters, AAT genes or portions thereof, or regulatory elements. Nucleic acid molecules can be single-stranded or double-stranded.

[0281] "AAT nucleic acid" refers to a nucleic acid containing the AAT gene or a portion thereof, or a functional variant of the AAT gene or a portion thereof. Functional variants of the gene include gene variants with minor variations, such as silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly alter gene function.

[0282] As used herein, the term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule isolated from a naturally occurring gene, or modified to contain nucleic acid segments in a manner not found in nature, or is synthetic. When a nucleic acid construct contains a control sequence required for expression of the coding sequence of this disclosure, the term "nucleic acid construct" is synonymous with the term "expression cassette."

[0283] The DNA sequence that “encodes” a specific AAT protein (including its fragments and portions) is a nucleic acid sequence that is transcribed into a specific RNA and / or protein. DNA polynucleotides can encode RNA (mRNA) that is translated into a protein, or DNA polynucleotides can encode RNA that is not translated into a protein (e.g., tRNA, rRNA, or RNA that targets DNA; also known as “non-coding” RNA or “ncRNA”).

[0284] As used herein, the term "gene" or "coding sequence" is intended in a broad sense to refer to the DNA region (transcribed region) that encodes a protein. When a coding sequence is placed under the control of appropriate regulatory regions (such as promoters), it is transcribed (DNA) and translated (RNA) into a polypeptide. A gene may contain several operable linked segments, such as promoters, 5′-leader sequences, coding sequences, and 3′-untranslated sequences, containing polyadenylation sites. The phrase "gene expression" refers to the process in which a gene is transcribed into RNA and / or translated into an active protein.

[0285] As used herein, “protein” or “polypeptide” should mean both peptide and protein. In this invention, a polypeptide may be naturally occurring or recombinant (i.e., produced by recombinant DNA technology) and may contain mutations (e.g., point mutations, insertion mutations, and deletion mutations) and other covalent modifications (e.g., glycosylation and labeling (by biotin, streptavidin, fluorescein, and radioisotopes)) or other molecular bonds with other components. For example, PEGylated proteins are included within the scope of this invention. PEGylation has been widely used as a post-modification methodology for improving the biomedical efficacy and physicochemical properties of therapeutic proteins. The suitability and safety of this technique have been demonstrated over many years of use with various PEGylated pharmaceuticals (see Jevsevar et al., Biotechnol J. January 2010; 5(1):1 13-28). In some embodiments, the polypeptides described herein are modified to exhibit a longer in vivo half-life and resistance to degradation compared to unmodified polypeptides. Such modifications are known to those skilled in the art, such as cyclized peptides, peptides fused to vitamin B12, pinned peptides, protein esterification, and replacement of native L-amino acids with D-amino acids (see Bruno et al., Ther Deliv. 2013 Nov; 4(11):1443-1467).

[0286] Production of recombinant AAT in yeast:

[0287] This invention relates to a method for producing recombinant AAT or fragments thereof from genetically modified yeast, the method preferably comprising the following steps: culturing genetically modified yeast containing a foreign nucleic acid molecule having an AAT coding region operatively linked to a promoter or promoter / enhancer combination; expressing recombinant AAT in the cultured yeast; and isolating recombinant AAT from the culture.

[0288] Yeast expression platforms suitable for use in the context of this invention include any yeast strain used to produce large quantities of protein (here, AAT) for research or industrial applications. While yeast tends to require more resources to maintain than, for example, bacteria, certain products can only be produced by eukaryotic cells (such as yeast), thus necessitating the use of yeast expression platforms. Yeasts differ in productivity and their ability to secrete, process, and modify proteins. Therefore, different types of yeast (i.e., different expression platforms) are better suited to different research and industrial applications. Importantly, expressing and producing proteins in yeast is advantageous because yeast can grow rapidly in large containers, produce proteins efficiently and safely, and produce and modify protein products to meet human-use standards in the case of cosmetic compositions.

[0289] The manufacture of recombinant therapeutic agents is a rapidly developing field in therapeutic medicine. Yeast is a mature eukaryotic host for the production of heterologous proteins and offers unique benefits in the synthesis of recombinant drugs. Yeast thrives on inexpensive culture media, is easily genetically manipulated, and can incorporate post-translational variations characteristic of eukaryotes. A variety of yeasts, including *Saccharomyces cerevisiae*, *Pichia pastoris*, *Hansenula polymorpha*, *Yarrowia lipolytica*, *Arxula adeninivorans*, *Kluyveromyces lactis*, and *Schizosaccharomyces pombe*, are exemplary yeast hosts for the production of recombinant proteins and are employed accordingly.

[0290] Yeasts are a common host for producing proteins from recombinant DNA. They offer relatively easy genetic manipulation and can grow rapidly to high cell densities on inexpensive culture media. As eukaryotes, they are capable of protein modifications such as glycosylation, which are common in eukaryotic cells but relatively rare in bacteria. Therefore, yeasts can produce complex proteins that are identical or very similar to natural products from mammals, particularly humans. The most commonly used yeast expression platform is based on baker's yeast, also known as brewer's yeast. However, other yeast expression platforms, such as those in the genus *Pichia* (particularly *Pichia pastoris*), have been studied and are widely used in various applications based on their different characteristics and capabilities. For example, some of them grow on a wide variety of carbon sources and are not limited to glucose like baker's yeast. Several of them are also used in genetic engineering and the production of heterologous proteins, and are applicable in the context of this invention.

[0291] In the implementation scheme, the yeast related to the present invention belongs to the family Saccharidae, preferably the genus Pichia, and more preferably Pichia pastoris.

[0292] Pichia pastoris is an excellent expression host for the production of heterologous proteins, including industrial enzymes and biopharmaceuticals. To date, this methyltrophic expression system has been successfully used to produce numerous recombinant proteins, including human erythropoietin, phospholipase C, phytase, human superoxide dismutase, trypsin, human serum albumin, collagen, and the 3H6 Fab fragment of a human monoclonal antibody. Compared to any other yeast species, Pichia pastoris is highly efficient in the secretory production of recombinant proteins.

[0293] Industrial interest in this host is also attributed to its robust methanol-regulated alcohol oxidase promoter (AOX1), efficient secretion mechanism, post-translational modification capabilities, and ability to achieve high cell densities on well-defined culture media. Various recombinant proteins have been expressed using *Pichia pastoris*. Several successful cases of producing therapeutic proteins in *Pichia pastoris* have been documented. In a previous study (2016), a nanobody (VHH) targeting Clostridium botulinum neurotoxin (BoNT / E) was demonstrated in *Pichia pastoris*, achieving a product yield of 16 mg / L, higher than that produced in *Escherichia coli*. Furthermore, Xia et al. demonstrated the production of 30 mg / L recombinant angiopoietin in this yeast. In addition, productivity of 111 mg / L human adiponectin, 8.1 g / L recombinant xylanase, and 260 mg / L anti-HIV antibody has been reported in *Pichia pastoris*.

[0294] The average concentration of AAT in plasma is 1.3 mg / ml, with a half-life of 3 to 5 days. Protein size, glycosylation patterns, metastable inhibitory properties of AAT, and production costs represent challenges in recombinant AAT production. The methyltrophic yeast *Pichia pastoris* is an attractive host for the production of human AAT. It possesses the ability to introduce various post-translational modifications, such as glycosylation and proteolytic processing, acquired via transmissibility along the secretory pathway. For glycoproteins such as AAT, these modifications are crucial for appropriate function and / or structure.

[0295] In the implementation scheme, AAT is expressed in *Pichia pastoris* as a fusion protein with a histidine tag (His tag) to facilitate purification. A signal sequence from the *Saccharomyces cerevisiae* α-factor secretion signaling pathway is included to guide protein secretion into the extracellular culture medium. This robust expression system utilizes a highly inducible alcohol oxidase 1 (AOX1) promoter to express a large amount of glycosylated protein. The activity and characterization of AAT produced from *Pichia pastoris* were evaluated using an elastase inhibition assay.

[0296] In the implementation scheme, the genetically modified yeast belongs to the family Saccharomycetes, preferably the genus Saccharomyces, and more preferably Saccharomyces cerevisiae. In the implementation scheme, Saccharomyces cerevisiae is a preferred host compared to bacteria. The Saccharomyces cerevisiae expression system is one of the most commonly used eukaryotes and has been used as a model for studying various biological phenomena and for the recombinant production of therapeutic proteins. Potential problems with protein glycosylation using Saccharomyces cerevisiae have been resolved, making it a viable yeast for the production of therapeutic proteins. For example, disruption of the Mnn2p and Mnn11p genes associated with glycosylation modification pathways has been shown to increase the yield of recombinant cellulase. Furthermore, removal of the α-1,6-mannosyltransferase Och1p increases the yield of the active form of human tissue plasminogen activator. These studies indicate that N-glycosylation modification also leads to increased protein secretion. Recombinant proteins can be expressed intracellularly or secreted using a secretion signal peptide-guided secretion device. A commonly used signal sequence that functions in all yeast expression systems is the precursor-propeptide sequence of mating factor α1 (MFα1).

[0297] From a safety perspective, *Saccharomyces cerevisiae* also possesses several significant advantages, encouraging its use in various industrial processes. It is Generally Recognized As Safe (GRAS) because it is non-pathogenic and has historically been used in various nutritional industries and the production of biopharmaceuticals. Furthermore, current knowledge of yeast genetics, physiology, and fermentation facilitates the use of this organism in the production of useful products. Examples of commercially available products derived from *Saccharomyces cerevisiae* include hepatitis B surface antigen, hirudin, insulin, glucagon, uricase, macrophage colony-stimulating factor, and platelet-derived growth factor. Alternative expression systems include the methyl-trophic yeasts *Pichia pastoris* and *Hansenula polymorpha*, and the non-methyl-trophic yeasts *Yersinia lipolytica*, *Kluyveromyces lactis*, and *Akistaniella adenine*.

[0298] As used herein, the term “promoter” should refer to a DNA sequence to which a protein binds to initiate the transcription of an RNA transcript that begins in the DNA downstream of that promoter.

[0299] As used herein, the terms "AOX1 promoter" and "alcohol oxidase I promoter" should refer to methanol-inducible promoters used for protein expression in *Pichia pastoris*. In the context of this invention, the AOX1 promoter includes the WT AOX1 promoter (the native AOX1 promoter) or any AOX1 promoter having alternative and similar functional sequences derived from the native AOX1 promoter. AOX1 promoter libraries have been developed using deletion or insertion methods according to the prior art. In one embodiment, the AOX1 promoter is specified according to SEQ ID NO:5.

[0300] In the implementation scheme, the promoter for rhAAT may be an inducible AOX1 promoter, a constitutive GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter, or an ADH3 (alcohol dehydrogenase) promoter. Other suitable promoters from Pichia pastoris are, for example:

[0301] Inducible promoters: DAS (dihydroxyacetone phosphate), FLD1 (formaldehyde dehydrogenase), PEX8 (peroxisome matrix protein), ICL1 (isocitrate lyase), LRA3(L) (L-rhamnolate dehydratase), LRA4 (L-KDR aldolase), THI11 (thiamine biosynthesis protein), GTH1 (high affinity glucose transporter), and PPLCC1 (laccase);

[0302] Constitutive promoters: YPT1 (a secreted GTPase), TEF1 (translation elongation factor-1α), GCW14 (glycosylphosphatidylinositol), and PGK1 (phosphoglycerate kinase).

[0303] In this implementation, the *Pichia pastoris* strain *Muts* is used to produce rhAAT as described herein. The recombinant protein is typically expressed in the wild-type *Mut+* host strain by the methanol-inducible promoter PAOX1. However, other host strains, such as *Muts(AOX1-*)* and *Mut-(AOX1-AOX2-*)*, have been developed that consume less methanol and express the recombinant protein at higher levels than *Mut+*. The *MutS* strain of *Pichia pastoris* is known and available to those skilled in the art.

[0304] Post-translation editing:

[0305] In one implementation, the method used herein includes post-translational modification of the recombinant AAT protein.

[0306] In the implementation scheme, the post-translational modification is N-glycosylation, O-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation, or any combination thereof.

[0307] In the implementation scheme, the recombinant AAT protein has one or more human-like glycoform patterns.

[0308] In the implementation scheme, the method as used herein includes modifying the recombinant AAT protein in vitro after isolating it from yeast.

[0309] In some implementations, the modification is a covalent linking of the recombinant AAT protein to a biocompatible polymer.

[0310] The term "post-translational modification" refers to any alteration of the translated polypeptide chain of AAT that occurs after or during translation. This includes modifications to the amino acid side chains of the polypeptide, or modifications to the terminal amino or carboxyl groups. Post-translational modification refers to the attachment of biochemical groups (such as acetate, phosphate, carbohydrate moieties, and lipids) to the amino acid side chains, which alters the biochemical and physical properties of the protein after translation. Many proteins undergo post-translational modification shortly after translation, and some undergo it after protein folding, while others undergo it after localization. The most common protein post-translational modifications are phosphorylation, glycosylation, methylation, ubiquitination, S-nitrosylation, and N-acetylation.

[0311] Such modifications can be covalent modifications carried out enzymatically by the expression system after protein biosynthesis. Post-translational modifications also include enzymatic cleavage of peptide bonds and processing of the translated protein, with specific chemical groups, polymers, lipids, carbohydrates, or even the entire protein covalently added to the amino acid side chains. These chemical modifications of the polypeptide chain after its biosynthesis expand the range of amino acid structures and properties, thus diversifying the structure and function of the protein. In a preferred embodiment, the recombinant AAT protein undergoes post-translational modifications such as phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, lipidation, and proteolysis.

[0312] In one aspect of the invention, the secreted or released AAT protein undergoes glycosylation, including a variety of options for adding a sugar moiety to the protein, ranging from simple monosaccharide modifications of nuclear transcription factors to highly complex branched-chain polysaccharide changes in cell surface receptors in yeast expression systems, preferably O-glycosylation and / or N-glycosylation. N-glycosylation is the binding of a carbohydrate to asparagine in a polypeptide. O-glycosylation is the binding of a carbohydrate to serine / threonine.

[0313] In the implementation scheme, modification of the recombinant AAT protein involves the covalent linking of recombinant AAT to a biocompatible polymer, such as polyethylene glycol (PEG). As used herein, the term "PEGylation" refers to the process of covalently and non-covalently linking a polyethylene glycol polymer chain to molecular and macroscopic structures, such as drugs or bioactive proteins or vesicles. PEGylation is typically performed by co-incubating a reactive derivative of PEG with the target molecule. The covalent linking of PEG to the protein can "mask" the agent from the host's immune system (thus reducing immunogenicity and antigenicity) and increase its hydrodynamic size (size in solution), which prolongs its circulation time by reducing renal clearance. In one aspect, PEGylation can occur at the N-terminus of the recombinant AAT protein.

[0314] As used herein, the term "biocompatible polymer" refers to polymers suitable for exposure to the body and bodily fluids. Biocompatible polymers can be synthetic or natural and work closely with or near living cells in the vicinity of living systems. These polymers are used to evaluate, treat, enhance, or replace any tissue, organ, or function of the body. Biocompatible polymers improve bodily function without altering its normal function or causing allergies or other side effects. This encompasses advancements in tissue culture, tissue scaffolds, implants, artificial grafts, wound creation, controlled drug delivery, bone filling materials, and more. Biocompatible polymers include polystyrene (PS), polypropylene (PP), polyvinyl chloride (PVC), polyethylene (PE), polyurethane (PU), polycarbonate (PC), polyethylene terephthalate (PET), and polyetheretherketone (PEEK).

[0315] As used in this article, "polyethylene glycol" is a polyether compound with a wide range of applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide or polyoxyethylene, depending on its molecular weight.

[0316] In some respects, the recombinant AAT protein is conjugated with a water-soluble polymer. This can be done by any of a variety of chemical methods known in the art. For example, in one embodiment, the recombinant AAT protein is modified by conjugating PEG to the free amino group of the protein using an N-hydroxysuccinimide (NHS) ester. In another embodiment, a water-soluble polymer (e.g., PEG) is coupled to a free SH group using maleimide chemistry, or, after pre-oxidation, PEG hydrazide or PEG amine is coupled to the carbohydrate moiety of the recombinant AAT protein.

[0317] According to one embodiment of the invention, PEGylation is performed using PEG of 20 kDa or greater. PEG conjugates with AAT and provides a protein-encapsulating effect, resulting in defense against loss after binding and clearing of the receptor or degradation by inactive proteases.

[0318] PEG is a polymer in linear or branched form, and low molecular weight PEG cannot completely encapsulate proteins and therefore cannot fully protect them. Therefore, the molecular weight should be equal to or higher than a critical level sufficient to encapsulate the protein to be protected. The PEGylation of the recombinant AAT protein of this invention can be carried out by replacing the amino acid at the PEGylation site with a cysteine ​​residue, followed by conjugation with a PEG containing an acryloyl, sulfone, or maleimide group at one end specific to cysteine.

[0319] Recombinant human AAT isolation / purification:

[0320] According to the invention, AAT is preferably purified or separated using methods known to those skilled in the art, for example from culture supernatants or other preparations obtained from yeast cultures. Harvesting the material obtained by Pichia pastoris fermentation using standard laboratory equipment is possible in order to process the product from the supernatant.

[0321] For example, suitable methods include centrifugation and / or deep filtration, followed optionally by (sterile) membrane filtration. Low-speed centrifugation, sufficient to precipitate the yeast cells, can also be used, leaving a clear, transparent supernatant, which can also serve as evidence of the absence of bacterial contamination. Such procedures produce a supernatant ready for subsequent purification steps, such as chromatography.

[0322] For example, technicians can effortlessly select from a variety of methods suitable for separating the target protein, including liquid chromatography. For instance, the solid phase used in chromatography, called the chromatographic medium or resin, is typically an engineered porous inert support functionalized with various chemical groups that determine the interaction with the molecules to be separated. Common separation modes applicable to rAAT described herein include, but are not limited to, those based on specific binding interactions (affinity chromatography), charge (ion exchange chromatography), size (size exclusion chromatography / gel filtration chromatography), hydrophobic surface area (hydrophobic interaction chromatography and reversed-phase chromatography), and / or multiple properties distinguished by size and / or aggregation (multi-mode or mixed-mode chromatography), which can be selected to provide glycoprotein formulations with one or more desired glycan properties.

[0323] For example, normal-phase liquid chromatography can be used to separate proteins, glycans, and / or glycoproteins based on polarity. Reversed-phase chromatography can be used, for example, for derivatized sugars. Anion-exchange columns can be used to purify proteins with sialylated, phosphorylated, and / or sulfated sugars, or modified proteins. Other methods include high-pH anion-exchange chromatography and size exclusion chromatography, which can be used in this invention for size-based separation.

[0324] Affinity-based methods can be used to selectively bind to certain protein and / or glycan structures. Matrices such as m-aminophenylboronic acid, immobilized lectins, and antibodies can bind to specific protein and / or glycan structures. The m-aminophenylboronic acid matrix can form a temporary covalent bond with any molecule containing a 1,2-cis-diol group (such as carbohydrates). This covalent bond can then be broken to elute the target protein. Lectins are a class of carbohydrate recognition proteins that exhibit affinity for a variety of monosaccharides. Lectins bind to carbohydrates specifically and reversibly. Major monosaccharides recognized by lectins include mannose / glucose, galactose / N-acetylgalactosamine, N-acetylglucosamine, fucose, and sialic acid (QProteome Glycoarray Handbook, Qiagen, September 2005, available at http: / / wolfson.huji.ac.il / purification / PDF / Lectins / QIAGEN_GlycoArrayHandbook.pdf) or similar monosaccharides.

[0325] According to the present invention, anion exchange chromatography (AEX) is an option for purifying rhAAT produced by Pichia pastoris. AEX is a method for separating substances based on their charge, using an ion exchange resin containing positively charged groups (such as diethylaminoethyl (DEAE)). In solution, the resin surface is coated with positively charged counterions (cations). The anion exchange resin binds to the negatively charged molecules and displaces the counterions. Anion exchange chromatography is commonly used to purify proteins, amino acids, sugars / carbohydrates, and other acidic substances that are negatively charged at higher pH levels. The tightness of the binding between the substance and the resin is based on the strength of the substance's negative charge.

[0326] In this implementation, rhAAT produced by Pichia pastoris can be purified by size exclusion chromatography (SEC). SEC is a chromatographic method in which molecules in solution are separated by their size, and in some cases by molecular weight. It is commonly used for macromolecules or macromolecular complexes, such as proteins and industrial polymers. Typically, when samples are transported through a column using an aqueous solution, this technique is called gel filtration chromatography, while when an organic solvent is used as the mobile phase, it is called gel permeation chromatography. The chromatographic column is packed with tiny porous microspheres, typically composed of dextran, agarose, or polyacrylamide polymers. The pore size of these microspheres is used to estimate the size of the macromolecules. SEC is a widely used polymer characterization method because it provides good molar mass distribution (Mw) results for polymers.

[0327] In this implementation, rhAAT produced by *Pichia pastoris* can be purified by affinity chromatography. Affinity chromatography typically employs antibodies or other binding reagents specific to the target protein rhAAT. Affinity chromatography is generally considered to be any separation method based on the specific binding interaction between an immobilized ligand and its binding partner. Examples include antibody / antigen, enzyme / substrate, and enzyme / inhibitor interactions. Affinity chromatography has the advantage of a specific binding interaction between the target analyte (typically dissolved in the mobile phase) and the binding partner or ligand (immobilized on the stationary phase). In a typical affinity chromatography experiment, the ligand is attached to a solid, insoluble matrix, typically a polymer (such as agarose or polyacrylamide), chemically modified to introduce reactive functional groups to which the ligand can react, thereby forming a stable covalent bond. Typically, the stationary phase is first packed into the column, and then the mobile phase is introduced into the column. Molecules bound to the ligand remain bound to the stationary phase. A wash buffer is then applied to remove non-target biomolecules while the target biomolecule remains bound. The target biomolecule can then be removed by applying a so-called elution buffer. Therefore, the target molecule is recovered in the eluent. Highly selective affinity chromatography resins for the efficient industrial purification of α-1 antitrypsin (AAT) are known and are available, for example, from Cytiva (Alpha-1 Antitrypsin Select). Alpha-1 Antitrypsin Select is a packed-bed affinity chromatography resin with high selectivity for AAT. It can be used to purify human AAT from plasma as well as from recombinant or transgenic sources.

[0328] The method for purifying rhAAT produced according to the present invention can be carried out by any suitable method in the art, depending on the cost and the required degree of purification.

[0329] Medical indications:

[0330] In one aspect of the invention, pharmaceutical compositions or combinations as described herein are provided for use in the treatment and / or prevention of nonviral lung diseases associated with inflammation and / or pathological immune responses. Preferred treatments involve treating nonviral lung diseases associated with inflammation and / or pathological immune responses, or related diseases that are those described herein.

[0331] Generally, the term "inflammation" is used in its accepted meaning in the field, referring to a local or systemic protective response resulting from tissue damage, infection, or destruction, which protects the body from damaging factors and harmed tissues. Typical characteristics of inflammation include the flocculentization of the microvascular system, leakage of blood components into the interstitial space, and migration of leukocytes into inflamed tissues, which can lead to a range of uncontrolled pain, fever, redness, swelling, and loss of function.

[0332] Inflammation can be classified as acute or chronic. Acute inflammation is the body's initial response to harmful stimuli, achieved through increased movement of plasma and white blood cells (especially granulocytes) from the blood into damaged tissue. A cascade of biochemical events amplifies and matures the inflammatory response, a process involving the local vascular system, the immune system, and various cells within the damaged tissue. Long-term inflammation, known as chronic inflammation, leads to a progressive shift in the cell types present at the site of inflammation, characterized by the simultaneous destruction and healing of tissues during the inflammatory process.

[0333] The terms “adverse inflammation” or “pathological inflammation” preferably refer to inflammation in the subject’s body that exceeds the physiologically beneficial level of inflammation and leads to damage to cells, tissues and / or organs at the site of inflammation.

[0334] The terms “adverse immune response” or “pathological immune response” preferably refer to alterations in the reactivity of the immune system in a subject that have detrimental effects on their health and may involve the stimulation and / or production of cytokines and the recruitment of immune cells. Adverse immune responses occur in, for example, autoimmune diseases, transplant rejection, allergies, or inflammatory diseases. Cytokines involved in “adverse inflammation” and / or “pathological immune responses” and contributing to further progression of inflammation in nonviral lung diseases are referred to as “inflammatory cytokines” and include, or include, the pro-inflammatory cytokines IL-1AB (also known as IL-1Aβ), IL-4, IL-5, IL-6, IL-8, CXCL8, IL-33, IFN-γ, TNF-α, and the anti-inflammatory cytokines IL-1 receptor antagonists (IL-1R antagonists, IL-1Ra, IL-1RA) and IL-10.

[0335] Examples of inflammatory lung diseases include, but are not limited to: lung injury, chronic obstructive pulmonary disease (COPD) (including chronic bronchitis, emphysema, bronchiectasis, and bronchiolitis), acute respiratory distress syndrome (ARDS), asthma, sarcoidosis, hypersensitivity pneumonitis and / or pulmonary fibrosis, and bronchiolitis obliterans syndrome (BOS) (such as BOS (chronic graft-versus-host disease) associated with lung transplantation and / or allogeneic hematopoietic stem cell transplantation).

[0336] Asthma, also known as bronchial asthma, is a chronic inflammatory disease of the respiratory tract characterized by bronchial inflammation and hyperresponsiveness, as well as variable airway obstruction. Due to persistent inflammation of the bronchial mucosa, bronchial wall remodeling can further occur, including smooth muscle hypertrophy, thickening of the subepithelial basement membrane, glandular hyperplasia, and subepithelial fibrosis. Asthma can be classified as allergic (exogenous) asthma and non-allergic (endogenous) asthma, but a mixed form is observed in most patients. Allergic asthma is triggered by external stimuli (sensitizing substances in the environment, i.e., allergens) and is often associated with an atopic genetic susceptibility. Immunoglobulin E (IgE) is formed, which interacts with external stimuli (such as specific allergens), leading to the release of messenger substances (such as histamine, leukotrienes, and bradykinin) from mast cells. These substances trigger airway constriction. In addition to this immediate type I reaction following exposure to the allergen, a delayed-type reaction, triggered by immunoglobulin G (IgG), may occur 6 to 12 hours later. Both reactions often occur simultaneously (double reaction). Non-allergic asthma can be triggered by other irritants, including: infections (usually respiratory infections); drug intolerance, also known as analgesic asthma (pseudo-allergic reactions to analgesics, usually nonsteroidal anti-inflammatory drugs, such as acetylsalicylic acid); drug side effects (e.g., beta-blockers and cholinesterase inhibitors); exposure to toxic or irritating substances (solvents, cold air, additives, etc.); specific physical activity (exercise-induced asthma); and reflux disease (acid reflux). Asthma can also be classified by the severity of symptoms and corresponding lung function (usually assessed by spirometry, which measures volume in one second (FEV1), peak expiratory flow (PEF), forced vital capacity (FVC), and intermediate or mean expiratory flow (MEF)). These categories include intermittent asthma, low-grade persistent asthma, moderate-grade persistent asthma, and severe persistent asthma. Asthma can also be classified by treatment control and treatment response as controlled asthma, partially controlled asthma, and uncontrolled asthma. To date, treatment for asthma has included the administration of beta-2 agonists (such as salbutamol, bambuterol, and indacaterol), glucocorticoids (such as beclomethasone, budesonide, and fluticasone), theophylline, leukotriene antagonists (such as montelukast), cromoglycine, nedocromil, and loxamethasone, usually administered by inhalation.

[0337] Bronchiolitis obliterans (BO) is a rare chronic lung disease characterized by inflammation and fibrotic thickening of the bronchioles, leading to narrowing of the lumen and restricting air circulation. BO often occurs secondary to lung infections or as a comorbidity of related conditions such as lung transplantation or allogeneic hematopoietic stem cell transplantation (chronic graft-versus-host disease). BO secondary to lung transplantation or allogeneic hematopoietic stem cell transplantation (chronic graft-versus-host disease) is termed bronchiolitis obliterans syndrome (BOS). In addition, several autoimmune diseases can be associated with BO, such as polymyositis, dermatomyositis, scleroderma, systemic lupus erythematosus (SLE), chronic inflammatory bowel disease, paraneoplastic pemphigus, non-Hodgkin's lymphoma, paraneoplastic autoimmune multiple organ syndrome (PAMS), CML, Castleman's disease, thymoma, and Waldenström. Post-infectious bronchitis (PiBO), also known as Swyer-James syndrome, can be secondary to bacterial (such as Mycoplasma pneumoniae) or viral (such as adenovirus, RSV, influenza virus, or parainfluenza virus) infections. Obliterative bronchiolitis can cause shortness of breath and a dry cough. These symptoms often progressively worsen over weeks to months. Pulmonary function tests show obstructive ventilatory impairment: decreased force-evident volume in one second (FEV1), increased residual volume (RV) and RV / TLC, while carbon monoxide diffusing capacity (DLCO) is normal. To date, treatment for BO typically includes the administration of azithromycin, cyclophosphamide, cyclosporine, azathioprine, anti-CD3 antibodies, tacrolimus, mycophenolate mofetil, or statins.

[0338] Acute respiratory distress syndrome (ARDS) is a life-threatening condition. It is characterized by tissue edema due to impaired pulmonary capillary permeability and an inflammatory response to neutrophil elastase, leading to severe disturbances in gas exchange. The severity of the disease depends on the degree of excessive inflammation. ARDS is often accompanied by multiple organ failure in the context of systemic inflammatory response syndrome (SIRS) and has a high mortality rate. The most common cause of ARDS is pneumonia. Other pulmonary causes include: pulmonary contusion, aspiration of gastric contents or water, inhalation injury (smoke inhalation, burns), high-concentration oxygen ventilation (so-called mechanical lung), fat embolism, amniotic fluid embolism, and e-cigarette-induced lung injury (EVALI, possibly due to the addition of vitamin E to the fluid). Systemic causes of ARDS include SIRS, sepsis, shock, disseminated intravascular coagulation, multiple injuries, preeclampsia, eclampsia, burns, acute pancreatitis, drugs and toxins (e.g., salicylates, tricyclic antidepressants, bleomycin, organophosphates, paraquat, anesthetics), metabolic disorders (e.g., uremia, liver failure, diabetic ketoacidosis), lung transplantation, traumatic brain injury, massive transfusion (TRALI), and radiation.

[0339] In this context, the term "nonviral lung disease associated with inflammation and / or a pathological immune response" refers to a disease that can be triggered by a variety of factors as disclosed above, subsequently leading to a persistent, often self-reinforcing inflammatory and pathological immune response independent of the initial cause. For example, the inflammation occurring in bronchiolitis obliterans (BO) leads to fibrosis of the bronchial walls, narrowing of the lumen, and restriction of air circulation. This inflammation is present in both post-infectious BO (PiBO) and bronchiolitis obliterans syndrome (BOS), and is therefore unrelated to the initial cause of the disease. In this context, the use of rhAAT in the treatment and / or prevention of such nonviral lung diseases characterized by persistent, often self-reinforcing inflammatory and pathological immune responses means that the anti-inflammatory function of rhAAT acts on the inflammatory process rather than the initial cause of the disease (such as viral infection), for example, by preventing viral entry into cells.

[0340] Any or more of the aforementioned nonviral lung diseases associated with inflammation and / or pathological immune responses may be susceptible to the inventive treatments described herein, and the treatment of each disease itself represents an embodiment of the invention.

[0341] The term "serum half-life" refers to the time elapsed after intravenous administration, from the peak concentration of a drug in plasma until it drops to half of that value. The term "pulmonary half-life" refers to the time elapsed after administration to the lungs, for example by inhalation, from the peak concentration of a drug in lung tissue (e.g., bronchial or alveolar tissue) until it drops to half of that value. The term "bronchial half-life" refers to the time elapsed after administration to the lungs, for example by inhalation, from the peak concentration of a drug in bronchial tissue until it drops to half of that value. The term "alveolar half-life" refers to the time elapsed after administration to the lungs, for example by inhalation, from the peak concentration of a drug in alveolar tissue until it drops to half of that value.

[0342] As used herein, a “patient” or “subject” may be a vertebrate. In the context of this invention, the term “subject” includes both humans and animals, particularly mammals, as well as other organisms.

[0343] As used herein, “subjects in need” refers to subjects who have an illness (specifically, coronavirus disease) or are at risk of developing it. Subjects in need may be those who are hospitalized due to symptoms of coronavirus disease, those who have symptoms of coronavirus disease and are in an outpatient or home setting, or those who have asymptomatic coronavirus disease and are in an outpatient or home setting.

[0344] In this invention, "treatment" or "therapy" generally means achieving the desired pharmacological and / or physiological effects. In one embodiment, "treatment" or "therapy" means slowing, stopping, or reversing the progression of a disease. The effect may be preventative, intended to completely or partially prevent the disease and / or symptoms, for example by reducing the risk of the subject developing the disease or experiencing symptoms; or the effect may be therapeutic, intended to partially or completely cure the disease and / or eliminate the adverse effects of the disease.

[0345] In this invention, "treatment" includes any treatment of a disease or symptom in mammals, particularly humans, such as the following treatments (a) to (c): (a) preventing the onset of a patient's disease, symptom, or symptoms; (b) suppressing the symptoms of the symptom, i.e., preventing the progression of the symptoms; (c) alleviating the symptoms of the symptom, i.e., inducing the resolution of the disease or symptoms.

[0346] Composition and application:

[0347] The AAT protein or composition containing the protein described herein may contain different types of carriers, depending on whether they are applied in solid, liquid or aerosol form and whether sterility is required for routes of administration such as injection.

[0348] The active agent AAT can be administered via: intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intratumoral, intramuscular, intraperitoneal, subcutaneous, subconjunctival, intracystic, transmucosal, intraperitoneal, intraumbilical, intraocular, oral, topical, local, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, direct, via catheter, via irrigation, as a cream, as a lipid composition (e.g., liposome), via sponge or by other methods, or any combination of the above as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, incorporated herein by reference).

[0349] This invention includes treating a patient by introducing a therapeutically effective amount of a peptide into a subject or the subject's bloodstream. As used herein, "introducing" a peptide into the subject's bloodstream includes, but is not limited to, introducing such a peptide into the subject's vein or artery by injection. Such administration may also be performed, for example, once, multiple times, and / or over one or more extended periods. A single injection is preferred, but in some cases, repeated injections may be necessary over time (e.g., quarterly, semi-annually, or annually). Such administration is also preferably performed using a mixture of the peptide and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01M-0.1M, and preferably 0.05M, phosphate-buffered saline or 0.8% saline.

[0350] As used herein, the term “therapeutic effective amount” may be interchanged with “therapeutic effective dose” or “sufficient / effective amount or dose” and refers to a dose that produces the desired therapeutic effect. Specifically, an effective dose generally refers to an amount of the compositions disclosed herein sufficient to achieve: inducing immunity, preventing and / or improving coronavirus infection, or alleviating at least one symptom associated with coronavirus infection, and / or enhancing the efficacy of another therapeutic composition. An effective dose may refer to an amount of a composition sufficient to delay or minimize the onset of infection. An effective dose may refer to an amount of a composition sufficient to prevent viral infection or reduce the risk of viral infection. An effective dose may also refer to an amount of a composition that provides a therapeutic benefit in the treatment or management of an infection. Furthermore, an effective dose may be an amount that provides a therapeutic benefit in the treatment or management of a viral infection, either alone or in combination with other therapies. An effective dose may also be an amount sufficient to enhance the autoimmune response of a subject (particularly a human) against subsequent exposure to a coronavirus. The exact effective dose depends on the purpose of treatment and may be determined by those skilled in the art using known techniques.

[0351] As used herein, "pharmaceutically acceptable carriers" include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers. The use of such media and agents is well known in the art for pharmaceutically active substances. Their use in therapeutic compositions is considered unless any conventional media or agent is incompatible with the active ingredient. Complementary active ingredients may also be incorporated into the composition.

[0352] Additionally, pharmaceutically acceptable carriers of this type can be aqueous or non-aqueous solutions, suspensions, and emulsions, with aqueous solutions being the most preferred. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, and suspensions, including saline and buffer media. Parenteral carriers include sodium chloride solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, and fixed oil. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements, such as Ringer's dextran, and those based on Ringer's dextran. Commonly used fluids for intravenous administration can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th edition, p. 808, Lippincott, Williams, S.-Wilkins (2000). Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0353] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic reactions or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well known in the art. Typically, such compositions are prepared as injectables, as liquid solutions or suspensions; they may also be prepared in solid forms suitable for dissolving or suspending in a liquid prior to injection. Formulations may also be emulsions.

[0354] Examples of parenteral dosage forms include aqueous solutions of the active agent in isotonic saline, 5% glucose, or other known pharmaceutically acceptable liquid carriers (such as liquid alcohols, glycols, esters, and amides). Parenteral dosage forms according to the invention may be in the form of a reconfigurable lyophilized composition comprising a dose of a composition containing particulate proteins. In one aspect of this embodiment, any of a variety of long- or sustained-release dosage forms known in the art may be administered, such as biodegradable carbohydrate matrices as described in U.S. Patent Nos. 4,713,249, 5,266,333, and 5,417,982, the disclosures of which are incorporated herein by reference.

[0355] In one illustrative embodiment, a pharmaceutical formulation typically used with AAT for parenteral administration is described, comprising: a) a pharmaceutically active amount of particulate protein; b) a pharmaceutically acceptable pH buffer to provide a pH in the range of about pH 4.5 to about pH 9; c) an ionic strength modifier in a concentration range of about 0 mmol to about 250 mmol; and d) a water-soluble viscosity modifier in a concentration range of about 0.5% to about 7% of the total formulation weight, or any combination of a), b), c) and d).

[0356] In the various illustrative embodiments, the pH buffers used in the compositions and methods described herein are those reagents known to those skilled in the art, and include, for example, acetate, borates, carbonates, citrates and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, potassium dihydrogen phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethylbarbituric acid and proteins, and various biological buffers such as TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, Cacodylate, MES.

[0357] In another illustrative embodiment, the ionic strength modifier includes those reagents known in the art, such as glycerol, propylene glycol, mannitol, glucose, dextran, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

[0358] The compositions of the present invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intra-arterial), intraperitoneal, or intramuscular administration. The term "parenteral administration" refers to a mode of administration other than through the digestive tract, such as by injection or infusion, and also refers to subcutaneous, intramuscular, or intravenous injection; intraperitoneal injection is also covered herein. Intramuscular administration includes intravenous or intra-arterial administration. Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may include one or more of the following suitable excipients: sterile diluents, such as water for injection, saline solutions (preferably physiological saline), Ringer's solution, or isotonic sodium chloride; fixed oils that can be used as solvents or suspension media, such as synthetic monoglycerides or diglycerides; polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for regulating tonicity, such as sodium chloride or dextran. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile. In addition, if necessary, the pharmaceutical composition to be applied may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, pH buffers, stabilizers, dissolution enhancers and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrin.

[0359] The daily unit dose of compositions containing AAT protein can vary significantly depending on the patient's condition, the disease state being treated, the route of administration and tissue distribution of AAT, and the possibility of co-administration with other therapeutic treatments. The effective dose of AAT to be administered to a patient is based on body surface area, patient weight, physician assessment of the patient's condition, etc.

[0360] In one illustrative embodiment, the effective dose range of AAT may be from about 1 mg / kg of patient body weight to about 500 mg / kg of patient body weight, more preferably from about 10 mg / kg of patient body weight to about 300 mg / kg of patient body weight, and even more preferably from about 50 mg / kg of patient body weight to about 200 mg / kg of patient body weight, such as about 100 mg / kg of body weight or about 180 mg / kg of body weight.

[0361] According to the invention, the composition is preferably administered by inhalation onto the mucosal surface of the subject's respiratory tract. "Inhalation administration" refers to both oral and nasal inhalation. "Oral inhalation" involves medication administered via oral inhalation and must be atomized into smaller droplets than medication administered via the nasal route, allowing the medication to pass through the airways (trachea) and enter the lungs. The depth to which they penetrate the lungs depends on the droplet size. Smaller droplets penetrate deeper, increasing the amount of medication absorbed. Within the lungs, they are absorbed into the bloodstream.

[0362] Drugs administered via this route can be delivered in dry powder form through a metering container (inhaler).

[0363] The medication can also be atomized from a solution containing the medication into a mist or appropriately sized droplets or aerosols for inhalation via a nebulizer (such as the Pari Boy Pro).

[0364] Inhaled medications are rapidly absorbed and act both locally and systemically. The inhaler device delivers the correct dosage. Generally, when inhaled orally, only 20%–50% of the pulmonary delivery dose in powder form will deposit in the lungs. The remaining 50%–70% of the undeposited aerosolized particles are cleared from the lungs upon exhalation. In embodiments, inhaled powder particles >8 μm in diameter structurally tend to deposit in the central and conduction airways via inertial impaction. In embodiments, inhaled powder particles with diameters between 3 μm and 8 μm tend to deposit primarily in the transitional zones of the lungs via sedimentation. In embodiments, inhaled powder particles <3 μm in diameter structurally tend to deposit primarily in the respiratory zones of the peripheral lungs via diffusion.

[0365] The term "nasal inhalation" refers to a pharmaceutical product or formulation, including delivery devices (where applicable), whose intended deposition site is the respiratory tract or the nasal or pharyngeal region. Nasal inhalation does not include topical nasal sprays or rinsings that deposit primarily in the nasal cavity. Medications may also be inhaled via a nebulizer, such as the Pari Boy Pro, by atomizing a solution containing the medication into a mist or appropriately sized droplets or aerosol.

[0366] In the context of this invention, simultaneous and / or subsequent administration of the pharmaceutical composition via different routes of administration is also contemplated. In embodiments, the route of administration of the composition includes inhalation. In a preferred embodiment, parenteral administration of the pharmaceutical composition according to the invention or a pharmaceutical composition containing human plasma-derived AAT protein is performed simultaneously or subsequently. In another preferred embodiment, nasal and / or oral administration of the pharmaceutical composition according to the invention or a pharmaceutical composition containing human plasma-derived AAT protein is performed simultaneously or subsequently.

[0367] As used in this article, the "respiratory tract" is generally considered to include the nose, pharynx, larynx, trachea, and lungs, which have their different compartments. The respiratory tract is involved in the respiratory process in mammals and is part of the respiratory system, and is lined with respiratory mucosa or respiratory epithelium.

[0368] As used in this article, the "mucosal surface" is characterized by the presence of an overlying mucus, such as saliva, tears, nasal mucus, gastric mucus, cervical mucus, and bronchial mucus. The functions of this mucus include supplying and delivering a range of immunomodulatory and healing-promoting substances, including growth factors, antimicrobial proteins, and immunoglobulins.

[0369] As used in this article, “drug excipient” is essentially any substance other than the active pharmaceutical ingredient in the composition.

[0370] In the formulation of pharmaceutical compositions, excipients are usually added together with the active pharmaceutical ingredient. The purpose of this is to: 1) protect, support or enhance the stability of the formulation; 2) increase the volume of the formulation in the case of potent drugs, which helps to formulate accurate dosage forms; 3) improve patient acceptability; 4) help improve the bioavailability of the active pharmaceutical ingredient; and 5) enhance the overall safety and efficacy of the formulation during storage and use.

[0371] Excipients used in the formulation of pharmaceutical compositions are subdivided into various functional categories based on their intended role in the resulting formulation. Some excipients may have different functional roles in different formulation types; furthermore, depending on those different functional roles, excipients may have different grades, types, and sources. Possible types of excipients commonly used in the formulation of pharmaceutical suspensions include: solvents / carriers, cosolvents, buffers, preservatives, antioxidants, wetting agents / surfactants, defoamers, flocculants, suspending agents / viscosity modifiers, flavoring agents, sweeteners, colorants, humectants, and chelating agents.

[0372] As used herein, “cryopreservation” is the process of preserving organelles, cells, tissues, extracellular matrix, organs, or any other biological construct susceptible to damage by unregulated chemical kinetics by freezing to extremely low temperatures (typically -20°C to -80°C using solid carbon dioxide, or -196°C in some cases using liquid nitrogen). At sufficiently low temperatures, any enzymatic or chemical activity that could damage the biological material involved is effectively stopped. Cryopreservation methods aim to achieve low temperatures without causing additional damage due to the formation of ice crystals during freezing.

[0373] As used herein, an "aerosol" is a suspension of liquid and solid particles, such as those generated by aerosol generators, like small volume nebulizers (SVNs), pressure-controlled dose-dispensing inhalers (pMDIs), or dry powder inhalers (DPIs). Aerosol deposition is the process by which aerosol particles are deposited on an absorbent surface.

[0374] Clinically used aerosol devices produce heterodisperse (also known as polydisperse) particle sizes, meaning that a mixture of multiple particle sizes exists within the aerosol. Specific particle sizes and / or particle size ranges are provided in the above description of the invention. Monodisperse aerosols consisting of a single particle size are relatively rare. Polydisperse aerosols can be defined by the median mass diameter (MMD). This metric determines the particle size (in μm) into which 50% of the mass is larger or smaller than the particle size. This parameter represents the particle size that equally divides the mass or amount of drug within the particle size distribution. Due to the way particle size is measured, this parameter is typically given as the median mass aerodynamic diameter (MMAD). The higher the MMAD, the more particles with larger diameters there are.

[0375] Dry powder formulations offer advantages, including greater stability than liquid formulations and the possibility of not requiring preservatives. Powders tend to adhere to the moist surface of the nasal mucosa before dissolving and being cleared. Using bioadhesive excipients or agents that slow ciliary movement can reduce clearance and improve absorption. Multiple factors, such as humidity sensitivity, solubility, particle size, particle shape, and flow properties, will affect deposition and absorption.

[0376] According to some embodiments, the drug composition administered via inhalation is applied once daily for at least two days. According to some embodiments, the drug composition administered via inhalation is applied once daily for at least three days. According to some embodiments, the drug composition administered via inhalation is applied once daily for at least four days. According to some embodiments, the drug composition administered via inhalation is applied once daily for at least five days. According to some embodiments, the drug composition administered via inhalation is applied once daily for at least six days. According to some embodiments, the drug composition administered via inhalation is applied once daily for at least one week. According to some embodiments, the drug composition administered via inhalation is applied once daily for at least eight days. According to some embodiments, the drug composition administered via inhalation is applied once daily for at least nine days. According to some embodiments, the drug composition administered via inhalation is applied once daily for at least ten days. According to some embodiments, the drug composition administered via inhalation is applied once daily for ten days.

[0377] Examples of dry powder nasal sprays:

[0378] SBNL Pharma (www.snbl.com) recently reported Phase 1 study data on enhanced absorption using zolmitriptan powder cyclodextrin formulations (μco™ system), which has been described in vitro. Zolmitriptan is rapidly absorbed and has higher relative bioavailability than commercially available tablets and nasal sprays. The company offers a capsule-based single-dose powder device (Fit-lizer) suitable for administering powder formulations according to the invention. Upon insertion of the chamber, the top and bottom of the capsule are cut off by sharp blades. Manual compression of the plastic chamber causes compressed air to pass through a one-way valve and the capsule during actuation, ejecting the powder.

[0379] Bespak's (www.bespak.com) Unidose-DP™ works on a similar principle to the Fit-lizer device. The inflatable compartment is compressed until a needle punctures the membrane, releasing the pressure and ejecting a powder atom. Delivery of the powder formulation of a model antibody (human IgG) has been tested in a nasal cast model based on human MRI images. Approximately 95% of the dose was delivered to the nasal cavity, but most of the deposition did not extend beyond the nasal vestibule; only about 30% deposited in deeper compartments of the nasal cavity.

[0380] Examples of dry powder inhalers:

[0381] Astra Zeneca has launched the Turbuhaler multi-dose inhaler (Rhinocort), a device modified for nasal inhalation. Budesonide powder delivered by www.az.com. In some markets, this powder formulation is marketed as an alternative to liquid sprays for allergic rhinitis and nasal polyps.

[0382] Aptar Group (www.aptar.com) offers a simple blister-based powder inhaler. Before use, the blister is punctured, and the nasal inhaler tip is placed in one nostril. The subject closes the opposite nostril with their finger and inhales the powder into the nose. Apomorphine powder formulations using this blister-based powder inhaler (BiDose™ / Prohaler™) from Pfeiffer / Aptar are being clinically developed for the treatment of Parkinson's disease; the original developer, Britannia (a UK company), was recently acquired by Stada Pharmaceutical (www.stada.de). Such delivery devices are considered for the administration of compositions as described herein.

[0383] Examples of inhalers using liquid formulations:

[0384] Inhalation solutions and suspensions are typically aqueous formulations containing a therapeutically active ingredient and may also contain additional excipients. Aqueous oral inhalation solutions and suspensions must be sterile. Inhalation solutions and suspensions are intended for oral delivery to the lungs to produce local and / or systemic effects and must be used with a designated nebulizer, such as the PariBoy Pro, which generates flexible, tunable droplets with a variable droplet size profile for the treatment of severe chronic respiratory diseases. Unit-dose packaging is recommended for these medications to prevent microbial contamination during use. The container closure system for these medications consists of a container and a closure and may include protective packaging (such as a foil outer packaging).

[0385] For example, amikacin liposome inhalation suspension (ALIS); Amikacin (ALIS) is a liposomal formulation of the aminoglycoside antibacterial drug. ALIS formulations are administered via inhalation after nebulization and are designed to promote targeted and local drug delivery to the lungs while minimizing systemic exposure. Such delivery devices are considered for use in administering compositions as described herein.

[0386] Examples of nasal sprays using liquid formulations:

[0387] Nasal spray dispensers are typically atmospheric pressure dispensers that deliver a spray containing a metered dose of the active ingredient. The dose can be metered using a spray pump or pre-metered during manufacturing. Nasal spray units can be designed for unit dosage or to dispense metered sprays of formulations containing the drug substance up to hundreds of times. Nasal sprays are applied to the nasal cavity to achieve local and / or systemic effects.

[0388] Liquid nasal preparations are primarily aqueous solutions, but suspensions and emulsions can also be delivered. Liquid preparations are considered convenient, especially for topical indications, where the moisturizing effect counteracts the dryness and crusting often associated with chronic nasal conditions.

[0389] For example, dispensers readily available from companies such as Nemera (La Verpillière, France) or Aptar Pharma (Illinois, USA) are preferred. Nemera, for instance, offers... The nasal spray dispenser features a high-performance pump for excellent dosage consistency and initial retention, an anti-clogging actuator, no metal contact with the formulation, and a hygienic snap-on cap to prevent accidental contact. As another example, Aptar Pharma's nasal pump technology eliminates the need for pharmaceutical companies to add preservatives to nasal spray formulations. The Advanced Preservative-Free (APF) system uses tip-sealing and filtration technology to prevent formulation contamination. A spring-loaded tip-sealing mechanism with a filter membrane is used in the airway.

[0390] Metering and spray generation (e.g., orifice, nozzle, jet) pump mechanisms and components are used for the reproducible delivery of drug formulations, and these mechanisms and components can consist of multiple parts with different designs that are precisely controlled in size and composition. Dispersing the formulation into a spray requires energy. This is typically achieved by driving the formulation through a nasal actuator and its orifice. The formulation and container closure system (container, closure, pump, and any protective packaging) together constitute the drug product. The design of the container closure system affects the drug's administration performance.

[0391] In some embodiments, the multiple-use dispenser employs a membrane (preferably a silicone membrane) to prevent bacteria or viruses from entering the reservoir or pump device. In some embodiments, the multiple-use dispenser employs a metal-free flow path to prevent oxidation of the formulation. In some embodiments, the multiple-use dispenser employs a spring-loaded tip seal mechanism to prevent microorganisms from entering the device between two spray events.

[0392] In one embodiment, the dispenser is configured for multiple individual spray events of 5 μL to 1000 μL, preferably 5 μL to 500 μL, more preferably 10 μL to 300 μL, and even more preferably 20 μL to 200 μL.

[0393] In one embodiment, the dispenser contains a total volume of composition of 0.1 mL to 500 mL, preferably 1 mL to 100 mL, more preferably 2 mL to 50 mL, such as about 5 mL, 10 mL or 15 mL.

[0394] Examples of throat sprays used in liquid formulations:

[0395] Bona (Shenzhen, China) is a supplier of medical packaging and accessories. The company offers a range of long-necked nebulizers that provide excellent dispensing and administration, specifically designed for throat sprays and other treatments requiring targeted throat coverage. The dispenser arm swings 360 degrees for easy consumer use. The nebulizers are suitable for throat medication treatments as well as other topical treatments, such as protecting the throat from viral infections. The dispensers are preferably made of pharmaceutical-grade PP and PE, and are compatible with bottles of any size. Currently, the nebulizers are available in various mainstream sizes (18 / 410, 18 / 415, 20 / 410, 24 / 410) and can be set to dispense 220 microliters to 50 microliters.

[0396] Simultaneous and sequential application

[0397] Simultaneous administration of one or more active agents should be understood as administration of one or more agents at the same time. For example, in one embodiment, AAT may be administered together with other active agents that can be used to treat nonviral lung diseases associated with inflammation and / or pathological immune responses. In other embodiments, AAT may be administered by inhalation via other routes of administration, including oral, nasal, parenteral (including intravascular, intraperitoneal, or intramuscular) administration. In some embodiments, at least one route of administration of the recombinant AAT according to the invention is required, preferably inhalation. Other routes of administration may alternatively be compositions comprising human plasma-derived AAT or any other recombinantly produced AAT. In embodiments, administration in a simultaneous manner may also be sequential.

[0398] Sequential administration of one or more active agents should be understood as administering the therapeutic agents in a sequential manner. In one embodiment, the therapeutic agents are administered at different times. In other embodiments, it is done by administering two or more therapeutic agents, wherein at least two of the therapeutic agents are administered sequentially, in a substantially simultaneous manner. Substantially simultaneous administration can be achieved, for example, by administering a single dose of each therapeutic agent in a fixed proportion to the subject, or by administering multiple single doses of each of the therapeutic agents. One or more therapeutic agents as described herein comprise at least a pharmaceutical composition comprising recombinant AAT protein according to the invention. In one embodiment, the therapeutic agent comprises an additional pharmaceutical composition comprising human plasma-derived AAT and / or other recombinantly produced AAT protein. In a preferred embodiment, the therapeutic agent further comprises a pharmaceutical composition comprising xanthohumol.

[0399] As used herein, the term "simultaneously" means the administration of one or more agents at the same time. For example, in some embodiments, the administration of AAT in combination with other active agents that can be used to treat viral infections or medical conditions related to lung inflammation is simultaneous. Simultaneous administration includes concomitant administration, i.e., administration within the same time period. In some embodiments, one or more agents are administered simultaneously within the same hour or on the same day. The sequential or substantially simultaneous administration of the therapeutic agents can be achieved by any suitable route, including but not limited to oral, intravenous, subcutaneous, intramuscular, direct absorption through mucosal tissues (e.g., nose, mouth, vagina, and rectum), and ocular routes (e.g., intravitreal, intraocular, etc.). Therapeutic agents can be administered via the same route or via different routes. For example, one component of a particular combination can be administered by inhalation, while other components of the combination can be administered intravenously. Components can be administered in any therapeutically effective sequence.

[0400] The decision to administer AAT concurrently or sequentially may be made based on the severity of the disease. For example, for patients with severe symptoms of BO, asthma, or ARDS who are receiving AAT inhalation, it may be decided to administer AAT protein concurrently and / or sequentially intravenously.

[0401] Culture medium and treatment:

[0402] The production methods described herein may include determining and / or selecting culture medium components or culture conditions to result in the production of one or more glycan properties of the desired rhAAT. In embodiments, the determined culture parameters include culture medium components, pH, feeding conditions, osmotic pressure, carbon dioxide level, agitation rate, temperature, cell density, inoculation density, timing, and bubbling rate.

[0403] Variations in production parameters, such as the agitation rate of cell cultures, the temperature of cultured cells, the components in the culture medium, the start and stop times of culture, and the timing of nutrient supply, can be appropriately selected. Therefore, the methods described herein may include one or more of the following: increasing or decreasing the agitation rate of cells, increasing or decreasing the temperature of cultured cells, adding or removing culture medium components, and changing the start and / or stop times of culture. As used herein, sequential selection of production parameters or combinations thereof means selecting a first parameter (or combination), and subsequently selecting a second parameter (or combination), for example, based on constraints imposed by the selection of the first production parameter.

[0404] The methods described herein may include determining and / or selecting culture medium components and / or their concentrations that are positively correlated with desired glycan properties. Depending on the culture conditions, culture medium components may be added or applied during glycoprotein production or when the culture medium changes. Culture medium components include components added directly to the culture and components that are byproducts of cell culture.

[0405] Culture medium components include, for example, buffers, amino acid content, vitamin content, salt content, mineral content, serum content, carbon source content, lipid content, nucleic acid content, hormone content, trace element content, ammonia content, cofactor content, indicator content, small molecule content, hydrolysis product content, and enzyme regulator content.

[0406] Various culture medium components can be selected, including amino acids, vitamins, carbon sources (natural and non-natural), salts, sugars, serum, plant-derived hydrolysates, sodium pyruvate, surfactants, ammonia, lipids, hormones or growth factors, buffers, non-natural amino acids, sugar precursors, indicators, nucleosides or nucleotides, butyrates or organic matter, DMSO, animal-derived products, gene inducers, non-natural sugars, intracellular pH regulators, betaine or osmotic protectants, trace elements, minerals, non-natural amino acids, and non-natural vitamins. Exemplary amino acids that may be included in or removed from the culture medium include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, proline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0407] Examples of vitamins that may be present in or removed from a culture medium include vitamin A, retinoids, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B0 (pyridoxine), vitamin B131 (biotin), vitamin B9 (folic acid), vitamin B12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D, vitamin E, and vitamin K.

[0408] Minerals that may be present in or removed from the culture medium include bismuth, boron, calcium, chlorine, chromium, cobalt, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, rubidium, selenium, silicon, sodium, strontium, sulfur, tellurium, titanium, tungsten, vanadium, and zinc. Exemplary salts and minerals include: CaCl2 (anhydrous), CuSO4·5H2O, and Fe(NO3). . 9H2O, ICI, KNO3, KH2PO4, MgSO4 (anhydrous), NaCl, NaH2PO4H2O, NaHCO3, Na2SE3 (anhydrous), ZnSO4.7H2O; linoleic acid, lipoic acid, D-glucose, hypoxanthine 2Na, phenol red, putrescine 2HCl, sodium pyruvate, thymidine, pyruvic acid, sodium succinate, succinic acid, succinic acid, glutathione (reduced), para-aminobenzoic acid (PAHA), methyl linoleate, bacterial peptone G, adenosine, cytidine, guanosine, 2'-deoxyadenosine HCl, 2'-deoxycytidine HCl, 2'-deoxyguanosine and uridine.

[0409] In some implementations, ammonia content can be selected as a production parameter to produce one or more desired glycan properties. For example, ammonia can be present in the culture medium in the range of 0.001 mM to 50 mM. Ammonia can be added directly to the culture and / or produced as a byproduct of glutamine or glucosamine. Another production parameter is butyrate content. The presence of butyrate in the culture medium can lead to increased galactose levels in the resulting glycoprotein formulation. Butyrate provides increased sialic acid content in the resulting glycoprotein formulation.

[0410] In some implementations, components such as enzymes, sugars, and / or sugar precursors may be added to the culture medium or fed to cells in batches to influence sugar synthesis. For example, enzymes and substrates (such as sugar precursors) may be added to the culture medium or fed to cells in batches to produce one or more desired glycan properties. These methods may utilize monosaccharide substrates that are absorbed by cells, converted in vivo into “activated” monosaccharide substrates, and incorporated by the cells into expressed proteins. This method is applicable to any cell that can be manipulated to produce the desired glycoprotein. Cells may use, for example, endogenous biochemical treatment pathways, or may be genetically engineered, to convert or process exogenously added monosaccharides into an activated form for use as a substrate for in vivo or in vitro coupling with target glycoproteins.

[0411] Monosaccharides added to polysaccharide chains can be incorporated in an activated form. Activated monosaccharides that can be added include UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-xylose, GDP-mannose, GDP-fucose, CMP-N-acetylneuraminic acid, and CMP-N-acetylhydroxyacetylneuraminic acid. Other monosaccharide precursors that can be added to the culture medium or fed to cells in batches include: N-acetylglucosamine, glucosamine, glucose, galactose, N-acetylgalactosamine, fructose, fucose, glucose-6-phosphate, mannose-6-phosphate, mannose-1-phosphate, fructose-6-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine, N-acetylneuraminic acid-6-phosphate, fucose-1-phosphate, ATP, GTP, GDP, GMP, CTP, CDP, CMP, UTP, UDP, UMP, uridine, adenosine, guanosine, cytidine, lactose, maltose, sucrose, fructose 1,6-bisphosphate, phosphoenolpyruvate, 2-oxaloacetic acid, and pyruvate.

[0412] Some aspects include the presence of glucosamine in the culture medium. Glucosamine can be added to the culture medium or fed to cells in batches, or appropriate enzymes and / or substrates can be added to the culture medium or fed to cells in batches to produce glucosamine. In some embodiments, cells (e.g., Pichia pastoris) can be cultured in the presence of glucosamine when elevated levels of high-mannose or heteropolysaccharide structures are desired. Glucosamine can be present, for example, at a concentration of about 0.001 mM to 40 mM. The method may also include adding uridine to the culture medium or feeding it to cells in batches, for example, to reduce the level of high-mannose structures associated with proteins produced by the cells. Adding cytidine, UTP, OMP, and / or aspartate to the culture medium or feeding it to cells in batches can also result in the production of uridine during culture. Preferably, uridine is present at a concentration of about 0.001 mM to 10 mM.

[0413] Other aspects include selecting one or more culture medium components that do not affect one or more glycosylation characteristics. For example, the presence of glucosamine and uridine in the culture does not significantly alter the galactosylation, fucosylation, high-mannose production, heterozygous production, or sialylation of glycoproteins produced by cells cultured in this combination (e.g., Pichia pastoris). When the presence of mannose is a selected production parameter, mannose can be added to the culture medium, fed to cells in batches, or produced by cells exposed to a suitable substrate (such as fructose or mannan). Preferably, mannose is present at a concentration of about 0.001 mM to 50 mM.

[0414] Fermentation parameters and bioreactor:

[0415] The methods described herein may include selecting culture conditions related to desired one or more glycan properties. These conditions may include temperature, pH, osmotic pressure, shear force or agitation rate, oxidation, bubbling rate, growth vessel, tangential flow, DO, CO2, nitrogen, feed batching, redox potential, cell density, and feeding strategy.

[0416] Examples of physicochemical parameters can be selected from temperature, pH, osmotic pressure, shear force (or agitation rate), oxidation, bubbling rate, growth vessel, tangential flow, batching, DO, CO2, nitrogen, fed-batch, redox potential, cell density, perfusion culture, and feeding strategy.

[0417] In other embodiments, the carbon dioxide level can be selected to produce one or more desired polysaccharide properties. The CO2 level can be, for example, about 5%, 6%, 7%, 8%, 9%, 1.0%, 11%, 13%, 15%, 17%, 20%, 23%, and 25% (and ranges therebetween).

[0418] A wide variety of culture flasks, tanks, reactors, and controllers allow for the establishment and scale-up of cell culture systems. The choice of system can be based at least in part on its relevance to one or more desired glycan properties. Cells can be grown, for example, as batch, fed-batch, perfusion, or continuous cultures, depending on their relevance to specific glycan properties.

[0419] Various culture media or growth media can be used. For high-cell-density fermentation of Pichia pastoris, the most commonly used medium is basal salt medium (BSM), as mentioned in the Pichia fermentation process guidelines published by Invitrogen (USA). Non-limiting examples of BSM medium components are described in the examples below.

[0420] In other embodiments, YPD medium, which is available in liquid (broth) form, can be used for the growth and propagation of yeast cultures. It primarily contains bacteriological peptone, yeast extract, and glucose. This medium is non-selective for Candida, Pichia pastoris, Saccharomyces, and Zygosaccharomyces.

[0421] In other embodiments, a yeast nitrogen-based (YNB containing amino acids) medium can be used, which is a commonly used growth medium for culturing yeast. This nutrient-rich microbial medium contains nitrogen, vitamins, trace elements, and salts. It is suitable for use in classifying yeast based on amino acid and carbon source requirements.

[0422] Batch culture typically involves placing cells to be cultured in a fixed volume of culture medium and allowing the cells to grow. The cell number usually increases exponentially until it reaches a maximum, after which growth stops and the cells die. Batch culture is characterized by: it is carried out at a fixed volume (because no substances are added after the cells are placed in the medium), for a fixed duration (depending on the length of time the cells survive), and for a single harvest.

[0423] Fed-batch culture is a variation of batch culture and involves adding feed to the batch. Cells are cultured in a fixed volume of culture medium. Specific supplemental nutrients are added to the culture before maximum cell concentration is reached. The volume of the feed is much smaller than the volume of the culture. Fed-batch culture involves periodically or continuously adding substrate in solid or concentrated liquid form to batches of cell cultures during growth. Fed-batch culture is described, for example, in U.S. Patent No. 5,672,502.

[0424] In perfusion culture, the culture medium is perfused through a reactor at a high rate, while cells are retained or recycled back into the reactor through sedimentation, centrifugation, or filtration. A key function of perfusing such a large volume of culture medium is the removal of metabolites (primarily lactate) from the culture medium. Perfusion culture is described, for example, in U.S. Patent No. 6,544,788.

[0425] In continuous culture, cells are initially grown in a fixed volume of culture medium. To prevent them from entering the apoptosis phase, a pump is started to replenish the fresh culture medium before the maximum cell concentration is reached. To maintain a constant volume, the culture containing a portion of the cells is continuously removed from the container. Continuous cultures and bioreactors are described, for example, in U.S. Patent Nos. 4,764,471, 5,135,853, and 6,156,570.

[0426] A bioreactor is a device or system that supports a biologically active environment, such as a device or system intended to grow cells or tissues in a cell culture setting (e.g., mammalian, plant, yeast, bacterial cells). The process can be aerobic or anaerobic. Bioreactors are typically cylindrical, ranging in size from several liters to tens of cubic meters, and are often made of stainless steel. Based on their operating mode, bioreactors can be classified as batch, fed-batch, or continuous-batch (e.g., continuous stirred tank reactor model).

[0427] For example, the Heraeus miniPERM bioreactor combines an autoclaved external nutrient container with a disposable internal bioreactor chamber. (CELLIGEN, New Brunswick Scientific) It is a highly flexible system for culturing virtually all eukaryotic cell lines. Wave Bioreactor TM Wave Biotech, LLC uses an adjustable-speed shaking platform and an electric air pump to aerate cultures. Quark Enterprises offers a full range of bioreactors, including those for high-density cultures. Culture flask.

[0428] Stirred tanks (and culture flasks) can provide cell cultures at increased density. Examples include disposable stirred tank bioreactors (such as Xcellerex) or scalable disposable stirred tank bioreactors (XDR). TM Applikon offers a full range of stirred tanks, from 2.3L benchtop systems to 10,000L production units. Batch bioreactors are also available from companies such as Rockland Immunochemicals, Inc.

[0429] Routine culture of Pichia pastoris in a stirred tank bioreactor (non-limiting example):

[0430] Pichia pastoris is a methyltrophic yeast that provides a unique expression system for producing high levels of recombinant proteins, including various levels of enzymes, protease inhibitors, single-chain antibodies, and regulatory proteins. The following workflow is provided as a non-limiting example of Pichia pastoris fermentation, which may be used as an alternative to or in combination with the methods described in the examples below. Details are provided in “Introduction to Pichia pastoris culture in a stirred-tank bioreactor” (Application note, 253, October 2011, Eppendorf).

[0431] The culture medium consists of heat-sensitive and non-heat-sensitive materials. Therefore, the inoculum can be prepared in two steps. First, the following substances are mixed and autoclaved:

[0432]

[0433] After autoclaving and allowing the container to cool completely, heat-sensitive materials can be added:

[0434] Culture medium composition quantity Trace metal solution, PTM1* 4.6 mL / L Alkali, to adjust the initial pH 25mL / L Inoculation 200mL glycerin** 400mL Methanol** <2L An alkali that maintains its pH at a set point <250mL

[0435] *: 6 g / L copper sulfate pentahydrate, 0.08 g / L sodium iodide, 3 g / L manganese sulfate monohydrate, 0.5 g / L cobalt chloride (anhydrous), 20 g / L zinc chloride (anhydrous), 0.02 g / L boric acid, 0.2 g / L sodium molybdate dihydrate, 65 g / L ferrous sulfate heptahydrate, 0.2 g / L biotin, 30 mL / L 6N sulfuric acid. **: Add as needed.

[0436] Inoculum was prepared using Pichia pastoris shake flask growth medium. The inoculum was incubated at 28°C for 40 hours in a shaker (New Brunswick G25) running at 240 rpm.

[0437] The control set point for Pichia pastoris should be entered and reached before inoculation, and the culture medium should be equilibrated before inoculation, except that dissolved oxygen (DO) is usually kept high. An initial DO value of approximately 100% is acceptable; this value will decrease as the culture metabolizes oxygen. Agitation limits may vary depending on the system and can be set to respond automatically to oxygen demand.

[0438] The preferred culture conditions are as follows:

[0439] parameter Set point temperature 30℃ pH 5.0 Dissolved oxygen 30％ agitation 300rpm-1200rpm*

[0440] Controlling dissolved oxygen (DO) often involves cascading, controlling agitation, gas flow, or oxygen individually or in combination. Cascading allows the controller to maintain the setpoint by automatically adjusting other process loops. Including oxygen in a cascade can help increase overall culture density. pH control often uses the addition of liquid acid and liquid alkali solutions to maintain pH at the setpoint, but often relies on the acid- / alkali-producing properties of the culture or medium to allow for natural fluctuations. pH control in Pichia pastoris is typically achieved by adding alkali (30% NaOH).

[0441] Glycosylated AAT produced by yeast

[0442] Over the past decade, Pichia pastoris expression systems have been widely used for the production of recombinant proteins, and the nature of the processing occurring in yeast has become a topic of interest. Yeast can glycosylate the amide nitrogen of asparagine residues in proteins, a process that occurs in the consensual sequence Asn-Xaa-Thr / Ser, providing N-linked glycosylation. Furthermore, glycosylation of the hydroxyl groups of threonine and / or serine residues in proteins occurs in yeast cells, resulting in O-linked glycosylation. For a review, see Bretthauer, Biotechnol. Appl. Biochem. (1999) 30, 193–200.

[0443] As described primarily in *Saccharomyces cerevisiae*, protein glycosylation involves the events outlined below. N-linked oligosaccharides originate from oligosaccharides (Glc3Man9GlcNAc2) assembled on polyterpenoids (pyrophosphates) in the endoplasmic reticulum (ER), and these oligosaccharides are transferred to the appropriate Asn of the nascent protein during co-translational events. This is a common eukaryotic glycosylation pathway for N-linked glycoproteins. However, the subsequent processing of newly glycosylated proteins differs in yeast compared to higher eukaryotic cells (plants, insects, and higher animals).

[0444] In most yeasts studied, three glucose residues and a specific α-1,2-linked mannose residue are removed by a specific glycosidase in the ER, resulting in an N-linked Man8GlcNAc2 core structure, which is further processed in the Golgi complex. This involves adding α-1,6-linked mannose residues to the α-1,3-linked mannose residues in the Mana-1,3Manb-1,4GlcNAc core sequence. The 1,6-linked residues can then be elongated into an α-1,6-linked backbone (50-100 residues), which can be branched with disaccharides or trisaccharides of the α-1,2-linked mannose, which can then be capped with α-1,3-linked mannose units. Another processing event that occurs in most yeasts is the addition of phosphate groups (in the form of mannose 1-phosphate) to specific α-1,2- or α-1,6-linked mannose residues in the core or side chain of N-linked oligosaccharides, thereby forming an acidic phosphodiester component present in a variety of yeast glycoproteins.

[0445] Complex oligosaccharides containing sialic acid, galactose, fucose, and N-acetylgalactosamine have not been found in Saccharomyces cerevisiae glycoproteins and most other yeast-derived glycoproteins. Therefore, when considering the structure and subsequent use (function) of glycoproteins, it is essential to focus on the assembly and processing of high-mannose-type N-linked oligosaccharides on the expressed recombinant protein.

[0446] Although some Pichia pastoris strains have been found to contain β-1,2-linked mannose residues in their cell wall mannans (as seen in structures such as Man(b-1,2)Man(b-1,2)Man(a-1,2)Man), β-linked mannose residues have not been reported in recombinant glycoproteins from Pichia pastoris. O-linked mannose oligosaccharides are prevalent in most yeasts, except for N-linked oligosaccharides. This biosynthesis is unique compared to animals because the mannose residues linked to the hydroxyl groups of serine or threonine residues in proteins via α-glycosidic bonds originate from the polyterpene phosphate carrier (polyterpene phosphate mannose) in the ER, rather than the glyconucleotides in the Golgi complex. These short oligosaccharides can also be phosphorylated, thus contributing to the acidity and potential other functions of glycoproteins.

[0447] Compared to known glycosylations of Pichia pastoris-derived proteins, rhAAT produced herein contains a unique HexNAc1 glycosylation, preferably comprising 50%-100%, more preferably 60%-90%, and more preferably 70%-80% of total N-glycans. Unexpectedly, the rhAAT protein described herein exhibits enhanced biological and cellular activity compared to the activity of plasma-purified human AAT (Prolastin).

[0448] In the embodiments, the post-translational modifications are one or more of N-glycosylation, O-glycosylation, N-terminal methionine removal, N-acetylation, and / or phosphorylation, or any combination thereof. In the embodiments, the recombinant AAT protein has one or more human-like glycosylations.

[0449] In implementation methods, the yeast used for AAT production, as described herein, allows AAT to be modified into a human-like structure. The term "human-like" structure or "human-like" glycoform pattern relates to a glycosylation pattern that, for example, prolongs the serum half-life and / or stability of recombinant AAT to a level comparable to human plasma-derived AAT. The "human-like" glycoform pattern is preferably different from but similar to a human glycosylation pattern to the extent that it prolongs the serum half-life of recombinant AAT compared to unmodified AAT protein. For example, the "human-like" glycoform pattern may comprise one or more polysaccharides selected from the group consisting of mannose, N-acetylglucosamine (GlcNAc), galactose, N-acetylneuraminic acid (Neu5Ac), N-hydroxyacetylneuraminic acid, and xylose.

[0450] The glycosylation pattern of recombinant AAT produced in yeast as described herein provides a novel form of recombinant AAT. This can be verified by a technician using SDS-page analysis. An upward or downward shift of the band of recombinant AAT produced by yeast (such as Pichia pastoris) compared to the band of plasma-derived AAT can be evidence of a novel recombinant AAT. Alternatively, the distinct peak distribution exhibited in comparative analyses between reference AAT (e.g., Prolastin) and AAT produced according to the present invention, using size exclusion HPLC or anion exchange HPLC, highlights the novel rhAAT.

[0451] Human AAT typically comprises up to three N-linked glycans attached to asparagine residues 46, 83, and 247. Naturally occurring AAT exhibits significant heterogeneity in glycosylation patterns and N-glycan structures. Therefore, several isoforms of AAT exist, also known as glycoforms. Glycoforms are protein isoforms that differ only in the number or type of glycans they are linked to. Glycoproteins are often composed of several different glycoforms, in which the linked sugars or oligosaccharides vary.

[0452] N-linked glycosylation refers to the process called N-glycosylation, in which an oligosaccharide (a carbohydrate composed of several sugar molecules, sometimes also called a glycan) is linked to a nitrogen atom (the amide nitrogen of the asparagine (Asn) residue in a protein). The resulting protein can be called an N-linked glycan, or simply an N-glycan.

[0453] In one embodiment, the rhAAT glycoprotein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 major N-glycan structures. The terms "major N-glycan structure" or "major glycoform" shall mean the specific N-glycan structure or glycoform that constitutes the largest proportion of all N-glycan structures or glycoforms of the protein (i.e., the one associated with the protein). In the embodiment, the major glycoform constitutes at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% or higher of the total group of glycoforms on the protein.

[0454] In the embodiments, the main N-glycan may be HexNAc1 glycosylated, wherein HexNAc1 glycosylation accounts for 50%-100%, preferably 60%-90%, and more preferably 70%-80% of the total N-glycan. In the embodiments, the main N-glycan may be Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, and Hex15HexNAc2 glycosylated.

[0455] As used herein, “HexNAc1” should refer to an amide derivative of a hexose and may also be referred to as N-acetylhexosamine (HexNAc), which is an acetylated derivative of hexosamine (HexN). Examples include N-acetylglucosamine (GlcNAc), which is an acetylated derivative of glucosamine (GlcN).

[0456] In the context of this invention, HexNAc1 refers to a single monosaccharide, not an oligosaccharide component. This applies particularly well to the occupancy of any given glycosylation site. For example, occupancy data for HexNAc1 refers only to the occupancy of a single HexNAc1, and not to HexNAc1 components that can be included as oligosaccharide components (e.g., Hex9HexNAc2).

[0457] As used herein, the term "hexose" refers to a monosaccharide (simple sugar) having six carbon atoms. The chemical formula of a hexose is C6H12O6. Hexoses can be glucose, mannose, galactose, allose, arbutin, gulose, idole, tarose, and fructose. Hexose also refers to units derived from a selection of hexoses: glucose (L- or D-form), maltose (L- or D-form), sucrose, mannose (L- or D-form), fructose (L- or D-form), lactose (L- or D-form), or mixtures thereof; more preferably, said compound is glucose (L- or D-form).

[0458] In the oligosaccharide according to the invention in the form Hex(n)HexNAc(m), "n" can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. "m" can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The oligosaccharide can be branched or linear.

[0459] In one embodiment, the oligosaccharide of Hex(n)HexNAc(m) may be Hex9HexNAc2, Hex10HexNAc2, Hex11HexNAc2, Hex12HexNAc2, Hex13HexNAc2, Hex14HexNAc2, Hex15HexNAc2, and / or Hex16HexNAc2. In another embodiment, Hex(n)HexNAc(m) is Man(n)HexNAc(m). In another embodiment, Hex(n) comprises mannose and other hexoses such as galactose, glucose, GlcNac, or Neu5Ac. In yet another embodiment, Hex(n) comprises other hexoses such as galactose, glucose, GlcNac, or Neu5Ac.

[0460] In embodiments, HexNAc or HexNAc(m) may be N-acetylglucosamine (GlcNAc). GlcNAc is an amide derivative of the monosaccharide glucose. In embodiments, HexNAc or HexNAc(m) may be an amide derivative of other hexoses. Non-limiting examples of Hex9HexNAc2 are Man9GlcNAc2 or Glc3Man6GlcNAc2.

[0461] Determination of glycosylation sites and site-specific heterogeneity in glycoproteins

[0462] Mass spectrometry (MS) has become the primary tool for the analysis of oligosaccharides or glycosylated proteins. It provides structural information with high sensitivity. Analysis of glycans or glycosylated proteins is typically performed using matrix-assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI), yielding ions that are sodium-coordinated or protonated in positive MS mode and deprotonated in negative MS mode, respectively. The fragmentation behavior of these two types of ions differs slightly in tandem MS, but glycans generally produce fragment ions resulting from glycosidic bond breakage.

[0463] Peptide mapping is a standard method for determining site-specific glycosylations and employs a combination of specific enzymatic proteolysis (usually with trypsin), glycopeptide fractionation (most commonly by liquid chromatography or affinity chromatography), and glycopeptide analysis by MS. Peptide mapping is a bottom-up protein characterization method that maps the primary structure of proteins and their post-translational modifications (PTMs, such as glycosylation).

[0464] A common workflow for confirming protein sequences and PTMs is peptide mapping, which combines proteolytic digests with RP-LC-MS / MS analysis. In this approach, for example, trypsin peptides are separated by reversed-phase chromatography and further analyzed using high-resolution tandem mass spectrometry. N-linked glycans are then analyzed in a second experiment, which includes enzymatic decomposition to release the glycans, chemical labeling, LC separation on graphitized carbon or HILIC columns, and MS analysis. Glycopeptide analysis is commonly used in glycoproteomics experiments to identify proteins and glycans using specific fragmentation involving collisional energy stepping. The resulting glycopeptide MS / MS spectra provide both glycan and peptide fragments and allow for the specification of peptide sequences and corresponding glycan structures on a single spectra.

[0465] An alternative method has been developed using nonspecific proteases to overcome several limitations of trypsin digestion. In this method, streptoprotein is used, a mixture of exonucleases and endonucleases capable of cleaving virtually any peptide bond. Glycoproteins are digested, with the exception of small peptide “footprints” around glycosylation sites, which are protected from digestion due to steric hindrance of the glycan moiety. The digestion products are glycopeptides with short peptides, small (unglycosylated) dipeptides, and amino acids. Purification is performed by solid-phase extraction, followed by MS analysis, yielding mass spectra containing only the glycopeptides. This method is now routinely used to obtain site heterogeneity for a wide variety of glycoproteins.

[0466] Tandem mass spectrometry (MS) can provide peptide and glycan sequences, as well as glycosylation sites. Studies of glycopeptide fragmentation reactions have almost entirely focused on protonated (via MALDI single-protonation or ESI multi-protonation) trypsin cleavage of glycopeptides. Typical fragmentation corresponds to the loss of the glycan portion, while information about peptide sequences and glycan attachment sites is often minimal. Tandem MS is complicated by the size of trypsin peptides (which tend to be larger than the useful mass range of collision-induced dissociation (CID)) and the unstable nature of the glycan-peptide bond. Therefore, a common strategy is to determine the total mass of the glycopeptide and perform tandem MS to obtain the peptide mass.

[0467] Those skilled in the art may choose either the method described above or the method described herein, depending on their needs in determining the presence, type, and / or location of glycosylation in rhAAT as described herein.

[0468] Attached Figure

[0469] The following figures are provided to describe specific embodiments of the invention, but are not intended to limit the scope.

[0470] Brief description of the attached diagram:

[0471] Figure 1 Exemplary expression plasmids carrying the PAOX1 promoter

[0472] Figure 2 Detailed view of superimposed electrophoretic patterns of simulated strain supernatants treated with EndoH (blue) and untreated with EndoH (red) relative to example supernatants treated with EndoH (brown) and untreated with EndoH (green).

[0473] Figure 3 Detailed view of the superimposed electrophoretic patterns of EndoH-treated screening supernatants of muts strains 1B3 (blue) and 1F8 (red) that secrete rAAT, relative to BSA (brown) diluted to 125 mg / L in a simulated strain matrix.

[0474] Figure 4 : Superimposed electrophoretic patterns from the supernatant reservoir of the secondary screening.

[0475] Figure 5 :mut s An exemplary chromatogram of the elution portion of the affinity chromatography of clone ID 1B6 (reactor S2).

[0476] Figure 6 :mut s Chromatogram of SEC for clone ID 1B6 (reactor S2).

[0477] Figure 7 : From strain mut s Affinity chromatography and SDS-PAGE (left) and Western blot analysis (right) of crude material from the bioreactor of clone ID 1B6 and SEC.

[0478] Figure 8 SDS-PAGE (left) and Western blot analysis of SEC eluent fractions (right).

[0479] Figure 9 :mut s An exemplary chromatogram of the elution portion of the affinity chromatography of clone ID 1B6 (reactors B1, B2, and S2).

[0480] Figure 10 :mut s Chromatograms of SEC runs of clone ID 1B6 (reactors B1, B2, and S2). UV signal is blue, conductivity is brown. Fractions are indicated in red along the x-axis.

[0481] Figure 11 : From strain mut s Affinity chromatography and SDS-PAGE (left) and Western blot analysis (right) of crude material from the bioreactor of clone ID 1B6 and SEC.

[0482] Figure 12 Changes in cell wet weight over time.

[0483] Figure 13 Superimposed electrophoretic patterns of supernatant and filtrate from selected sampling points of strain 1B6 cultured in reactor S2.

[0484] Figure 14 : Using strain mut in a 10L bioreactor S2 s SDS-PAGE (left) and Western blot (right) analysis of 1B6 culture.

[0485] Figure 15 : strain mut s Historical curve of AAT 1B6 cultured in a 10L reactor S2 (pH 6.0, 28°C, slow partial co-feeding process).

[0486] Figure 16 Size exclusion (SE)-HPLC of DS(I).

[0487] Figure 17 Size exclusion (SE)-HPLC of DS(II).

[0488] Figure 18 Quantitative results of monomer-related peak areas in the stability of the DS autosampler at 5±1℃.

[0489] Figure 19 Size exclusion (SE)-HPLC linearity check of DS.

[0490] Figure 20 Size exclusion (SE)-HPLC elution curve of monomer-related area of ​​DS(III).

[0491] Figure 21 Anion exchange-HPLC of DS(I).

[0492] Figure 22 Anion exchange-HPLC of DS(II).

[0493] Figure 23 Quantitative results of monomer-related peak areas in the stability of the DS autosampler at 5±1℃.

[0494] Figure 24 Linearity of DS anion exchange (AEX)-HPLC.

[0495] Figure 25 Anion exchange (AEX)-HPLC elution curve of monomer-related area of ​​DS(III).

[0496] Figure 26 Pierce TM BSA standard curve for the Rapid Gold BCA protein assay kit.

[0497] Figure 27 RecA1AT inhibits neutrophil elastase activity, similar to...

[0498] Figure 28 RecA1AT inhibits TMPRSS2 activity, similar to...

[0499] Figure 29 A study of inflammatory parameters in sputum of patients with post-infectious bronchiolitis obliterans (PiBO).

[0500] Figure 30 : miRNA expression in PiBO patients compared to the control group.

[0501] Figure 31 Inflammation and miRNA expression in human lung epithelial cells (A549).

[0502] Figure 32 Experimental design and setup of a cell culture system for stimulation of human lung epithelial cells (A549).

[0503] Figure 33 Analysis of the effects of rhAAT on cellular inflammation and IL-8 levels in A549 cells stimulated with a mixture of cytokines.

[0504] Figure 34 A schematic diagram of balanced inflammation and immune response.

[0505] Figure 35 : A schematic diagram of inflammation and pathological immune response.

[0506] Figure 36 Exemplary extracted ion chromatograms of the WP15 strain mutS clone ID 6B2 in the final AAT library, showing the presence and absence of the most abundant glycosylation at site N131.

[0507] Figure 37 Exemplary extracted ion chromatograms of the WP15rep strain mutS clone ID 6E2 in the final AAT library, showing the presence and absence of the most abundant glycosylation at site N131.

[0508] Figure 38 Affinity runs #2 and #4 for SDS-PAGE and Western blot analysis.

[0509] Figure 39Changes in secretion production (mg / L, measured by mCE, in both forms) with induction time

[0510] Figure 40 Changes in cell wet weight (g / L) over time

[0511] Figure 41 Detailed views of superimposed electrophoretic patterns of 2-fold diluted EndoH-treated supernatants from various sampling points.

[0512] Figure 42 (A) Full View and (B) Detailed View of superimposed electrophoretic patterns of 2-fold diluted EndoH-treated supernatants from various sampling points.

[0513] Figure 43 SDS-PAGE and Western blot analysis of supernatant from shake-flask cultures of strain muts pre-LS1-pro-aMF-rAAT 6E2 supplemented with protease inhibitors.

[0514] Figure 44 Quantitative results of glycosylation sites N46, N83 and N247 in strains IB6, 6B2 and 6E2.

[0515] Figure 45 Quantitative results of site-specific N-glycosylation analysis at N46 in strains IB6, 6B2 and 6E2.

[0516] Figure 46 Quantitative results of site-specific N-glycosylation analysis at N83 in strains IB6, 6B2 and 6E2.

[0517] Figure 47 Quantitative results of site-specific N-glycosylation analysis at N247 in strains IB6, 6B2, and 6E2.

[0518] Figure 48 Standard curve for protease assay.

[0519] Figure 49 Results of protease assay.

[0520] Figure 50 SDS-PAGE (left) and Western blot (right) analysis of gel migration assay samples: trypsin and neutrophil elastase.

[0521] Figure 51 SDS-PAGE (Coomassie staining; top) and Western blot analysis of gel migration assay: CatG, NE, PR3.

[0522] Figure 52SDS-PAGE (Coomassie staining; top) and Western blot analysis of gel migration assay (bottom): V8.

[0523] Detailed description of the attached diagram:

[0524] Figure 1 Example plasmid map of pPZ-PAOX1-aMF- containing rAAT. 5′AOX Prom represents the remainder of the original clone, the chromosomal sequence of the 5′ of the natural PAOX1 sequence; aMF-pre-pro represents α-mating factor (Saccharomyces cerevisiae), without the precursor-propeptide form of EAEA; rAAT is the gene sequence of the target gene; AOX1TT represents the transcription terminator of PpAOX1; pILV5 represents the eukaryotic promoter controlling the transcription of the resistance gene; EM72 represents the prokaryotic promoter controlling the transcription of the resistance gene; Zeocin resistance gene; AOD TT represents the transcription terminator of PpAOD; pUC ORI represents the origin of replication of E. coli, from the pUC plasmid series.

[0525] Figure 2 Detailed view of superimposed electrophoretic patterns of simulated strain supernatants treated with EndoH (blue) and untreated with EndoH (red) relative to example supernatants treated with EndoH (brown) and untreated with EndoH (green); Arrow 1 indicates the putative rAAT peak produced by deglycosylation, Arrow 2 indicates the rAAT peak present under both conditions, Arrow 3 shows the EndoH signal (blue and brown lines only), and Arrow 4 depicts analytical artifacts caused by bubbles in the microfluidic system.

[0526] Figure 3 : Mutants that secrete rAAT s Detailed views of the superimposed electrophoretic patterns of EndoH-treated screening supernatants of strains 1B3 (blue) and 1F8 (red) relative to BSA (brown) diluted to 125 mg / L in a simulated strain matrix; arrow 1 indicates the putative target protein peak of deglycosylated rAAT, arrow 2 indicates the putative signal of non-glycosylated rAAT, arrow 3 shows the EndoH peak, arrow 4 highlights the BSA signal, and arrow 5 points to an analytical artifact (caused by air bubbles in the microfluidic system).

[0527] Figure 4 : mut processed by EndoH sDetailed view of the supernatant reservoirs of strains (1F8 (blue), 1B3 (red), and 1E2 (brown)) relative to the superimposed electrophoretic patterns of 1B6 in EndoH-treated (green) and untreated (pink) forms. All strains secrete rAAT; arrow 1 indicates the putative target protein peak of deglycosylated rAAT, arrow 2 indicates the putative signal of non-glycosylated rAAT, arrow 3 shows the EndoH peak, and arrow 4 points to an analytical artifact (caused by bubbles in the microfluidic system).

[0528] Figure 5 :mut s An exemplary chromatogram of the elution fraction of the affinity chromatography for clone ID 1B6 (reactor S2). UV signal is blue, conductivity is brown, and the elution gradient (% of buffer B) is green. Fractions are indicated in red along the x-axis. The inset in the upper right corner shows the chromatogram of the complete run.

[0529] Figure 6 :mut s Chromatograms of SEC run of clone ID 1B6 (reactor S2). The top plot shows the first SEC run (elution library of affinity run 1+2), and the bottom plot shows the second SEC run (elution library of affinity run 3+4). UV signal is blue, and conductivity is brown. Fractions are indicated in red along the x-axis.

[0530] Figure 7 : From strain mut s Affinity chromatography and SDS-PAGE (left panel) and Western blot (right panel) analyses of crude bioreactor material from clone ID 1B6 were performed. All samples were analyzed with and without EndoH glycosidase (top panel) (bottom panel). 10 μL of sample was loaded per lane; MES run buffer was used.

[0531] Swimming lane S MW standard

[0532] Lane 2 starting sample (3-fold dilution for SDS-PAGE, 30-fold dilution for Western blot)

[0533] Lane 3 affinity chromatography flow-through buffer (3-fold dilution for SDS-PAGE, 30-fold dilution for Western blotting)

[0534] Lane 4 capture step library run 1 (40x dilution for SDS-PAGE, 400x dilution for Western blot)

[0535] Lane 5 capture step library run 2 (40x dilution for SDS-PAGE, 400x dilution for protein blot)

[0536] Lane 6 capture step library run 3 (40x dilution for SDS-PAGE, 400x dilution for Western blot)

[0537] Lane 7 capture step library run 4 (40x dilution for SDS-PAGE, 400x dilution for Western blot)

[0538] Flow-through buffer for 8-lane centrifuge apparatus (3-fold dilution for SDS-PAGE, 30-fold dilution for Western blotting)

[0539] 9 SEC lanes for starting sample run 1 (150-fold dilution for SDS-PAGE, 1500-fold dilution for Western blot)

[0540] 10 SEC lanes for starting sample run 2 (150-fold dilution for SDS-PAGE, 1500-fold dilution for Western blot)

[0541] Lane 11 SEC library run #1 9-10 (20-fold dilution for SDS-PAGE, 200-fold dilution for protein blot)

[0542] The 12-lane SEC library was run in run #11 (50-fold dilution for SDS-PAGE, 500-fold dilution for protein blot).

[0543] Lane 13 SEC library run #12-14 (25-fold dilution for SDS-PAGE, 250-fold dilution for protein blot)

[0544] Lane 14 SEC library run 2 (50-fold dilution for SDS-PAGE, 500-fold dilution for Western blot)

[0545] Lane 15 reference material (1 μg for SDS-PAGE, 0.2 μg for Western blotting)

[0546] Figure 8 SDS-PAGE (left) and Western blot (right) analysis of SEC eluent fractions. All samples were analyzed with or without EndoH glycosidase (lanes 2-6 or 7-12). 10 μL of sample was loaded into each lane; MES run buffer was used.

[0547] Swimming lane S MW standard

[0548] Lane 2 / 7 SEC fraction #9 (15-fold dilution for SDS-PAGE, 150-fold dilution for Western blot)

[0549] Lane 3 / 8 SEC fraction #10 (25-fold dilution for SDS-PAGE, 250-fold dilution for Western blot)

[0550] Lane 4 / 9 SEC grade #11 (20-fold dilution for SDS-PAGE, 200-fold dilution for Western blot)

[0551] Lane 5 / 10 SEC grade #12 (10-fold dilution for SDS-PAGE, 100-fold dilution for Western blot)

[0552] Reference material for lanes 6 / 11 (1 μg for SDS-PAGE, 0.1 μg for Western blotting)

[0553] Figure 9 :mut s Exemplary chromatograms of the elution fractions of affinity chromatography for clone ID 1B6 (reactors B1, B2, and S2). UV signal is blue, conductivity is brown, and elution gradient (% of buffer B) is green. Fractions are indicated in red along the x-axis. The inset in the upper right corner shows the chromatogram of the complete run.

[0554] Figure 10 :mut s Chromatograms of SEC runs of clone ID 1B6 (reactors B1, B2, and S2). UV signal is blue, conductivity is brown. Fractions are indicated in red along the x-axis.

[0555] Figure 11 : From strain mut s Affinity chromatography and SDS-PAGE (left panel) and Western blot (right panel) analyses of crude bioreactor material from clone ID 1B6 were performed. All samples were analyzed with or without EndoH glycosidase (top panel) (bottom panel). 10 μL of sample was loaded per lane; MES run buffer was used.

[0556] Figure 12 Changes in cell wet weight over time during fermentation.

[0557] Figure 13 Detailed view of the superimposed electrophoretic patterns of 2-fold dilutions from each sampling point, EndoH-treated supernatants (blue for 45h induction, red for 67h induction, and brown for 90h induction) relative to 2-fold dilutions of filtrate (green for EndoH treatment, pink for untreated) and BSA reference (turquoise); green arrows indicate putative signals of deglycosylated target proteins, red arrows indicate major byproducts, blue arrows show EndoH signals, and black arrows point to BSA signals.

[0558] Figure 14 : Using strain mut in a 10L bioreactor S2 sSDS-PAGE (left) and Western blot (right) analyses of 1B6 culture. Orange arrows indicate bands of putative full-length target proteins; red arrows indicate bands of deglycosylated AAT reference material.

[0559] SeeBlue Plus 2 (WB) protein standards for lane 1 (PAGE) / 2 (WB); indicating the molecular weight (MW) of standard proteins.

[0560] Samples from lanes 2-5 (PAGE) / 3-6 (WB) were TP3-5 (induced for 45h, 67h, and 90h) and filtrate; treated with EndoH (8-fold dilution for PAGE, 40-fold dilution for Western blot).

[0561] Lane 6 (PAGE) / 7 (WB) AAT reference, untreated (1 μg sample loaded).

[0562] Lanes 7-10 (PAGE) / 8-11 (WB) samples TP3-5 (induction at 48h, 68h, and 91.5h) and filtrate; no EndoH added (8-fold dilution for PAGE, 40-fold dilution for Western blot).

[0563] Lane 11 (PAGE) / 12 (WB) AAT reference, deglycosylated (using PNGase F; 1 μg sample), marked with a red arrow.

[0564] Lane 12 (PAGE only) BSA (1 μg)

[0565] Figure 15 : strain mut s Historical curves of AAT 1B6 cultured in a 10L reactor S2 (pH 6.0, 28℃, with a slow partial co-feeding process); stirring rate (red); pressure (light blue); dissolved oxygen (dark blue); pH (light green); alkali addition (magenta); aeration (orange); glycerol feeding (dark green); methanol feeding (purple); temperature (yellow); due to a software issue during the induction phase, the methanol feeding rate was manually adjusted to conform to the curve defined by the feeding strategy.

[0566] Figure 16 Size exclusion (SE)-HPLC chromatogram of DS(I). A 10 μL volume of 1 mg / mL DS(AATec) was diluted in the mobile phase and injected (sample volume = 10 μg). A significant shift in elution was observed compared to the reference and Prolastin.

[0567] Figure 17 Size exclusion (SE)-HPLC chromatograms of DS samples under forced and unforced degradation. Samples were stored at 40°C for 5 days and subjected to 5 freeze-thaw cycles for forced degradation.

[0568] Figure 18 Stability of DS monomer-related peak areas in an autosampler at 5±1℃. Time points for the HPLC autosampler were 0h, 24h, and 48h. The relative peak area percentage remained stable at the specified time points.

[0569] Figure 19 Linearity check of DS size exclusion (SE)-HPLC linearity test. Linearity of absolute peak area of ​​monomer was observed in the range of 5 μg to 20 μg.

[0570] Figure 20 Size exclusion (SE)-HPLC chromatograms of DS(III) show absolute peak areas of monomers of 5 μg, 7.5 μg, 10 μg, 15 μg and 20 μg, respectively.

[0571] Figure 21 The anion exchange-HPLC chromatogram of DS(I) shows the elution profiles of Prolastin (DP), the commercial reference (Ref), and the AATec drug substance (DS). The elution profiles of DP and Ref show a main peak and a shoulder peak, while the elution profile of DS shows a non-uniform curve.

[0572] Figure 22 Anion exchange-HPLC of DS(II) was performed. Samples were stored at 40°C for 5 days and subjected to 5 freeze-thaw cycles to force degradation.

[0573] Figure 23 Quantitative results of monomer-related peak areas in the stability of the DS autosampler at 5±1℃. Time points for the HPLC autosampler were 0h, 24h, and 48h. The relative peak area percentages at the indicated time points were stable.

[0574] Figure 24 Linearity of DS was checked using anion exchange (AEX)-HPLC. Linearity of the absolute peak area of ​​monomers was observed in the range of 10 μg to 80 μg.

[0575] Figure 25 The anion exchange (AEX)-HPLC chromatogram of DS(III) shows that the absolute peak areas of the monomers are 10 μg, 20 μg, 40 μg, 60 μg and 80 μg, respectively.

[0576] Figure 26 Pierce TM BSA standard curve for the Rapid Gold BCA protein assay kit. (Based on Pierce) TM The Rapid Gold BCA protein assay kit is used to prepare a dilution of BSA at a predetermined concentration in TM buffer to obtain a standard curve.

[0577] Figure 27 RecA1AT inhibits neutrophil elastase activity, similar to... recA1AT、 Serial dilutions of carmustine mesylate were mixed with human neutrophil elastase, followed by the addition of a fluorescent reporter substrate. Fluorescence intensity was measured at excitation wavelength of 380 nm and emission wavelength of 460 nm. The mean ± SEM values ​​from three independent experiments are shown, with each experiment performed in triplicate. The half-maximal inhibition concentration (IC50) was calculated using a nonlinear regression model.

[0578] Figure 28 RecA1AT inhibits TMPRSS2 activity, similar to... recA1AT、 NIBSC standards and serial dilutions of carmosstatin mesylate were mixed with recombinant TMPRSS2, followed by the addition of the fluorescent reporter substrate. Fluorescence intensity was measured at excitation wavelength of 380 nm and emission wavelength of 460 nm. The mean ± SEM values ​​from three independent experiments are shown, with each experiment performed in triplicate. The half-maximum inhibition concentration (IC50) was calculated using a nonlinear regression model.

[0579] Figure 29 A study of inflammatory parameters in sputum from patients with post-infectious bronchiolitis obliterans (PiBO). (A) Persistent neutrophilic inflammation is associated with (B) impaired lung function. Elevated levels of neutrophil-driven (C) IL-6, (D) IL-8 and lung epithelial cell-driven (E) IL-33, (G) IL-10, and (H) TGF-β were analyzed by flow cytometry bead array (CBA). (F) Analysis of the percentage of macrophages in lung tissue from PiBO patients and healthy controls.

[0580] Figure 30 (A) Volcano plot showing significant miRNA dysregulation at nominal levels P<0.05 and Padj<0.001. (B) Expression levels of miR-335-5p, miR-30b-5p, miR-186-5p, and miR-30c-5p confirmed by TaqMan qPCR analysis. (C) Correlation between validated miRNA expression (readings) and the pulmonary function parameter FEV1 analyzed in peripheral blood of PiBO patients and healthy controls. (D) NGS of induced sputum samples from PiBO patients showing readings of miR-335-5p and miR-186-5p compared to control samples (n=3).

[0581] Figure 31Inflammation and miRNA expression in human lung epithelial cells (A549). (A) IL-6 production in A549 cells analyzed by flow cytometry bead array after stimulation with elevated concentrations of a mixture of cytokines (CM) containing IL-1β, IFN-β, and TNF-α. (B) Expression levels of miR-335-5p, miR-30b-5p, miR-186-5p, and miR-30c-5p as measured by qPCR after stimulation with A549 cells as described above.

[0582] Figure 32 Experimental design and setup of a cell culture system using a mixture of cytokines to stimulate human lung epithelial cells (A549) and optionally HL60 and THP-1 cells. A549 cells (optionally HL60 and THP-1 cells) were stimulated with a mixture of cytokines (CM, IL-1β, IFN-γ, and TNF-α) to activate the NFKB pathway. Inflammation readings were IL-6 and IL-8.

[0583] Figure 33 Analysis of the effects of rhAAT on cellular inflammation and IL-8 levels in A549 cells stimulated with a cytokine mixture. IL-8 production in A549 cells was analyzed by flow cytometry microbead array after stimulation with elevated concentrations of a cytokine mixture (CM) containing IL-1β, IFN-β, and TNF-α and incubation with different concentrations of rhAAT.

[0584] Figure 34 A schematic diagram of balanced inflammatory and immune responses compared to an overreaction that leads to non-resolving and / or chronic inflammation. A balanced response to inflammation begins in the inflammatory phase, followed by a resolution phase in which inflammation subsides, thus protecting inflamed tissue. In contrast, an overreaction to inflammation results in a non-resolving chronic inflammation without a resolution phase following the inflammatory phase.

[0585] Figure 35 Schematic diagram of inflammatory and pathological immune responses at the cellular level in lung tissue of patients with PiBOS. Pro-inflammatory mediators involved in PiBOS include IL-33, IL-6, IL-8, CXCL8, and TNF-α.

[0586] Figure 36 Exemplary extracted ion chromatograms of the WP15 strain mutS clone ID 6B2 in the final AAT library, showing the presence and absence of the most abundant glycosylation at site N131.

[0587] Figure 37 Exemplary extracted ion chromatograms of the WP15rep strain mutS clone ID 6E2 in the final AAT library, showing the presence and absence of the most abundant glycosylation at site N131.

[0588] Figure 38 Affinity runs #2 and #4 were performed for SDS-PAGE and Western blot analysis. All analyte samples were deglycosylated and reduced, and evaluated using MOPS run buffer. Lanes: 1. MW standard; 2. Sample loaded for affinity run, 5-fold dilution (Coomassie) / 50-fold dilution (Western blot); 3. Flow-through buffer (library for 4 runs), 5-fold dilution (Coomassie) / 50-fold dilution (Western blot); 4-9. Elution fractions from affinity run #2, dilutions: 5-fold, 50-fold, 50-fold, 20-fold, 10-fold, 5-fold (Coomassie); 50-fold, 500-fold, 500-fold, 200-fold, 100-fold, 50-fold (Western blot); 10-15. Elution fractions from affinity run #4, dilutions: 5-fold, 50-fold, 50-fold, 20-fold, 10-fold, 5-fold (Coomassie); 50-fold, 500-fold, 500-fold, 200-fold, 100-fold, 50-fold (Western blot).

[0589] Figure 39 : Changes in secretion production (mg / L, measured by mCE, in both sheared and full-length forms) with induction time.

[0590] Figure 40 Changes in cell wet weight (g / L) over time.

[0591] Figure 41 Detailed views of superimposed electrophoretic patterns of 2-fold dilutions of EndoH-treated supernatants (0h induction, shown in the data line with no central peak and a large left-hand peak; 24h induction, shown in the data line with two lower central peaks; and 47h induction, shown in the data line with two higher central peaks) relative to BSA diluted to 125 mg / L (data line with a large right-hand peak) from various sampling points for strain 6E2 cultured in reactor S2; two central arrows indicate the putative signal of the (deglycosylated) target protein, the leftmost arrow indicates the EndoH signal, and the rightmost arrow points to the BSA signal.

[0592] Figure 42(A) Full view and (B) Detailed view of superimposed electrophoretic patterns of strain 6E2 cultured in reactor S2, from various sampling points, including 2-fold dilutions, EndoH-treated supernatants (0h induction, shown in the data line with no central peak and a large left-hand peak; 24h induction, shown in the data line with two lower central peaks; 47h induction, shown in the data line with two middle central peaks; and 53h induction, shown in the data line with the two highest central peaks) and filtrate (2-fold dilution, EndoH-treated, pink) relative to BSA diluted to 125 mg / L (data line with a large right-hand peak); two central arrows indicate the putative signal of the (deglycosylated) target protein, the leftmost arrow indicates the EndoH signal, and the rightmost arrow points to the BSA signal.

[0593] Figure 43SDS-PAGE and Western blot analysis of supernatant from shake-flask cultures of strain muts pre-LS1-pro-aMF-rAAT 6E2 supplemented with protease inhibitors. Coomassie staining gel (left) and anti-AAT Western blot (right). Lanes: 1. MW standard (SeeBlue Plus 2); 2. Prolastin, deglycosylated (using PNGaseF), 50 μg / mL (Coomassie), 10 μg / mL (WB); 3. Prolastin without protease inhibitor, deglycosylated (using EndoH), undiluted (Coomassie), 1:5 (WB); 4. 5 mM EDTA, deglycosylated (using EndoH), undiluted (Coomassie), 1:5 (WB); 5. 5 mM (in feed) PMSF, deglycosylated (using EndoH), undiluted (Coomassie), 1:5 (WB); 6. 1 μM pepsin A, deglycosylated (using EndoH), undiluted (Coomassie), 1:5 (WB); 7. Prolastin without protease inhibitor, +NE, deglycosylated (using EndoH), undiluted (Coomassie), 1:5 (WB); 8. 5 mM EDTA, +NE, deglycosylated (using EndoH), undiluted (Coomassie), 1:5 (WB); 9.5 mM (feeding in progress) PMSF, +NE, deglycosylated (using EndoH), undiluted (Coomassie), 1:5 (WB); 10.1 μM pepsin A, +NE, deglycosylated (using EndoH), undiluted (Coomassie), 1:5 (WB); 11. No protease inhibitor, untreated, undiluted (Coomassie), 1:5 (WB); 12.5 mM EDTA, untreated, undiluted (Coomassie), 1:5 (WB); 13.5 mM PMSF (in feed), untreated, undiluted (Coomassie), 1:5 (WB); 14.1 μM pepsin A, untreated, undiluted (Coomassie), 1:5 (WB); 15. Prolastin, untreated, 50 μg / mL (Coomassie), 10 μg / mL (WB).

[0594] Figure 44 Quantitative results of glycosylation sites N46, N83 and N247 in strains IB6, 6B2 and 6E2.

[0595] Figure 45 Quantitative results of site-specific N-glycosylation analysis at N46 in strains IB6, 6B2 and 6E2.

[0596] Figure 46 Quantitative results of site-specific N-glycosylation analysis at N83 in strains IB6, 6B2 and 6E2.

[0597] Figure 47Quantitative results of site-specific N-glycosylation analysis at N247 in strains IB6, 6B2, and 6E2.

[0598] Figure 48 Standard curve for protease assay. Plot the fluorescence signal relative to the amount of trypsin in each well. Apply point-to-point curve fitting.

[0599] Figure 49 Results of the protease assay. The protease activity (average of two repeated measurements) for each sample dilution is shown as trypsin equivalents (in ng). Note that the values ​​are not recalculated with each dilution, but are displayed individually for each dilution.

[0600] Figure 50 SDS-PAGE (left) and Western blot (right) analyses of gel migration assay samples: trypsin and neutrophil elastase. All samples were deglycosylated using EndoH (rAAT) or PNGaseF (Prolastin), with enzyme bands indicated by yellow dashed lines. Samples were reduced to 10 μL per lane and loaded with MOPS run buffer. Top: AAT incubated with trypsin; Bottom: AAT incubated with neutrophil elastase. Lane: 1 / 7MW standard (SeeBlue Plus2); 2 / 1 single protease (trypsin / neutrophil elastase); 3 / 2rAAT, protease-free; 4 / 3rAAT, containing 25% protease; 5 / 4rAAT, containing 50% protease; 6 / 5rAAT, containing 75% protease; 7 / 6rAAT, containing 100% protease; 8Prolastin, protease-free; 9Prolastin, containing 25% protease; 10Prolastin, containing 50% protease; 11Prolastin, containing 75% protease; 12Prolastin, containing 100% protease.

[0601] Figure 51SDS-PAGE (Coomassie staining; top) and Western blot analysis (bottom) for gel migration assays: CatG, NE, PR3. All analyte samples were deglycosylated (using EndoH or PNGaseF) and reduced; MOPS run buffer was used. The orange dashed lines on the Coomassie stained gel highlight the corresponding enzymes added for deglycosylation. Left: rAAT 6E2, Right: Prolastin. Lanes: 1(2) MW standard (SeeBlue Plus2); 2(1) rAAT / Proalstin without added protease; 3, 8, 13 individual proteases (cathepsin G, neutrophil elastase, protease 3); 4-7 rAAT / Prolastin with incremental Cat G (0.1, 0.5, 1, 2 molar equivalents); 9-12 rAAT / Prolastin with incremental NE (0.1, 0.5, 1, 2 molar equivalents); 14-17 rAAT / Prolastin with incremental PR3 (0.1, 0.5, 1, 2 molar equivalents).

[0602] Figure 52 SDS-PAGE (Coomassie staining; top) and Western blot analysis for gel migration assay: V8. All analytes were deglycosylated (using EndoH or PNGaseF) and reduced; MOPS run buffer. The orange dashed lines on the Coomassie stained gel highlight the corresponding enzymes added for deglycosylation. Left: rAAT 6E2, Right: Prolastin. Lanes: 1 (2) MW standard (SeeBlue Plus 2); 2 (1) rAAT / Proalstin, without protease; 3 protease alone (protease V8); 4-8 rAAT / Prolastin, with increments of V8 (0.1, 0.25, 0.5, 1, 2 molar equivalents).

[0603] Example

[0604] The following methods were used to develop and validate a Pichia pastoris expression strain for recombinant production of human α-antitrypsin (rhAAT).

[0605] Examples 1-13 illustrate a detailed exemplary workflow for generating recombinant human AAT. The workflow includes: transformation of Pichia pastoris muts strain with a recombinant AAT expression plasmid (Examples 1-4), microscale culture (Example 5), target protein expression analysis (Example 6), clone screening (Examples 7 and 8), purification of AAT from selected clones at a 2L fermentation scale (Examples 9 and 10), bioreactor AAT production (up to a 10L fermentation scale; Example 11), and target protein expression analysis (Examples 12 and 13).

[0606] Examples 14-19 involve comparative analyses of Prolastin (DP), a commercial reference (Ref), and the rhAAT formulation of the present invention (DS) using various chromatographic techniques to identify differences in yeast-derived protein formulations compared to commercial products.

[0607] Further functional and structural characterization of the rhAAT of the present invention is shown, including measuring the anti-proteolytic activity of recombinant rhAAT (Example 20) and analyzing the rhAAT glycosylation pattern (Example 21).

[0608] Examples 22-24 provide experimental evaluations of rhAAT in the treatment of acute respiratory distress syndrome (ARDS), bronchiolitis obliterans (BO), and asthma.

[0609] Example 25 outlines a research plan to test the efficacy of AAT in a mouse model of endotoxin (LPS)-induced lung inflammation.

[0610] In Examples 26 and 27, polysaccharide analysis was performed on batches produced from clone 1B6 (Example 26) and 6E2 / 6B2 (Example 27).

[0611] Example 28 describes the identification of the shear / cut variant.

[0612] Example 29 describes a manufacturing process with a shortened induction time.

[0613] Example 30 describes a manufacturing method using different protease inhibitors and their effect on reducing protein cleavage variants.

[0614] Example 31 describes the evaluation of the potential immune response to ATL-105 in mice after oropharyngeal administration.

[0615] In Examples 32-34, analytical tests were performed to characterize the purified rAAT compared to Prolastin.

[0616] Example of a workflow for reorganizing AAT:

[0617] Example 1: Recombinant human α-1-antitrypsin:

[0618] This embodiment includes the cloning and development of Pichia pastoris expression strains to achieve recombinant production of human α-1-antitrypsin (rAAT).

[0619] The amino acid sequence of the target protein (SEQ ID NO 4):

[0620]

[0621] The amino acid sequence of the target protein rAAT; potentially occupied N-glycosylation sites are underlined and shown in bold.

[0622] Table 1 compiles the theoretical molecular weight (without the contribution of N-glycans) and characteristics of the target proteins.

[0623]

[0624] Example 2: Workflow in vitro and in E. coli:

[0625] After developing and producing the optimized synthetic gene sequence, the target gene was cloned into the expression plasmid pPZ-α (digested with XhoI / NotI) using the XhoI / NotI restriction enzyme. This plasmid carries the α-mating factor precursor-propeptide (without EAEA) secretion guide sequence (Saccharomyces cerevisiae). This gene was also cloned into PAOX1 for methanol-induced production.

[0626] Table 2: Workflow from generating synthetic genes to final expression plasmids:

[0627]

[0628]

[0629] The nucleotide sequence of the inserted target gene containing a stop codon (without flanking regions for cloning purposes) (SEQ ID NO 3).

[0630]

[0631] After transformation into *E. coli* TOP10F′ cells (NEB-5α competent *E. coli*, C2987I, NEB), the transformants were recultured and plasmids were prepared. Restriction enzyme mapping analysis was used to check for correct target gene insertion, and sequencing (LGCGenomics, Berlin, Germany) confirmed the authenticity of specific expression cassettes. An example plasmid map is shown below. Figure 1 As shown.

[0632] Sufficient quantities of all plasmids were linearized (by BglII cleavage) and desalted (MCE membrane, 0.025 μM, Millipore VSWP01300) for transformation into Pichia pastoris. Additionally, a ready-made kanamycin / genimycin resistance plasmid pPK carrying PAOX1 was prepared for transformation, as described above. DNA concentration was determined spectrophotometrically and adjusted to approximately 1 μg / μL.

[0633] Example 3: Transformation into Pichia pastoris:

[0634] All culture medium components used in experiments during the growth, transformation, regeneration (during transformation), and colony storage of *Pichia pastoris* have been certified free of animal-derived ingredients in terms of direct contents or direct contact (e.g., the use of animal-derived proteases in the preparation of tryptone / peptone lysates) and potential contamination.

[0635] Linearized plasmid constructs are targeted and integrated into the host cell genome via homologous recombination of the PAOX1 sequence into the natural AOX1 locus, either as a single integrating construct (thus, only one promoter variant exists, also known as "single-copy integration") or as a tandem construct for subsequent integration into the locus. Alternatively, integration into other loci in the genome can occur once or multiple times via non-homologous integration.

[0636] Using the basic strain CBS7435 Mut s (Genotype Δaox1; Phenotype Mut) s The competent cells were electroporated using a modified standard procedure and a standard electroporator.

[0637] After regeneration at 28°C, the preparation was spread on an agar plate (YPhyD solid) containing the antibiotic Zeocin.

[0638] Example 4: Culture medium used for transformation, restrikeing, and culturing:

[0639] YPhyD liquid medium (1% yeast extract, 2% plant peptone, 2% dextrose)

[0640] YPhyD solid medium (same as above, with 2% w / v agar added, and antibiotics supplemented as needed).

[0641] BMD solution (1.34% YNB, 2% dextrose, 0.2M pH 6.0 sodium phosphate buffer, 4×10⁻⁶ ppm) -5 % Biotin)

[0642] Table 3 compiles the components of solid and liquid culture media.

[0643]

[0644] Examples of microscale fermentation and glycosylation analysis:

[0645] Example 5: Microscale culture in a 96-well deep-well plate:

[0646] For screening, single colonies were picked from transformation plates and placed into individual wells of 96-well deep-well plates filled with optimized culture medium. Some corner wells of the plates were inoculated with specific mimic strains to serve as the substrate for analysis.

[0647] After the initial growth phase of biomass generation (BMD solid), expression of the AOX1-promoter-variant was induced by adding an optimized liquid mixture containing a defined concentration of methanol. Further induction with methanol was performed at defined time points.

[0648] After a total of 72 hours of initial methanol induction, all deep-well plates were centrifuged, and the supernatant from all wells was collected in a reserve microtiter plate for subsequent analysis.

[0649] After selecting specific strains from the screening results, these strains are re-stripeded onto non-selective agar plates to ensure that each strain produces a single isolated colony.

[0650] During the secondary screening process, each strain was cultured, and six of these individual colonies were inoculated into a single well of a 96-well deep plate filled with optimized culture medium and treated as described above.

[0651] Table 4. Workflow of microscale culture.

[0652]

[0653] The cloning process can be performed as follows:

[0654] Step 1: Preparation of Pichia pastoris host cell line (Pp-mut) s )

[0655] Step 2: Transformation with plasmids

[0656] Step 3: Microscale culture

[0657] Step 4: Microscale reculturing

[0658] Step 5: Select a pilot clone

[0659] Example 6: Target protein expression analysis:

[0660] Microfluidic capillary electrophoresis (mCE):

[0661] A high-throughput screening method involving microfluidic capillary electrophoresis separation (GXII, CaliperLS, now part of Perkin Elmer) was established, followed by size-based identification of target proteins. In short, a few μL of the entire culture supernatant was fluorescently labeled and analyzed according to protein size using a microfluidic-based electrophoresis system. Internal standards (included in a supplier-provided solution) enabled approximate allocation of the detection signal to kDa-level size and concentration. Bovine serum albumin (BSA), diluted to a known concentration in a simulated bacterial matrix, was used as a calibrator for apparent molecular weight and concentration. Different allocations of protein peaks relative to apparent molecular weight (estimated from migration time) can occur between different analysis plates on the same microfluidic chip and across different chips due to systematic variability. Therefore, a deviation between apparent and nominal molecular weights (a shift towards higher apparent molecular weights) is commonly observed.

[0662] program:

[0663] 5 μL of sample (from deglycosylation, see below) was mixed with 8 μL of sample buffer (PerkinElmer, containing LDS, pH 7.58) containing an appropriate amount of reducing agent (final pH 7.37), and heated at 95 °C for 5 min. Subsequently, 32 μL of ddH2O was added, and the sample was loaded into mCE after centrifugation at 4,000 rpm for 3 min (to precipitate potential aggregates).

[0664] Deglycosylation:

[0665] Samples were treated with EndoH to analyze the putative glycosylation status of expressed target proteins and provide preliminary quantification of the amount of glycosylated target proteins. Due to the heterogeneity of glycosylation, N-glycosylated proteins tend to produce indistinct peaks on the mCE, making it difficult to assign and quantify the often broad and blunt peaks. Therefore, deglycosylation is used to produce clearer peaks for target proteins, enabling specific assignment and quantification of the target signal.

[0666] For deglycosylation using EndoH (NEB), mix 10 μL of the supernatant sample with 10x denaturing buffer and heat to 70°C for 10 minutes. Add 10x G3 reaction buffer and 0.5 μL of EndoH (for treated conditions) or 0.5 μL of G3 reaction buffer (for untreated conditions). Incubate at 37°C for 60 minutes, then use the sample for analysis.

[0667] Example 7: Result Screening:

[0668] Comparison of target supernatant and simulated strain supernatant:

[0669] The supernatant of a simulated strain and the supernatant of an exemplary strain designed to secrete rAAT were subjected to deglycosylation conditions with and without the addition of EndoH (brown line). Figure 2 ).

[0670] Presumed non-glycosylated rAAT (in) Figure 2 (Indicated by arrow 2 in the brown and green lines) appears to be secreted into the culture supernatant, next to which is N-glycosylated rAAT (in brown line) revealed by deglycosylation with EndoH. Figure 2 (Indicated by arrow 1), while it was not detected under conditions without EndoH treatment (green line).

[0671] Used to compare mut s Standard settings for an exemplary clone of a strain:

[0672] Figure 3 The study showed that mutagenesis secreted rAAT. s Detailed views of the superimposed electrophoresis patterns of EndoH-treated screening supernatants of strains 1B3 (blue) and 1F8 (red) relative to BSA (brown) diluted to 125 mg / L in a simulated strain matrix. Arrow 1 indicates the target protein peak of putatively deglycosylated rAAT, arrow 2 indicates the signal of putatively non-glycosylated rAAT, arrow 3 shows the EndoH peak, arrow 4 highlights the BSA signal, and arrow 5 points to analytical artifacts (caused by air bubbles in the microfluidic system). The signal ratio of putatively non-glycosylated to putatively deglycosylated rAAT varies slightly between the various supernatants. The selection of strains for rescreening was based on the peak area of ​​the putatively deglycosylated signal.

[0673] Example 8: Result Rescreening:

[0674] After methanol induction for 72 hours with or without EndoH treatment, the supernatant pools (approximately 1.8 mL in total) generated from all six wells of each strain were analyzed by mCE relative to BSA (diluted to 125 mg / L in the simulated strain matrix) as a calibrator.

[0675] Figure 4 The image shows the mutagenesis processed by EndoH. s A detailed view of the supernatant pools of strains 1F8 (blue), 1B3 (red), and 1E2 (brown) relative to the superimposed electrophoretic patterns of 1B6 in EndoH-treated (green) and untreated (pink) forms. All clones secrete rAAT. Arrow 1 indicates the peak of the target protein of the putative deglycosylated rAAT, arrow 2 indicates the signal of the putative non-glycosylated rAAT, arrow 3 shows the EndoH peak, and arrow 4 points to an analytical artifact (caused by bubbles in the microfluidic system). When the peak content in the green (EndoH-treated) and pink (untreated) lines was examined again, it appeared that the attribution of a putative non-glycosylated peak (present in both lines) and a putative deglycosylated signal (present only in the green line) matched the screening hypothesis. The signal ratios of the putative non-glycosylated and putative deglycosylated forms of rAAT differed slightly in the individual supernatants. The estimated concentrations of the putative deglycosylated and nonglycosylated rAAT fractions found in the supernatant are compiled in Table 5.

[0676] Table 5: Estimated concentrations of rAAT (as putative deglycosylated fraction and putative nonglycosylated fraction) in the supernatant of the rescreened sample pool, calculated by comparing the peak area of ​​a specific specific peak with the peak area of ​​BSA present at a known concentration.

[0677]

[0678] A common observation was obtained: the supernatant contained a (presumably) non-glycosylation peak (arrow 2) and a signal that appeared only after deglycosylation (arrow 1), the latter possibly representing the N-glycosylation fraction of the target protein revealed by deglycosylation.

[0679] Given the potential potency range of approximately 10 mg / L in the supernatant at the microscale, achieving deglycosylated rAAT concentrations of 150 mg / L–200 mg / L in bioreactor cultures is feasible.

[0680] Protein purification example (from a 2L fermentation):

[0681] Example 9: strain mut s First purification activity for clone ID 1B6:

[0682] The purpose of this embodiment is to obtain strain mut sPreparative two-step purification of rAAT was performed from bioreactor crude material of clone ID 1B6. The target protein was captured by affinity chromatography using a Vantage L column packed with approximately 18 mL of Alpha-1 Antitrypsin Select resin (Cytiva, product number 17547201). As a second step, SEC was performed to finely purify the sample, and the sample was then replaced with PBS (pH 7.4). In the initial run, 2 L of bioreactor crude material from reactor S2 was used for preparative purification; for the second purification, a mixture of bioreactor crude materials from reactors B1, B2, and S2, produced in WP4-rep, was used.

[0683] Affinity chromatography:

[0684] For initial capture of the target protein, affinity chromatography was used. Chromatographic operations were performed according to the resin manufacturer's (Cytiva, product number 17547201) recommendations. Prior to purification, the column was washed with 70% ethanol (as recommended by the manufacturer) for 48 hours, washed with water, and equilibrated with buffer A.

[0685] Table 6: Chromatographic system.

[0686]

[0687] Table 7: Procedure for affinity chromatography.

[0688]

[0689] CV: Column volume

[0690] *Dilution in buffer A at a 1:1 ratio

[0691] Size exclusion chromatography (SEC):

[0692] After capturing the target protein, size exclusion chromatography (SEC) was used as a fine purification step and the final sample was replaced with pH 7.4 PBS.

[0693] Table 8: Chromatographic system.

[0694]

[0695] Table 9: SEC steps.

[0696]

[0697] The first purification activity using material from reactor S2:

[0698] Affinity chromatography for the first activity:

[0699] 2L of crude bioreactor material (reactor S2 produced in WP4-rep) was diluted 2-fold in buffer A (i.e., to 4L) and loaded onto the column in 4 separate runs (1000 mL per run).

[0700] For all chromatograms (example chromatogram is shown in...), Figure 5 In the chromatograms, well-defined peaks were observed, and the main peak fractions (2A2, 2A3, and 2A4 in the example chromatogram) were combined and used for further analysis.

[0701] mut s An example chromatogram of the elution fraction of the affinity chromatography for clone ID 1B6 (reactor S2) is shown below. Figure 5 As shown. UV signal is blue, conductivity is brown, and elution gradient (% of buffer B) is green. Fractions are indicated in red along the x-axis. The inset in the upper right corner shows the chromatogram of the complete run.

[0702] Size exclusion chromatography for the first activity:

[0703] The eluent libraries from the first and second runs, as well as the third and fourth runs, were combined and concentrated to approximately 10 mL by ultrafiltration (centrifugation, 10 kDa cutoff). These libraries were then loaded onto the SEC column in two separate runs. Before sample loading, the column was washed with 0.5 M NaOH and equilibrated in buffer.

[0704] Chromatograms from two SEC runs are as follows Figure 6 As shown. The chromatogram of the second SEC run depicted a well-defined peak (as previously seen in WP10); while the chromatogram of the first SEC run showed a left shoulder peak with a lower measured UV signal than the second SEC run. Fractions #37-41 from the second SEC run were combined for further analysis, while fractions #9-10, #11, and #12-14 were analyzed separately to exclude potential adulterant fractions. Due to visible impurities for fractions #9-11, they were combined and concentrated to 10 mL, and then loaded back onto the SEC column to minimize protein loss. The chromatogram showed a well-defined peak, and all fractions were analyzed separately and stored frozen at -70°C as backups.

[0705] mut s The SEC chromatogram of clone ID 1B6 (reactor S2) is shown below. Figure 6 As shown. The top figure shows the first SEC run (elution libraries of affinity runs 1+2), and the bottom figure shows the second SEC run (elution libraries of affinity runs 3+4). UV signals are in blue, and conductivity is in brown. Fractions are indicated in red along the x-axis.

[0706] Following SEC and final analysis by Western blotting and SDS-PAGE, samples from the first SEC run (elution fractions #12-14) and the second SEC run (elution fractions #37-41) were combined, concentrated to 22 mg / mL (via BCA, 8 mL volume), and sterile filtered. The samples were then sterile filtered again and stored frozen at -70°C.

[0707] Final analysis and discussion of the first activity:

[0708] Starting materials, intermediates, and the final library (before concentration to 22 mg / mL) were analyzed by SDS-PAGE and Western blotting. Figure 7 The final protein concentration in the library was determined to be 14.4 g / L (BCA), equivalent to 107.7 mg of protein. Endotoxin levels were analyzed by LAL assay using the Charles River Endosafe PTS and a dedicated test chip (sample diluted 1:100). The summarized parameters are listed in Table 10.

[0709] Table 10: Summary parameters of the final sample from the first purification activity.

[0710] parameter Analytical methods unit result concentration BCA mg / mL 14.4 volume mL 7.5 quantity mg 107.7

[0711] SDS-PAGE and Western blot analysis showed that affinity chromatography based on AAT-specific resin was highly effective in capturing proteins from the culture, and multiple bands with molecular weights between 17 kDa and 40 kDa were cleared. The target protein loss was calculated to be 19% (surprisingly low considering the level of purification achieved).

[0712] To ensure no protein loss during the concentration step, the capture step eluate library was concentrated to 10 mL using a centrifuge with a 10 kDa cutoff. No protein loss was observed in SDS-PAGE, and only very small bands were visible in the protein blot (which were calculated as a 0.5% loss). Figure 7 SDS-PAGE and Western blot analysis showed no loss of target proteins during SEC because the final sample was calculated as 100% of the starting SEC sample. However, for the first SEC run, impurity bands (possibly dimers / multimers) were visible in the earlier fractions (#9-11), and therefore this fraction was excluded from subsequent experiments. Therefore, SEC is a very helpful step for further improving purity and replacing the sample with PBS. For the final sample, eluent fractions #12-14 (first SEC run) and eluent fractions #37-41 (second SEC run) were combined and concentrated to 22 mg / mL.

[0713] To minimize protein loss, SEC fractions #9-11 (which were initially excluded, see above) were combined, concentrated to 10 mL, and loaded back onto the SEC column. Figure 8 The analysis of each fraction by Western blotting and SDS-PAGE is shown, revealing again that the first two fractions (#9 and 10) showed impurity bands, while fractions #11 and 12 contained only the target protein. Therefore, fractions #11 and 12 were combined, sterile filtered, and frozen at -70°C for use as spare samples. The summary parameters of the final samples of fractions #11 and 12 are listed in Table 11.

[0714] Table 11: Summary parameters of the final samples of fractions #11 and #12.

[0715] parameter Analytical methods unit result concentration Protein blot mg / mL 13.5 volume mL 20 quantity mg 270

[0716] Example 10: Using combined materials from reactors B1, B2, and S2, strain mut was tested. s The second clone ID 1B6 Secondary purification activity:

[0717] Affinity chromatography for the second activity:

[0718] To demonstrate the reliability of the reproducible process, a second purification run was performed. For the second purification run, 280 mL of the remaining bioreactor crude material from reactor S2 was mixed with 345 mL from reactor B1 and 345 mL from reactor B2 (note that all three showed very similar performance). The resulting 970 mL was diluted to 2 L in buffer A and loaded onto the column in two separate runs (1000 mL each). Well-defined peaks were observed in both chromatograms (example chromatogram shown on...). Figure 9 (in the middle), and the main peak fractions (2A2, 2A3, 2A4 of the example chromatogram) were combined and used for further analysis.

[0719] Size exclusion chromatography for the second activity:

[0720] The eluents from the two runs were combined and concentrated to approximately 10 mL by ultrafiltration (centrifugation, 10 kDa cutoff). The sample was then reburied in 1x PBS (pH 7.4) and loaded onto Sartobind. PA. Then, in a single run, Sartobind will be... The eluent of PA (still >22 EU / mg) was directly loaded onto the SEC column. The column was washed with 0.5 M NaOH and equilibrated in buffer before sample loading. The chromatogram of the SEC run is shown below. Figure 10As shown, a well-defined peak was observed. Fractions #10-13 were aseptically filtered and analyzed by Western blotting and SDS-PAGE. After the final analysis by Western blotting and SDS-PAGE showed no impurity bands, the samples were pooled, concentrated to 51.7 mg / mL (via BCA, 1.9 mL), and aseptically filtered again. The samples were then aseptically filtered and stored frozen at -70°C until shipment.

[0721] Final analysis and discussion of the second activity:

[0722] Samples of starting materials, intermediates, and the final library (before concentration) were analyzed using SDS-PAGE and Western blotting. Figure 11 The final protein concentration in the library was determined to be 51.7 g / L (BCA), equivalent to 98.2 mg of protein. The summarized parameters are listed in Table 12.

[0723] Table 12: Summary parameters of the final sample

[0724] parameter Analytical methods unit result concentration BCA mg / mL 51.7 volume mL 1.9 quantity mg 98.2

[0725] SDS-PAGE and Western blot analysis showed that affinity chromatography based on AAT-specific resin was highly effective in capturing proteins from cultures, and multiple bands with molecular weights between 17 kDa and 40 kDa were cleared. The target protein loss was calculated to be only 13%.

[0726] To ensure no protein loss during the concentration step, the capture step eluate library was concentrated to 10 mL using a centrifuge with a 10 kDa cutoff. No protein loss was observed in either SDS-PAGE or Western blotting. Figure 11 The SDS-PAGE and Western blot results showed an 85% yield for the final sample compared to the SEC starting sample. No impurity bands were observed for the four main peak fractions; however, it should be noted that the sample exhibited high purity after the affinity step. The final sample (concentration determined by BCA: 51.7 mg / mL) was frozen until shipment.

[0727] Examples 11-13 relate to bioreactor methods for the secretion of recombinant AAT in Pichia pastoris strains.

[0728] Bioreactor examples (up to 10L):

[0729] Example 11: Bioreactor Cultivation Protocol:

[0730] Culture medium preparation:

[0731] All culture medium components were certified free of animal-derived contaminants. Purified H2O (via reverse osmosis, conductivity 6.9 μS / cm) was used in the preparation of all media.

[0732] Pre-culture medium:

[0733] Table 13: Pre-culture medium.

[0734]

[0735] 1 2127 is the order number, and 10, 20, and 30 are the container dimensions.

[0736] Distribute appropriate amounts of water to each component; heat all components separately to sterilize, then cool and mix them for use in a laminar flow cabinet.

[0737] Table 14: For example, for 1L YPG medium.

[0738]

[0739]

[0740] Bioreactor culture medium:

[0741] Table 15: Modified Basal Salt Medium (BSM), listed in order of addition. Each component is added after the previous component has completely dissolved.

[0742]

[0743] BSM has a pH of 1.8 ± 0.2 and a conductivity of 24 μS / cm.

[0744] Table 16: Trace elements in PTM1, listed in the order of addition. Slight turbidity is still present, which is normal.

[0745]

[0746] PTM1 has a pH of 1.6 ± 0.2 and a conductivity of 55 μS / cm.

[0747] Other materials:

[0748] Glycerin replenishment solution: 60% w / w + 12 ml / L PTM1

[0749] Methanol feed solution: MeOH (>99.85%, CHEMRES)

[0750] Alkali: 25% ammonia solution (Item 5460.3, Roth)

[0751] Defoamer: PPG2000 (automatically added using a defoaming probe)

[0752] Pre-culture in a 1L fermenter:

[0753] The strain was inoculated from an agar plate into a 300 mL wide-necked, baffled, capped shake flask filled with 50 mL of YPG medium using an inoculation loop (covering the biomass at the end of the loop), and incubated overnight at 28°C with shaking at 110 rpm (50 mm rotation diameter) (pre-culture 1). Ideally, pre-culture 1 reached an OD600 value of 15-20 after 20-24 hours of growth (depending on the strain and the biomass used for inoculation). OD600 is the optical density measured at 600 nm using a Genesys 10S VIS spectrophotometer (Thermo Scientific) relative to deionized water as a blank control. Pre-culture 2 was inoculated from pre-culture 1 into a 1 L wide-necked, baffled, capped shake flask filled with 100 mL of YPG medium, adjusted to OD600 = 2-3, and incubated at 28°C with shaking at 110 rpm (50 mm rotation diameter) for 9-10 hours. Ideally, the OD600 value of preculture 2 reaches 18-20 after 9-10 hours of growth (the exponential growth doubling time is about 2 hours), after which the preculture 2 material is used to inoculate the bioreactor.

[0754] Pre-cultivation in a 10L fermenter:

[0755] The strain was inoculated from an agar plate into a 300 mL wide-necked, baffled, capped shake flask filled with 50 mL of YPG medium using an inoculation loop (covering the biomass at the end of the loop), and cultured overnight at 28°C with shaking at 110 rpm (50 mm rotation diameter) (pre-culture 1). Ideally, pre-culture 1 reached an OD600 value of 15-20 after 20-24 hours of growth (depending on the strain and the biomass used for inoculation). OD600 is the optical density measured at 600 nm using a Genesys 10S VIS spectrophotometer (Thermo Scientific) relative to deionized water as a blank control. Pre-culture 2 was inoculated from pre-culture 1 into a 2 L wide-necked, baffled, capped shake flask filled with 200 mL of YPG medium, adjusted to OD600 = 2-3, and cultured at 28°C with shaking at 110 rpm (50 mm rotation diameter) for 9-10 hours. Ideally, the OD600 value of preculture 2 reaches 18-20 after 9-10 hours of growth (the doubling time of the exponential growth phase is approximately 2 hours), after which the bioreactor is inoculated using preculture 2 material. Three culture flasks are used for each strain and bioreactor to obtain sufficient inoculum.

[0756] Alternatively, pre-culture medium can be directly inoculated from the glycerol stock solution: inoculate 1 mL of stock solution (prepared to OD600 = 4) into 200 mL of medium in a 2 L baffled (4 baffles) shake flask and incubate at 110 rpm and 28 °C until OD600 = 20 ± 2 is reached after approximately 24-28 hours (OD600 monitoring is still recommended).

[0757] Bioreactor culture conditions:

[0758] Description of a bioreactor with a working volume of 1L:

[0759] The bioreactor was filled with 0.4 L of BSM medium, sterilized by heat, and the pH was corrected to 5 with ammonia solution. The bioreactor was inoculated with a calculated volume of pre-culture 2 and adjusted to an OD600 of 2.0. The bioreactor culture consisted of four stages:

[0760] 1) Batch growth using glycerol as a carbon source

[0761] 2) Feeding-in stage for generating additional biomass with glycerol

[0762] 3) Transition / Induction Stage of Co-feeding with Methanol and Glycerol

[0763] 4) Induction stage with methanol as the sole carbon source

[0764] Throughout the process, the temperature was set at 28°C. The pH was set at 5.0 for most of the initial batching and glycerol feeding batching phases. A gradient was used to gradually increase the pH setpoint to 6.0, starting two hours before the transition phase (18 hours in this case) and continuing for 1.5 hours, and maintained at this level thereafter.

[0765] Throughout the process, the oxygen saturation was set at 30%.

[0766] DO cascade control for a reactor with a maximum working volume of 1L:

[0767] Mixing speed: 700 rpm to a maximum of 1200 rpm

[0768] Ventilation rate: (Air) flow rate ranges from 1.0 L min⁻¹ to a maximum of 2.0 L min⁻¹

[0769] Oxygen supplementation: up to 60%

[0770] Description of a bioreactor with a working volume of 10L:

[0771] The bioreactor was filled with 4.5 L of BSM medium, heat-sterilized (please compensate for any volume loss during sterilization), and the pH was corrected to 5 with ammonia solution. The bioreactor was inoculated with a calculated volume of pre-culture 2 and adjusted to an OD600 of 2.0. The bioreactor culture consisted of four stages:

[0772] 1) Batch growth using glycerol as a carbon source

[0773] 2) Feeding-in stage for generating additional biomass with glycerol

[0774] 3) Transition / Induction Stage of Co-feeding with Methanol and Glycerol

[0775] 4) Induction stage with methanol as the sole carbon source

[0776] Throughout the process, the temperature was set at 28°C. The pH was set at 5.0 for most of the initial batching and glycerol feeding batching phases. A gradient was used to gradually increase the pH setpoint to 6.0, starting two hours before the transition phase (18 hours in this case) and continuing for 1.5 hours, and maintained at this level thereafter.

[0777] Throughout the process, the oxygen saturation was set at 30%.

[0778] Dissolved oxygen cascade control in a 10L reactor:

[0779] Stirring speed: between 250 rpm and 1500 rpm

[0780] Ventilation rate: (Air) flow rate range is 10.0 L / min -1 (Initial value) -20.0L min -1 Simultaneously adjust the pressure to a basic level of 0.3 bar and a maximum level of 1.5 bar while increasing the stirring rate.

[0781] Increase the stirring speed after it reaches 1,000 rpm.

[0782] Replenishment strategy:

[0783] Table 17 provides feeding protocols for the current fermentation process induced by methanol, with an initial batch culture volume of 4.5 L (e.g., applicable to a 10 L bioreactor).

[0784] Table 17: Feeding strategies for "partially co-feeding schemes that are slower" For an initial batch culture of 4.5L in a 10L reactor basal volume )

[0785]

[0786] For scaling up or down, the feed rate can be adjusted accordingly. It should be noted that glycerol feeding will automatically start 12 hours after bioreactor inoculation, and this strategy is beneficial when the initial OD600 of the bioreactor inoculation is 2 and the cells are not oxygen- or other parameter-limited. When the initial OD600 of the bioreactor inoculation is less than 2 and / or the cells are oxygen- or other parameter-limited, it is recommended to start glycerol feeding only after all the glycerol added to the batch medium has been consumed, indicated by a sharp increase in dissolved oxygen (DO spike). Preferably, the required amount of glycerol is added and consumed by the culture (DO spike) before starting methanol feeding to reduce the risk of overfeeding cells with methanol.

[0787] sampling:

[0788] Sample at the specified time points using the following procedure: Discard the initial 2-5 mL of fermentation broth. Transfer 1 mL of the freshly collected sample (2-5 mL) to a 1.5 mL centrifuge tube and centrifuge at 13,200 rpm (16,100 g) and 5 °C for 5 minutes in an Eppendorf 5415R centrifuge. Carefully transfer the supernatant to a separate vial and freeze or analyze the target protein content directly.

[0789] Determination of cell density (wet cell weight):

[0790] Transfer 1 mL of fermentation broth to a tare Eppendorf vial and centrifuge in an Eppendorf 5415R centrifuge at 13,200 rpm (16,100 g) and 5 °C for 5 minutes, carefully removing the supernatant. Weigh the vial (accurate to 0.1 mg) and subtract the tare weight of the empty vial to obtain the wet weight of the cells.

[0791] Harvesting and filtering:

[0792] After centrifuging the fermentation broth (20 min, 12,000 g, 5 °C, Thermo Scientific Sorvall Bios A centrifuge with an F6-10x1000 LEX rotor), the supernatant was recovered and filtered through a Sartopure PP3 0.45 μm filter (Sartorius #5051306P4-OO-B) (for the supernatant of a 1 L reactor) or a Sartopure PP3 Midicaps 0.45 μm filter (Sartorius #5055306P7-SO-A) (for the supernatant of a 10 L reactor). Suitable aliquots were prepared and frozen at -70 °C.

[0793] Example 12: Target protein expression analysis:

[0794] Microfluidic capillary electrophoresis (mCE):

[0795] Microfluidic capillary electrophoresis (GXII, CaliperLS, now part of Perkin Elmer) was used to separate proteins, and target proteins were subsequently identified based on their size. In short, a small amount (μL) of the total culture supernatant was fluorescently labeled and analyzed according to protein size using a microfluidic-based electrophoresis system. Internal standards allowed for approximate distribution of the detection signal with kDa-level size and concentration.

[0796] Different distributions of protein peaks relative to apparent molecular weight (estimated by migration time) may occur between different analysis plates on the same microfluidic chip and across different chips, all due to systematic variation.

[0797] Bovine serum albumin (BSA) reference material was used as a calibrator (diluted to 125 mg / L).

[0798] program:

[0799] 5 μL of sample (from deglycosylation, see below) was mixed with 8 μL of sample buffer (PerkinElmer, containing LDS, pH 7.58) containing an appropriate amount of reducing agent (final pH 7.37), and heated at 95 °C for 5 min. Subsequently, 32 μL of ddH2O was added, and the sample was loaded into mCE after centrifugation at 4,000 rpm for 3 min (to precipitate potential aggregates).

[0800] Deglycosylation:

[0801] For deglycosylation using EndoH (NEB), 10 μL of diluted supernatant sample was mixed with 10x denaturing buffer and heated to 70°C for 10 minutes. Then, 10x G3 reaction buffer and 0.7 μL of EndoH were added. After incubation at 37°C for 60 minutes, the sample was used for analysis.

[0802] SDS-PAGE and Western blot:

[0803] For SDS-PAGE, the diluted sample was deglycosylated as described above, or a non-deglycosylated sample (treated in the same manner but without EndoH) was used, mixed with 4x LDS sample buffer and 10x Novex reducing agent (both from ThermoScientific), and incubated at 70°C for 10 min. The sample was then loaded onto a Bolt Bis-Tris 4-12% gel and run with MES buffer. SeeBlue Plus2 pre-stained standards (all from ThermoScientific) were included as molecular weight standards.

[0804] For Coomassie staining, wash the gel in water, stain with SimplyBlueSafeStain (Thermo Scientific) according to the manufacturer’s protocol, and decolorize in water.

[0805] For protein blotting, proteins were transferred to the pre-run gel using a dry transfer method on an iBlot 2 gel transfer instrument equipped with a dedicated Novex PVDF transfer membrane stack. The membrane was then saturated with PBS, 0.1% Tween-20, and 5% BSA for 30 min with gentle shaking at RT.

[0806] For AAT-specific detection, the AAT primary antibody (SIGMA, SAB4200196-200 μL) diluted 1:750 in PBS, 0.1% Tween-20, and 3% BSA was used, and the membrane was incubated at RT with gentle shaking for 30 min. After washing the membrane three times (5 sec, 5 min, and 10 min, with vigorous agitation at approximately 200 rpm using PBS-0.1% Tween-20), the secondary antibody (abcam anti-mouse IgG H&L (HRP) ab2057-19; 5,000-fold diluted in PBS, 0.1% Tween-20, and 1% BSA) was added for 20 min. The membrane was then washed again and developed by adding TMB ultrasensitive substrate (Thermo Scientific). For all gels and blotting, the sample dilution for protein blotting was 5 times that for Coomassie-stained gels.

[0807] Example 13: 10L scale of non-glycosylated engineered strain Mut s Results of bioreactor culture in 1B6:

[0808] Overview:

[0809] The product titer and cell density estimated during the process and induction times are shown in Table 17 and... Figure 12 As shown.

[0810] Table 17: Cultured strain mut s During the process and induction time of 1B6, the target protein concentration (in mg / L; in the deglycosylated state, relative to BSA calibration) and cell wet weight (g / L) were measured from mCE.

[0811]

[0812] This calculation was performed based on the peaks at approximately 58 kDa and approximately 62 kDa of apparent molecular weight in mCE (see figure below).

[0813] like Figure 12 As shown, strain mut s1B6 grew well, reaching a cell density of 400g / L-500g / L.

[0814] Figure 13 This image shows a detailed view of the superimposed electrophoretic patterns of 2-fold dilutions of EndoH-treated supernatants (blue for 45 h of induction, red for 67 h, and brown for 90 h) relative to 2-fold dilutions of filtrate (green for EndoH-treated supernatants and pink for untreated supernatants) and BSA reference (turquoise) from various sampling points. Green arrows indicate the putative signal of the deglycosylated target protein, red arrows indicate major byproducts, light blue arrows show the EndoH signal, and black arrows point to the BSA signal. A bimodal pattern of the putative target protein was observed under deglycosylation conditions. Note that the left peak (migrating to a lower apparent molecular weight, approximately 58 kDa) was also present under untreated conditions, but at a lower abundance.

[0815] Figure 14 Based on SDS-PAGE and Western blotting, samples from the last three sampling points and the final filtrate, with and without deglycosylation, were compared.

[0816] Figure 15 The strain mut was shown s Historical curve of AAT 1B6 cultured in a 10L reactor S2 (pH 6.0, 28°C, partially fed process).

[0817] Table 18 compiles the full-length target protein relative to the AAT reference standard, estimated from SDS-PAGE by density measurement analysis of the 48 kDa band signal. Figure 3 The concentration of (orange and red arrows in the text).

[0818] Table 18: Culture of strain mut at a 10L scale s During the process and induction time of 1B6, the concentration of the full-length target protein relative to the AAT reference material was estimated from SDS-PAGE by density measurement analysis of the 48kDa band signal.

[0819]

[0820]

[0821] Examples of analytical comparisons between rhAAT, Prolastin, and commercial reference materials:

[0822] Examples 14-19 involve comparative analyses of Prolastin (DP), a commercial reference (Ref), and the rhAAT formulation of the present invention (DS).

[0823] Example 14: Size exclusion (SE)-HPLC analysis of DP, Ref, and DS(I)

[0824] Take 10 μL of 1 mg / mL DS (AATec) and dilute it in the mobile phase before injection, resulting in a sample loading of 10 μg. Figure 16 The study showed a significant shift in elution time for DS compared to the reference and Prolastin, indicating potential variation in post-translational modifications. To validate this observation, unconcentrated DS (thawed and directly injected) was used as a control, which also exhibited a shift in elution time. This finding suggests that this shift is not caused by a concentration effect. Additionally, as shown in Table 19, two distinct high molecular weight substances (HMWS) were detected.

[0825] Table 19: Two different HMWS materials

[0826] monomer HMWS LMWS DP 78.5％ 20.6％ 0.9％ Ref 89.2％ 9.9％ 0.9％ DS 92.2％ 7.8％ NA DS Unconcentrated 94.2％ 5.8％ NA

[0827] Example 15: Size exclusion (SE)-HPLC analysis of forced and unforced degradation DS samples

[0828] The samples were stored at 40°C for 5 days and subjected to 5 freeze-thaw cycles to undergo forced degradation. Figure 17 As shown, a new LMWS peak appeared after forced degradation at 40℃, indicating fragmentation caused by forced degradation. However, it can be concluded that freeze-thaw forced degradation has no significant effect on the size distribution. Table 20 provides information on the fragmentation distribution of samples with and without forced degradation.

[0829] Table 20: Fragmentation distribution of samples with and without forced degradation

[0830] Unforced degradation 5d 40℃ 5xFT monomer 92.2％ 86.4％ 92.7％ HMWS 7.8％ 6.3％ 7.3％ LMWS NA 7.3％ NA

[0831] Example 16: Size exclusion (SE)-HPLC analysis of monomer-related peak area stability

[0832] Prior to analysis, DS samples were stored in an autosampler at 5±1℃ for at least 48 hours. Figures 18-20 The absolute peak area of ​​the monomer is linear in the range of 5 μg to 20 μg.

[0833] Example 17: Anion exchange (AEX)-HPLC analysis of DP, Ref and DS

[0834] Take 40 μL of 1 mg / mL DS(rhAAT) and dilute it in mobile phase A before injection, resulting in a sample loading of 40 μg. Figure 21 The elution curves of DS revealed the absence of distinct main and shoulder peaks, and showed significant differences from those of Prolastin and the reference standard. The DS curves indicated a non-homogeneous sample, which may be due to post-translational modifications, such as changes in glycosylation patterns. The main and shoulder peaks were specified based on the elution time of the reference standard.

[0835] • Main peak: 15.2%, • Shoulder peak: 4.1%, • Basic variant: 64.3%, • Acidic variant: 16.4%

[0836] To verify this observation, unconcentrated DS (thawed and directly injected) was used as a control. The elution curves of unconcentrated DS also showed differences, indicating that the changes in the curve were not caused by a concentration effect.

[0837] Example 18: Anion exchange (AEX)-HPLC analysis of forced and unforced degradation DS samples

[0838] DS underwent forced degradation by being stored at 40°C for 5 days and subjected to 5 freeze-thaw cycles. Figure 22 The results show an increase in basic variants after incubation at 40°C, leading to a pattern change (indicated by blue arrows). Basic variants also increased slightly after freeze-thaw forced degradation. Table 21 presents the peak distributions for samples without forced degradation and those with forced degradation.

[0839] Table 21: Peak distribution of samples with and without forced degradation

[0840] Unforced degradation 5d 40℃ 5x FT Main Peak 15.2％ 8.7％ 15.3％ acromion 4.1％ 4.6％ 4.0％ basic variants 64.3％ 71.7％ 65.0％ Acid variants 16.4％ 15.0％ 15.8％

[0841] Example 19: Anion exchange (AEX)-HPLC analysis of monomer-related peak area stability

[0842] Before analysis, DS samples should be stored in an autosampler at 5±1℃ for at least 48 hours. Figures 23-25 The linearity of the absolute peak area of ​​the monomer is shown in the range of 10 μg to 80 μg. Therefore, the DS sample remained stable in the autosampler at 5 ± 1 °C for at least 48 h.

[0843] Discussion of Examples 14-19:

[0844] SE-HPLC analysis of prolastin and the reference revealed the presence of multiple HMWS and slight LMWS shoulders. In comparison, SE-HPLC analysis of DS showed an elution shift, suggesting a smaller size for DS, which may be attributed to diverse post-translational modifications.

[0845] Furthermore, AEX-HPLC was established, demonstrating enhanced resolution of charged variants compared to the original method. The curves for Prolastin and the reference showed a similar pattern, characterized by a prominent main peak accompanied by slight shoulder peaks and additional charged variants. In contrast, DS's AEX-HPLC revealed multiple peaks without a distinguishable main peak, indicating charge heterogeneity in the sample, which may be attributed to heterogeneous post-translational modifications.

[0846] The methods described above indicate the presence of heterogeneity in the samples, particularly in terms of charge and size, with DS showing more pronounced variations.

[0847] Examples of rhAAT activity and glycosylation:

[0848] Example 20: Characterization of the anti-proteolytic activity of recombinant rhAAT

[0849] The purpose of this experiment was to evaluate the anti-proteolytic activity of rhAAT compared to Prolastin (a human plasma-derived A1AT formulation).

[0850] Materials and methods:

[0851] Protein concentration measurement : Receive RecAAT (rhAAT) according to the present invention and use Pierce TM The Rapid Gold BCA Protein Assay Kit (Thermo Fisher #A53226) measures protein concentration according to the manufacturer's instructions. This assay is based on the protein-dependent reduction of copper ions, detectable colorimetrically at 480 nm using a VERSAMax microplate reader equipped with SoftMax Pro 7.0.3 software. For this purpose, a concentration-determining dilution of bovine serum albumin (BSA) standards for the kit was prepared in TM buffer (150 mM NaCl, 50 mM Hepes, 5 mM EDTA, and 1% Triton (v / v)). RhAAT and 5 mg / ml were also included. Control samples were diluted 1:1 and 1:4 in TM buffer for measurement. Concentrations were calculated based on the obtained kit BSA standards and dilution factors.

[0852] Neutrophil elastase activity assay Neutrophil elastase activity was measured by the following steps: 25 μl of serially diluted... (Grifols), carmostat mesylate (Sigma Aldrich #SML0057), or rhAAT were mixed with 25 μl of 2 ng / μl of recombinant neutrophil elastase (Merck Millipore #324681) in 25 μl of analysis buffer (50 mM Tris, 1 M NaCl, 0.05% (w / v) Brij-35, pH 7.5) and incubated at 37 °C for 15 min. Next, 50 μl of 200 μM MEOSUC-Ala-Ala-Pro-Val-AMC substrate (Bachem #4005227) was added and incubated at 37 °C. After 5 min, fluorescence intensity was measured at 380 nm excitation and 460 nm emission wavelengths using a Synergy™ H1 microplate reader (BioTek) equipped with Gen5 3.04 software.

[0853] TMPRSS2 activity assayTo assess the activity of recombinant human TMPRSS2, serially diluted 25 μl of... (Grifols), plasma-derived NIBSC standard α-1-antitrypsin (International Standard No. 1) (#05 / 162NIBSC), carmostat mesylate (Sigma Aldrich #SML0057) or rhAAT were co-incubated with 25 μl of 2 μg / ml recombinant TMPRSS2 enzyme (CusaBio #YP023924HU) in analytical buffer (50 mM Tris-HCl, 0.154 mM NaCl, pH 8.0) at 37 °C for 15 min. In the next step, 50 μl of 20 μM BOC-Gln-Ala-Arg-AMC protease substrate (Bachem #4017019) was added and incubated at 37 °C for 2 h. Fluorescence intensity was measured at 380 nm excitation and 460 nm emission wavelengths after 2 h using a Synergy™ H1 microplate reader (BioTek) equipped with Gen5 3.04 software.

[0854] result:

[0855] rhAAT sample and 5 mg / ml Control samples were diluted 1:1 and 1:4, with duplicate replicates. Concentrations were calculated based on the BSA standards and dilution factors in the kit. Table 22 describes the measured optical density (OD) and the corresponding calculated protein concentrations (mg / ml). This revealed that the protein concentration of the rhAAT sample was approximately 2.5 mg / ml.

[0856] Table 22: Measured optical density and corresponding protein concentration of the samples. Based on the BSA standard curve ( Figure 26 The protein concentration of the sample is calculated using the sample's dilution factor.

[0857]

[0858] rhAAT inhibits neutrophil elastase activity, similar to

[0859] The primary physiological function of AAT is the inhibition of neutrophil elastase (a protease released by immune cells). This invention provides a recombinant alternative to human plasma-derived antitrypsin for replacement therapy and for treating respiratory viral infections. To determine whether rhAAT possesses antiproteolytic activity, rhAAT, along with... The assay was evaluated against carmostatin mesylate (a small molecule inhibitor of the cellular protease TMPRSS2 and SARS-CoV-2 infection). The assay used a peptide substrate linked to 7-amino-4-methylcoumarin (AMC), which was quenched by the amide bond of an adjacent amino acid. Fluorescent AMC was released when this bond was cleaved by neutrophil elastase. Therefore, the addition of an inhibitor inhibited neutrophil elastase and reduced the fluorescence signal. Figure 27 As shown, rhAAT inhibits neutrophil elastase activity in a dose-dependent manner, with an IC50 of 16.6 nM, similar to... As expected, the small molecule TMPRSS2 inhibitor carmustine did not inhibit the activity of neutrophil elastase.

[0860] rhAAT inhibits TMPRSS2 activity, similar to and NIBSC standards:

[0861] In addition to its physiological role in regulating elastase activity, α-1-antitrypsin also exhibits inhibition of TMPRSS2 (a protease involved in viral entry, such as SARS-CoV-2). Therefore, similar to neutrophil elastase measurements, the ability of rhAAT to inhibit recombinant TMPRSS2 was assessed. As an additional control, the UK National Institute for Biological Standards and Control (NIBSC) antitrypsin was used as the international reference standard for the activity of α-1-antitrypsin preparations. Figure 28 As shown, rhAAT exhibits dose-dependent inhibition of TMPRSS2 activity, similar to... and NIBSC standards.

[0862] Table 23 summarizes the compound concentrations required to achieve 50% or 90% inhibition of enzyme activity (IC50 and IC90) in each assay. IC50 and IC90 were calculated using a nonlinear regression model in GraphPad Prism 9.

[0863] Table 23: Inhibitory concentrations (IC50 and IC90) of compounds tested in neutrophil elastase and TMPRSS2 activity assays, in nanomolars. - = no activity, nd = not measured

[0864]

[0865] Example 21: Glycosylation pattern analysis of rhAAT produced according to the present invention

[0866] The rhAAT protein produced in the aforementioned examples was analyzed using peptide mapping, a method combining trypsin-mediated proteolytic digestion and trypsin / GluC dual digestion with RP-LC-MS / MS analysis. The digested peptides were separated by reversed-phase chromatography and further analyzed using high-resolution tandem mass spectrometry. N-linked glycans were subsequently analyzed in a second experiment, which included enzymatic release of the glycans, chemical labeling, LC separation on graphitized carbon or HILIC columns, and MS analysis.

[0867] method:

[0868] Following enzymatic digestion, glycosylation sites were determined by HPLC-ESI-MS and N-MS / MS measurements. N-glycosylated peptides were screened from the HPLC-ESI-MS / MS data.

[0869] To remove interfering buffer components, the sample was reburied by ultrafiltration using a Nanosep centrifugal ultrafiltration device (Pall Life Science) according to the manufacturer's instructions, and the solution was changed to denaturing buffer (0.2M histidine-HCl, 5.6M guanidine-HCl, pH 6) for denaturation.

[0870] The sample was reduced with TCEP at 37℃.

[0871] According to the manufacturer's instructions, use Zeba TM The sample was desalted using a centrifugal desalting column (Thermo Scientific), and then reburied via ultrafiltration with a digestion buffer (0.02M histidine-HCl, 0.5mM TCEP, pH 6.0). Protein concentration was determined by UV measurement (absorbance at 280nm).

[0872] Protein samples were digested using the following digestion strategies: (1) trypsin and (2) trypsin / GluC under enzyme-specific conditions.

[0873] A portion of the digest was enzymatically deglycosylated with EndoH (NEB) under enzyme-specific conditions. Negative samples without the addition of EndoH were prepared.

[0874] HPLC-ESI-MS and -MS / MS mass spectra are obtained using Acquired using a 3000 system (Thermo Fisher Scientific) coupled with a Q Exactive Orbitrap Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated by reversed-phase (RP) chromatography on an Accucore RP-MS LC column (2.1 × 100 mm, 2.6 μm particle size, Thermo Fisher Scientific). The eluent was A: water containing 0.1% formic acid; B: acetonitrile containing 0.1% formic acid. Peptides were separated at 30 °C and a flow rate of 0.4 mL / min over 45 min using a segmented gradient from 3% B to 36% B. MS and MS / MS spectra (generated by high-energy collisional dissociation (HCD)) were recorded in positive ion mode and internally calibrated.

[0875] MS datasets were analyzed using the ProteinScape 2 bioinformatics platform (Bruker Daltonics, Protagen AG). Protein identification was achieved through database searches. Fragment mass spectra were matched against an internal database consisting of the NCBI Human Protein Database (http: / / www.ncbi.nlm.nih.gov / ) and manually inserted protein sequences (provided by the client).

[0876] The following protein modifications were considered:

[0877] • Deamidation (Asn)

[0878] • Oxidation (Met)

[0879] ·HexNAc(Asn)

[0880] • Pyroglutamate formation (Gln / Glu at the N-terminus of the peptide)

[0881] • Acetylation (N-terminus of protein)

[0882] For each peptide (sequence motif NXS / T, X≠P) covering potential N-glycosylation sites, extracted ion chromatograms (XICs) were generated showing Asn and Asn peptides with HexNAc. The modification level was specified by comparing the signal intensities of peaks matching those of peptides with and without HexNAc. The sum of the signal intensities of the two peptides was set to 100%. No correction was made for different ionization efficiencies of the peptides; the modification level expressed as a percentage does not accurately reflect the amount of peptide with or without glycosylation.

[0883] Glycosylated peptides were identified using an internally developed algorithm in HPLC-ESI-MS and -MS / MS data, enabling annotation of glycosylation sites and linker glycan types. Additionally, ProteinMetrics Byonic was used... TM The dataset is searched against a sequence database consisting of client sequences and common contaminants (e.g., sequences of proteases and keratins used), and N-glycosylation is allowed as a peptide modification.

[0884] Byonic TM The search results were entered into ProteinMetrics. Here, for Byonic TM For each glycopeptide identified by the internal algorithm, extracted ion chromatograms (XICs) are generated to determine the MS signal intensity. The quantitative level of each identified glycan structure is specified by comparing the MS signal intensities of the corresponding glycopeptides. The sum of the signal intensities of all glycopeptides with the same peptide sequence is set to 100%. The distribution of glycans connected to each site is calculated as the average glycan distribution of each peptide covering the glycosylation site.

[0885] result:

[0886] The occupancy rates of glycans at each N-glycosylation site are shown in Table 24. Site N46 is 100% occupied by glycans, while sites N83 and N274 show occupancy rates of 98.12% and 67.58%, respectively (Table 25).

[0887] Table 24: Glycosylation occupancy rate of N-glycosylation sites in rhAAT:

[0888]

[0889]

[0890] Table 25: Average glycosylation occupancy rate of N-glycosylation sites in rhAAT:

[0891]

[0892] Tables 26 and 27 show the percentage of glycans detected at each location. For example, HexNAc1 accounts for 100% at position N46, 39% at position N83, and 83% at position N247. Table 28 reveals the total amount of each glycosylation of the glycan, without distinguishing between glycosylation sites.

[0893] Table 26: Glycopeptide area at each glycosylation site of rhAAT

[0894]

[0895]

[0896] Table 27: Summary of glycopeptides at various glycosylation sites of rhAAT

[0897]

[0898] Table 28: Summary of glycopeptides based on the total amount of each glycosylation of the glycan, without distinguishing glycosylation sites.

[0899]

[0900]

[0901] Example 22: rhAAT for the treatment of acute respiratory distress syndrome (ARDS)

[0902] Research Design

[0903] in vitro: In vitro, the binding ability of recombinant α-1 antitrypsin to TNF-α and its ability to reduce IL-6 and IL-8 were demonstrated.

[0904] in vivo: This is the animal experimental setup using a mouse model. Acute lung failure was induced by inhalation of lipopolysaccharide (LPS). Different doses of rhAAT or comparative prolastine were administered at different time points. After sacrifice at different time points, the concentrations of cytokines in the blood, lung lining fluid, and lung tissue were measured. Simultaneously, histological examination was performed to quantify the migration of monocytes and macrophages. During this histological examination, morphological quantitative analysis of the extent of interstitial edema and lung tissue destruction was performed, as well as the detection of microthrombi in small vessels. A group of surviving animals was retained for six months. At this time, histological examination of the lung tissue was performed to quantify any pulmonary fibrosis that had developed.

[0905] Each group consists of six experimental animals.

[0906] Group 1: 50 μg rhAAT inhaled once daily / Data collected on the third day.

[0907] Group 2: 50 μg rhAAT inhaled twice daily / Data collected on the third day.

[0908] Group 3: Inhale 50 μg rhAAT once daily / Data collected on day 5.

[0909] Group 4: 50 μg rhAAT inhaled twice daily / Data collected on day 5.

[0910] Group 5: Control group (no rhAAT inhalation) / Data collected on day 3

[0911] Group 6: Control group (no rhAAT inhalation) / Data collected on day 5

[0912] Group 7: 50 μg rhAAT inhaled once daily for one week / data collected after 6 months.

[0913] Group 8: 50 μg rhAAT inhaled twice daily for one week / data collected after 6 months.

[0914] Group 9: Control group (no rhAAT inhalation), data collected after 6 months.

[0915] Primary endpoint: The primary endpoint is the laboratory and histological data collected at each study time point.

[0916] Secondary endpoints: Secondary endpoints are observations of the overall health of the study animals. This includes transdermal measurements of oxygen saturation.

[0917] Reduction of inflammatory cytokines

[0918] In vitro: Comparison of rhAAT treatment before and after rhAAT treatment, or comparison of rhAAT treatment with no rhAAT treatment or Prolastine treatment (control).

[0919] IL-1Ra: decreased from 6 ng / mL to 0.5 ng / mL

[0920] IL-6: decreased from 50 ng / mL to 10 ng / mL

[0921] IL-8: decreased from 70 ng / mL to 5 ng / mL

[0922] IL-1B: decreased from 1 ng / mL to 0.5 ng / mL

[0923] Treatment with rhAAT in vitro is expected to reduce the concentration of IL-1Ra in whole blood to 1 / 10 to 1 / 15, for example, from 6 ng / mL to 1 / 12, or 0.5 ng / mL. Treatment with rhAAT in vitro is expected to reduce the concentration of IL-6 in whole blood to 2 / 3 to 1 / 10, for example, from 50 ng / mL to 1 / 5, or 10 ng / mL. Treatment with rhAAT in vitro is expected to reduce the concentration of IL-8 in whole blood to 1 / 10 to 1 / 20, for example, from 70 ng / mL to 1 / 14, or 5 ng / mL. Treatment with rhAAT in vitro is expected to reduce the concentration of IL-1B in whole blood to 2 / 3 to 1 / 10, for example, from 1 ng / mL to 1 / 2, or 0.5 ng / mL.

[0924] The pathological mechanisms of ARDS and its treatment with rhAAT:

[0925] In the early stages of ARDS, the infiltration of pro-inflammatory cytokines and leukocytes into lung tissue is significantly increased. Here, a positive feedback loop exists between tumor necrosis factor-α (TNF-α) and IL-1β, IL-6, and IL-8. Rapid increases are associated with poor patient prognosis (Pugin et al., Am J Respir Crit Care Med. 1996). TNF-α and IL-1β stimulate the production of IL-6 and IL-8, which attract leukocytes into alveolar fissures. Simultaneously, TNF-α disrupts the alveolar barrier and leads to pulmonary edema (Dada et al., AdvExp Med Biol. 2007). Early in the disease, neutrophils accumulate in large numbers in the alveolar spaces, leading to cell death in lung tissue. The concentration of neutrophil elastase is directly related to disease severity and prognosis (Perl et al., Expert Rev Respir Med. 2011). The extracellular traps (NETs) of neutrophils accumulate in the extracellular fluid, acting as nuclei for microthrombus formation (Jarrahi et al., J Thromb Haemost. 2023). In subsequent processes, macrophages accumulate in the interstitium, their task being to transform lung tissue damaged by neutrophil elastase into scar tissue. If patients survive the early stages of the disease, this pathway can lead to persistent and severe lung dysfunction.

[0926] Therefore, inhaled therapy using α-1 antitrypsin for acute respiratory failure is a promising treatment approach, for example, by administering it through an inhaler (such as a vibrating mesh inhaler with defined droplet size and flow rate), thereby achieving sufficiently high concentrations of rhAAT even deep in the lungs. Pharmacological effects unfold at the cellular level and by influencing the immune cascade. IL-6, IL-8, and TNF-α, as core components of the inflammatory response, are affected. Thus, interstitial pulmonary edema is reduced in the early stages of the disease, thereby improving gas exchange. Simultaneously, the integrity of endothelial cells is protected. In the long term, the development of pulmonary fibrosis can be prevented or at least inhibited.

[0927] Example 23: rhAAT for the treatment of bronchiolitis obliterans syndrome (BOS)

[0928] A cytokine-induced inflammation system using human lung epithelial cells (A549) was established, and the effect of rhAAT on the expression of inflammatory cytokines was analyzed.

[0929] Cytokine-induced inflammatory systems using human lung epithelial cells (A549) include:

[0930] • A human lung epithelial cell (A549) stimulation system was established using a mixture of cytokines (IL-1β, IFN-γ, and TNF-α).

[0931] • Inflammatory cytokines (IL-6, IL-8, etc.) were measured using flow cytometry microbead arrays.

[0932] • Cell viability after stimulation was measured using the XTT assay.

[0933] • The effects of rhAAT on inflammatory cytokines and cell activity in the A549-stimulated system were analyzed in a time- and dose-dependent manner.

[0934] • The role of AAT in the overall miRNA expression profile was analyzed using next-generation sequencing technology.

[0935] Human lung epithelial cells (A549) were stimulated using a mixture of cytokines.

[0936] A549 cells were cultured in RPMI 1640 + 2mM glutamine + 10%-20% fetal bovine serum (FCS) and stimulated for 24 hours with an elevated concentration of a cytokine mixture (CM) containing IL-1β, IFN-γ, and TNF-α. Figure 32 Cytokines were administered at the following concentrations: IL-1β 0.01 U / ml, 0.05 U / ml, 0.1 U / ml, 0.5 U / ml, 1.0 U / ml, 5.0 U / ml, 10 U / ml, 50 U / ml, and 100 U / ml; IFN-γ 0.08 U / ml, 0.4 U / ml, 0.8 U / ml, 4.0 U / ml, 8.0 U / ml, 40 U / ml, 80 U / ml, 400 U / ml, and 800 U / ml; and TNF-α 0.004 ng / ml, 0.02 ng / ml, 0.04 ng / ml, 0.2 ng / ml, 0.4 ng / ml, 20 ng / ml, and 40 ng / ml. The supernatant was removed and stored at -80°C until analysis. Cell viability was analyzed by XTT assay.

[0937] Measurement of inflammatory cytokines using flow cytometry bead array (CBA):

[0938] Using BD TMThe CBA Flex Set system (Bioscience-PharMingen, San Diego, CA, USA) was used to analyze the inflammatory cytokines IL-6 and IL-8 in the supernatant of cell culture systems using flow cytometry bead array (CBA) technology. Samples were measured using a BD FACSVerse™ flow cytometer and analyzed using Array Software v3.0 (BD Bioscience-PharMingen, San Diego, CA, USA). A second aliquot of the sample was reserved for potential measurements of other cytokines (e.g., IL-10, IL-12, IL-15, IL-18, IP10, MCP1, RANTES, or others).

[0939] Cell viability after stimulation was measured using the XTT assay.

[0940] Following stimulation with CM and / or treatment with Prolastin and rhAAT, the viability of A549 cells was analyzed by XTT assay (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazonium-5-formylaniline) after stimulation, according to the manufacturer's instructions.

[0941] In a time- and dose-dependent manner, the effects of rhAAT on inflammatory cells in the A549-stimulated system compared to Prolastin were analyzed. The role of cytokines and cell activity:

[0942] A549 cells were cultured in RPMI 1640 + 2mM glutamine + 10%-20% fetal bovine serum (FCS) and stimulated with a selected concentration of cytokine mixture (CM) for 24 hours. To test the effects of AAT on inflammatory cytokines and cell viability, different doses of Prolastin or rhAAT were added to CM-stimulated A549 cells. Il-6 and IL-8 in the supernatant were analyzed by flow cytometry bead array (CBA), and cell viability was analyzed by XTT assay.

[0943] After stimulation with an elevated concentration of a cytokine mixture (CM) containing IL-1β, IFN-β, and TNF-α and incubation with different concentrations of rhAAT, IL-8 production in A549 cells was analyzed by CBA. Figure 33 When the concentration of the cytokine mixture (CM6 and CM8) was increased, a decrease in IL-8 concentration was observed after treatment with rhAAT compared to untreated. The decrease in IL-8 concentration was more pronounced at higher concentrations of rhAAT.

[0944] Decrease in inflammatory cytokines:

[0945] In vitro: rhAAT treatment compared to before rhAAT treatment, or rhAAT treatment compared to no rhAAT treatment or Prolastine (control):

[0946] IL-1Ra: decreased from 6 ng / mL to 0.5 ng / mL

[0947] IL-6: decreased from 50 ng / mL to 10 ng / mL

[0948] IL-8: decreased from 70 ng / mL to 5 ng / mL

[0949] IL-1B: decreased from 1 ng / mL to 0.5 ng / mL

[0950] In patients: comparison of rhAAT treatment before and after rhAAT treatment, or comparison of rhAAT treatment with no rhAAT treatment (control):

[0951] IL-1B: 1 pg / mL vs. 1.6 pg / mL

[0952] Il-6: 50 ng / L vs. 100 ng / L

[0953] IL-6: 17 pg / mL vs. 70 pg / mL

[0954] Treatment with rhAAT in vitro is expected to reduce the concentration of IL-1Ra in whole blood to 1 / 10 to 1 / 15, for example, from 6 ng / mL to 1 / 12, or 0.5 ng / mL. Treatment with rhAAT in vitro is expected to reduce the concentration of IL-6 in whole blood to 2 / 3 to 1 / 10, for example, from 50 ng / mL to 1 / 5, or 10 ng / mL. Treatment with rhAAT in vitro is expected to reduce the concentration of IL-8 in whole blood to 1 / 10 to 1 / 20, for example, from 70 ng / mL to 1 / 14, or 5 ng / mL. Treatment with rhAAT in vitro is expected to reduce the concentration of IL-1B in whole blood to 2 / 3 to 1 / 10, for example, from 1 ng / mL to 1 / 2, or 0.5 ng / mL.

[0955] Treatment with rhAAT is expected to reduce serum IL-1β concentrations in patients by 2 / 3 to 1 / 5, for example, from 1.6 pg / mL to 5 / 8, or 1 pg / mL. Treatment with rhAAT is also expected to reduce serum or plasma IL-6 concentrations in patients by 2 / 3 to 1 / 10, for example, from 100 ng / L to 1 / 2, or 50 ng / L, in serum, and from 70 pg / mL to 1 / 4, or 17 pg / mL, in plasma.

[0956] Analysis of the role of AAT in the overall miRNA expression profile using next-generation sequencing technology:

[0957] Total RNA (including miRNA) was isolated using the Qiagen miRNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA concentration was determined using a Nanodrop Lite spectrophotometer (Thermo Scientific, Dreieich, Germany), and RNA quality was assessed using an Agilent RNA 6000 Nano Kit and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Carla, CA, USA). miRNA libraries were prepared using the QIAseq miRNA Library Kit (Qiagen, Hilden, Germany). cDNA concentration was then measured using a Qubit dsDNA assay kit, Qubit assay tubes, and a Qubit 3.0 fluorometer (all from Thermo Fisher Scientific, Dreieich, Germany). DNA quality was verified using a high-sensitivity DNA chip, Agilent high-sensitivity DNA reagent, and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Carla, CA, USA). MiSeq Regent Kit v3, PhiX sequencing controls v3, and MiSeq were used. TM Next-generation sequencing (NGS) was performed using benchtop sequencers (all from Illumina Inc., San Diego, CA, USA). Data preprocessing was performed using the Quiagen web portal service (https: / / geneglobe.qiagen.com / de / analyze) to convert coverage files into raw data matrices. Differential expression analysis was conducted using RStudio 1.2.1335 (https: / / cran.r-project.org / ).

[0958] Research on the inflammatory pathological mechanism in patients with post-infectious BO (PiBO):

[0959] The pathological mechanism of bronchogenic inflammatory disease (BO), such as PiBO, may involve the infiltration of leukocytes into the airway submucosa. A cascade of inflammatory cytokines and mediators leads to the accumulation of inflammatory cells. Studies by Rosewich et al. ha...

Claims

1. A human recombinant AAT (rhAAT) protein or a fragment thereof, expressed in genetically modified yeast, for use in the treatment and / or prevention of nonviral lung diseases associated with inflammation and / or pathological immune responses.

2. The rhAAT protein or a fragment thereof used according to claim 1, wherein the lung disease associated with inflammation and / or pathological immune response is associated with: elevated levels of inflammatory cytokines such as IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-8, IFN-γ and / or TNF-α, elevated levels of one or more transcription factors such as NF-κB, and / or increased immune cell infiltration in the lung tissue of the subject compared to healthy subjects.

3. The rhAAT protein or a fragment thereof used according to any one of the preceding claims, wherein the lung disease is asthma.

4. The rhAAT protein or a fragment thereof used according to any one of claims 1-2, wherein the lung disease is acute respiratory distress syndrome (ARDS).

5. The rhAAT protein or a fragment thereof used according to any one of claims 1-2, wherein the lung disease is bronchiolitis obliterans (BO).

6. The rhAAT protein or a fragment thereof used according to the preceding claim, wherein the obliterative bronchiolitis (BO) is associated with lung transplantation and / or allogeneic hematopoietic stem cell transplantation (chronic graft-versus-host disease).

7. The rhAAT protein or a fragment thereof used according to any one of the preceding claims, wherein the rhAAT protein or the fragment thereof comprises the sequence according to SEQ ID NO 4, or is encoded by SEQ ID NO 1, 2 or 3, or a sequence having at least 80% identity with them.

8. The rhAAT protein or a fragment thereof used according to any one of the preceding claims, wherein the rhAAT protein or the fragment thereof has a serum half-life, pulmonary half-life and / or activity not less than that of AAT purified from human plasma.

9. The rhAAT protein or a fragment thereof used according to any one of the preceding claims, wherein the rhAAT protein or the fragment thereof is expressed in yeasts of the family Yeastae, preferably Pichia pastoris.

10. The rhAAT protein or a fragment thereof used according to any one of the preceding claims, wherein the rhAAT protein or the fragment thereof comprises post-translational modifications.

11. The rhAAT protein or a fragment thereof used according to any of the preceding claims, wherein the post-translational modification is N-glycosylation, O-glycosylation, N-terminal methionine removal, N-acetylation and / or phosphorylation or any combination thereof, preferably comprising one or more N-linked glycosylations, comprising at least one HexNAc1 glycosylation.

12. The rhAAT protein or a fragment thereof used according to any one of the preceding claims, wherein the rhAAT protein or the fragment thereof is administered by inhalation, preferably as a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation.

13. The rhAAT protein or a fragment thereof used according to any one of the preceding claims, wherein the rhAAT protein or the fragment thereof is administered by inhalation of an aerosol solution.

14. A pharmaceutical composition comprising the rhAAT protein or a fragment thereof according to any one of the preceding claims and a pharmaceutically acceptable carrier, for use in the treatment of nonviral lung diseases associated with inflammation and / or pathological immune responses.

15. The pharmaceutical composition according to the preceding claim, wherein the composition is a solution suitable for inhalation, a soluble powder suitable for inhalation, or a dry powder suitable for inhalation.

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