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Abstract
The discovery of numerous potent and broad neutralizing antibodies (bNAbs) against Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein has invigorated the potential of using them as an effective preventative and therapeutic agent. The majority of the anti-HIV-1 antibodies, currently under clinical investigation, are formulated singly for intra-venous (IV) infusion. However, due to the high degree of genetic variability in the case of HIV-1, a single broad neutralizing antibody will likely not be sufficient to protect against the broad range of viral isolates. To that end, delivery of two or more co-formulated bnAbs against HIV-1 in a single subcutaneous (SC) injection is highly desired. We, therefore, co-formulated two anti-HIV bnAbs, 3BNC117-LS and 10-1074-LS, to a total concentration of 150 mg/mL for SC administration and analyzed them using a panel of analytical techniques. Chromatographic based methods, such as RP-HPLC, CEX-HPLC, SEC-HPLC, were developed to ensure separation and detection of each antibody in the co-formulated sample. In addition, we used a panel of diverse pseudoviruses to detect the functionality of individual antibodies in the co-formulation. We also used these methods to test the stability of the co-formulated antibodies and believe that such an approach can support future efforts towards the formulation and characterization of multiple high-concentration antibodies for SC delivery.
Highlights
The number of approved monoclonal antibodies for therapy against various cardiovascular, cancer, respiratory, hematology, and autoimmune diseases is continuously on the rise [1]
Thereafter, as a first step towards formulation to aid subcutaneous administration, the antibodies were concentrated 7.5-fold in a new formulation buffer with optimal viscosity to enable drug injection volumes of 2 mL. This resulted in 3BNC117-LS and 10-1074-LS as individually formulated bnAbs, at 150 mg/mL in respective buffers, for subsequent co-formulation studies
These high-concentration individually formulated antibodies were extensively characterized using a wide range of analytical methods i.e., Enzyme-Linked Immunosorbent Assay (ELISA), Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC), Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), ion exchange (IEX)-HPLC, capillary isoelectric focusing, CE-SDS, Sialic Acid analysis, intrinsic Tryptophan fluorescence spectroscopy, Isoquant analysis, dynamic light scattering (DLS), far and near UV circular dichroism (CD), differential scanning calorimetry (DSC), second derivative UV spectroscopy, N-terminal amino acid sequencing, HILIC based glycan profiling, liquid chromatography coupled with mass spectrometry (LC/MS), and peptide mapping by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)
Summary
The number of approved monoclonal antibodies (mAbs) for therapy against various cardiovascular, cancer, respiratory, hematology, and autoimmune diseases is continuously on the rise [1]. In addition to therapy against non-infectious diseases, monoclonal antibodies are increasingly seen as potent prophylactic and therapeutic agents against several infectious pathogens [2,3,4,5], those against which effective vaccines do not exist or are under arduous development. The use of monoclonal antibodies as prophylactic and therapeutic options is attractive against Human Immunodeficiency Virus type 1 (HIV-1) [8,9], a viral pathogen for which the development timeline for a prophylactic vaccine is uncertain [10,11,12]. Protection using passive administration of broadly neutralizing antibodies (bNAbs) against HIV-1 is being evaluated through multiple human clinical studies to test the validity of the approach. Recent studies by Bar-On et al [22] and Mendoza et al [23]
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