The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal for many therapeutic applications, and this has led to the design of a vast number of unique multispecific antibody formats that enable targeting of multiple antigens or multiple epitopes on the same antigen. Despite the diversity of these formats, a common challenge in generating multispecific antibodies is that they display suboptimal physical and chemical properties relative to conventional IgGs and are more difficult to develop into therapeutics. Here we review advances in the design and engineering of multispecific antibodies with drug-like properties, including favorable stability, solubility, viscosity, specificity and pharmacokinetic properties. We also highlight emerging experimental and computational methods for improving the next generation of multispecific antibodies, as well as their constituent antibody fragments, with natural IgG-like properties. Finally, we identify several outstanding challenges that need to be addressed to increase the success of multispecific antibodies in the clinic.

Toward Drug-Like Multispecific Antibodies by Design.

http://www.cell.com/article/S0167779923001233/pdf

Challenges in scaling up AAV-based gene therapy manufacturing

The invention discloses a polypeptide with improved alkaline stability, which polypeptide comprises a mutant of a B or C domain of Staphylococcus Protein A (SpA), as specified by SEQ ID NO 1 or SEQ ID NO 2, or of Protein Z, as specified by SEQ ID NO 3, wherein at least the glutamine residue at position 9 has been mutated to an amino acid other than asparagine. The invention also discloses multimers of said polypeptide, as well as separation matrices comprising the multimers or polypeptides.

Mutated immunoglobulin-binding polypeptides

ABSTRACT Introduction Antibody therapies have made huge strides in providing safe and efficacious drugs for autoimmune, cancer, and infectious diseases. These bispecific antibodies can be assembled from the basic building blocks of IgGs, resulting in dozens of formats. Areas covered It is important to consider the manufacturability of these formats early in the antibody discovery phases. Broadly categorizing bispecific antibodies into IgG-like, fragment-based, appended, and hybrid formats can help in looking at early manufacturability considerations. Expert opinion Ideally, bispecific antibody manufacturing should contain a minimal number of steps, with processes that give high yields of protein with no contaminants. Many of these have been determined for the fragment-based bispecific blinatumomab and the IgG-like bispecifics from hybridomas. However, for new formats, these need to be considered early in the research and development pipeline. The hybrid formats offer an unusual alternative in generating high pure yields of bispecific molecules if the engineering challenges can be deciphered.

The manufacturing considerations of bispecific antibodies

This invention provides a method for purifying a monomeric monoclonal antibody which comprises contacting the sample, wherein the sample comprises the monomeric monoclonal antibody, host cell impurities, dimers, and higher order aggregates, with a Protein A affinity chromatography column; eluting the monomeric monoclonal antibody from the Protein A affinity chromatography column with an elution buffer; and collecting one or more fractions of the monomeric monoclonal antibody to form a Protein A product pool, wherein the product pool comprises less than 5% higher order aggregate, and has a pH from about 3.2 to about 4.5, thereby purifying the monomeric monoclonal antibody from the sample. This invention also provides a method for purifying a monomeric monoclonal antibody which comprises eluting with acetate or citrate, optionally in the presence of amino acids. This invention also provides a method for purifying a monomeric monoclonal antibody which comprises conducting the method within certain temperature ranges.

Methods for purifying antibodies using protein a affinity chromatography

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.

https://tandfonline.com/doi/pdf/10.1080/19420862.2019.1618675

Using bispecific antibodies in forced degradation studies to analyze the structure-function relationships of symmetrically and asymmetrically modified antibodies

Methods useful for producing a solution of purified Fc peptide chains are disclosed. The methods employ protein A chromatography to separate high molecular weight, aggregated peptide chains comprising antibody Fc domains from lower molecular weight unaggregated peptide chains that also comprise antibody Fc domains. The solutions of purified Fc peptide chains obtained by the methods of the invention contain less than 5% aggregate and greater than 70% of the Fc peptide chains subjected to purification.

Process for preparing unaggregated antibody fc domains

Methods of Producing Heterodimeric Antibodies

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Using bispecific antibodies in forced degradation studies to analyze the structure-function relationships of symmetrically and asymmetrically modified antibodies

Process for preparing unaggregated antibody fc domains

Methods of Producing Heterodimeric Antibodies