Small Molecules 

Small molecules refers to molecules which generally have a MW of less than about 6 kDa. Many companies have libraries of small molecules, often fungal, bacterial, or algal extracts, which can be screened. Tan described a library with over two million synthetic compounds which is compatible with miniaturized cell based assay (J. Am. Chem. Soc. 1998, 120: 8 565-8566). There are numberous commerically available compound libraries such as Chembridge DIVERSet. Libraries are also avilable from academic investigators, such as the Diversity set from the NCI developmental therapeutics program. Rational drug design can be achieved based on known compounds (e.g., a known inhibitor of C5 such as an antibody) or by the use of crystal or solution structural information. Peptidomimetics can be compounds in which at least a porition of a subject polypeptide is modified and the three D structure of the peptidomimetic remains substanitally the same as that of the subject polypeptide. 

 Protein M

Grover (US 15/115,773, published as 2017/0320921) discloses a method of purifying immunoglobulin molecules using an amino acid sequence from Mycoplasma genitalium (MG281, aka “Protein M”). The Protein M is 556 amino acids and 50 kDa in size. It has a large domain of 360 amino acid resiudes that binds primarily to the variable light chain of the immunoglobulin was well as a binding site called the LRR-like motif. It also ahs a C-terminal domain with 115 amino acid resiudes that protrudes over the antibody binding site. In addition, Protein M contains a 16-36 amino acid transmembrane domain. Grover showed that a number of Perotein M homologs or orthologs from mycoplasmas and other species share functional and structural properties with protein MG281 and also developed several variants of Protein M that have similar or improved Ig binding proterins. For example, Protein M variants lacking the C terminal doamin retain the ability to bind to immunoglobulins. Also conserved residues can be substituted with non-polar amino acid residuesbinds to immunoglobulins. Variants can also lack the N temrinal membrane spanning region and still be capable of generically binding to immunoglobulins. 

See also “Protein A – hydroxyapatite” under “protein A” and “chromatography”

Purification schemes typically include an affinity purification step, such as Protein A purificaiton in the case of antibodies. Despite a high binding affinity of Protein A chromatography for antibodies and ability to remove as much as 99.5% of impurities, it is an expensive purificaiton step on a commercial scale. Even if Protein A affinity chromatography is used, adequate purity is often not achieved unless several purificaiton steps are combined, thereby further increasing cost and reducing product yield.

I. Protein A/G Affinity Chromatography: See outline

II. Non Protein A/G Affinity Purification Schemes: 

Despite the fact that affinity chromatography is often employed as a capture step for antibody purification, the high cost and instability of affinity media as led to schemes using fewer steps and without the need for a costly affinity step.

A. Ion Exchange Chromatography: See outline

B. Mixed-Mode (Multimodal) Chromatography:

–Hydroxyapatite Chromatography: See outline

C. Filtration Methods: See outline

D. High Performance Liquid Chromatography (HPLC): Danielsson “”one-step purification of monoclonal IgG cantibodies from mouse ascities, J. Immunological methods, 115 (1988) 79-88 disclose purification of monoclonal antibodies in one step from mouse ascites by 4 different HPLC adsorption techniques; anion exchange (Mono Q), cation exchange (Mono S), chromatofocusing (Mono P) and hydrophobic interaction chromatography. They propose using cation exchange as a first choice for antibodies with high isoelectric points (>7.2) and hydrophobic interaction chromatography as a first choice when isoelectric point is below 7.2

E. Precipitation: See outline

F. Immobilized metal affinity chromatography (IMAC): can purify IgG molecuels due to the affinity binding of histidyl residues in the antibody molecules to a chelated nickel support. Mammalian IgGs ahve a highly conserved histidyl cluster at the junctures of the CH2 and CH3 domains. Under alkaline conditions, nickel loaded iminodiacetic acid (Ni-IDA) captures IgG through the histidyl residues electively and effectively. (Josic, Methods for purificaiton of antibodies, Food technol. biotechnol. 39 (3) 215-226 (2001).