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1.
Biotechnol Bioeng ; 110(11): 2928-37, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23740533

ABSTRACT

Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges.


Subject(s)
Antibodies/isolation & purification , Biological Products/isolation & purification , Chemical Fractionation/methods , Filtration/methods , Flocculation , Technology, Pharmaceutical/methods , Animals , CHO Cells , Cell Culture Techniques/methods , Cricetinae , Cricetulus , Humans , Recombinant Proteins/isolation & purification
2.
MAbs ; 5(3): 418-31, 2013.
Article in English | MEDLINE | ID: mdl-23567210

ABSTRACT

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.


Subject(s)
Antibodies, Monoclonal/metabolism , Immunoglobulin G/metabolism , Immunoglobulin Light Chains/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Antibody-Dependent Cell Cytotoxicity/genetics , CHO Cells , Cricetinae , Cricetulus , Cysteine/genetics , HEK293 Cells , Hot Temperature/adverse effects , Humans , Immunoglobulin G/genetics , Immunoglobulin Light Chains/genetics , Mutagenesis, Site-Directed , Mutation/genetics , Protein Binding/genetics , Protein Stability , Serine/genetics
3.
J Infect Dis ; 206(5): 706-13, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22732923

ABSTRACT

The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Clostridioides difficile/metabolism , Enterocolitis, Pseudomembranous/drug therapy , Enterotoxins/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Neutralizing/biosynthesis , CHO Cells , Cricetinae , Enterocolitis, Pseudomembranous/microbiology , Female , Mesocricetus , Mice , Mice, Inbred BALB C , Neutralization Tests , Survival Analysis
4.
Proc Natl Acad Sci U S A ; 108(28): 11440-5, 2011 Jul 12.
Article in English | MEDLINE | ID: mdl-21709254

ABSTRACT

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.


Subject(s)
HIV-1/chemistry , HIV-1/immunology , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/immunology , AIDS Vaccines/chemistry , AIDS Vaccines/immunology , Biophysical Phenomena , CD4 Antigens/chemistry , Cryoelectron Microscopy , HIV Antibodies/chemistry , HIV Antigens/chemistry , HIV Antigens/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , Humans , Immunoglobulin Fab Fragments/chemistry , Ligands , Models, Molecular , Protein Structure, Quaternary , env Gene Products, Human Immunodeficiency Virus/ultrastructure
5.
Vaccine ; 27(37): 5120-32, 2009 Aug 13.
Article in English | MEDLINE | ID: mdl-19567243

ABSTRACT

SOSIP gp140 trimers represent a soluble, stabilized, proteolytically cleaved form of the HIV-1 envelope (Env) glycoproteins. SOSIP gp140 derived from a subtype A HIV-1 isolate, KNH1144, forms exceptionally stable trimers that resemble virion-associated Env in antigenicity and topology. Here, we used electron microscopy to demonstrate that KNH1144 SOSIP gp140 trimers bound three soluble CD4 molecules in a symmetrical orientation similar to that seen for native Env. We compared the immunogenicities of KNH1144 SOSIP gp140 trimers and gp120 monomers in rabbits and found that the trimers were superior at eliciting neutralizing antibodies (NAbs) to homologous virus as well as neutralization-sensitive subtype B and C viruses. The NAb specificities for SOSIP antisera mapped in part to the CD4 binding site on gp120. We also observed adjuvant-dependent induction of antibodies to the residual levels of host cell proteins (HCPs) contained in the purified Env preparations. When present, HCP antibodies enhanced pseudovirus infection. Our findings are relevant for the further development of Env-based vaccines for HIV-1.


Subject(s)
HIV-1/chemistry , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/immunology , Animals , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/immunology , CD4 Antigens/metabolism , Cell Line , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , HIV Antibodies/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Humans , Microscopy, Electron , Models, Molecular , Neutralization Tests , Protein Structure, Quaternary , Rabbits
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