As to charge variants see outline

Therapeutic proteins can undergo a variety of degradation processess, including aggregation, deamidation, isomerization, oxidation, disulfide bond scrambling and truncation. These events can occur during fermentation, purificaiton, formulation, manufacturing and storage. The product’s clinical utility or shelf life can be compromised if degradation adversely affects the biologcial activity of the moelcule. (Pan “Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn” Protein Science, 2008, 18, 424-433). 

The main species is the antibody that elutes as the major peak on chromatograms. The main species does not necessarily correspond to the unmodified or non-degraded antibody. In fact, the main peak often consists of species of antibodies with three types of typical postranslational modifications: (2) cyclization of the N-terminal glutamine (Gln) to pyroGlu; (2) removal of the heavy chain C terminal lysine (Lys) and (3) glycosylation of the conserved asparagine (Asn) residue in the CH2 domain with neutral oligosaccharides. (Du, “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, mAbs 4-5, 578-585, 2012).

The US Code of Federal Regulations states that biologics must be free of extraneous material except that which is unavoidable. 25 CFR 610.33. 

Homodimeric impurities: (see also bispecific antibodies)

Homodimeric impurites are particularly challenging to evaluate with traditional antibody purity assays becasue the similarities between homidmer and heterodimer lead to difficult, if not impossible separations. Dixit “LC-MS characterization and purity assessment of a prototype bispecific antibody” mAbs 5-6, 711-722 (2013)

Low Molecular Weight (LMW) Species:

Examples of LMW protein drug product impruties includes precursors, degradation products, trancated speceis, proteolytic fragments including Fab, ligand or receptor fragments or H chain fragments, free L chain, half antibdoy, H2L (2 heavy chains and 1 light chain), H2 (2 heavy chains), HLeavy chain and 1 light chain), HC (1 heavy chain) and LC (1 light chain) .  (Wang, US 16/259095, published as US 2019/0234959)

LMW species of any therapeutic protein may result from host cell protease activity during production. LMW species often have low or substantially reduced activity relative  to the monomeric form of the antibody, while exposing novel epitopes that can lead to immunogenicity or potentially impact pharmacokinetic properties in vivo. (Wang, US 16/223,463, published as US 2019/0194298)

 Antibody Fragments

Antibody fragments are an impurity that is composed of part of a target protein that has a mass less than that of the target protein. The breakage of chemical bonds in the target protein results in the formation of one or more fragments. Fragments are common impurites requiring removal during the purificiaton of a taget prtoein. They are difficult impurities to separate form teh target prtoein becasue they often have properties very similar to the target protein, such as their hydrophobicity and isoelectric point. (Kozlov, US Patent No: 14/891,724, published as US 2016/0090399). 

Kashi (US 2015/0290325) disclsoes stable iquid formulations of human p75 TNF receptor fused to an Fc doamin of human immunoglobulin rpotein with percetnage of fragments ranging from 0 to over 10% fragments over a span of 0-6 weeks. The fragments were late eluting species in SEC. Kash teaches that size hterogeneity can be primarily attributed to fragmentation and aggregation. 

–Purification Methods used for ro reduce antibody fragments

—-Using Activated Carbon

Kozlov (US 14/891,724, published as (US2016/0090399) discloses that activated carbon can be used to absorbed and thereby reduce such fragments in antibody mixtures. For example, Kozlov demonstrate preparing a fragment spiked MAB solution from papain digested mAB, loading centrifuge tuabes with Nuchar HD activated carbon and loading the antibody fragment spiked solution, centrifugation and subsequent filtration. The amount of the MAB remaining int he samples is determeind using IG quantification by Protein A HPLC and the percentage of fragments in the samples is deteremind by size exclusion chromatography (SEC). 

—-Using Mixed Mode Chromatography

O’Connor (US 2016/0251441)  discloses a method for separating antibody fragments wehrein at least some of the antibody fragmentation product impurities are adsorbed to the mixed mode chromatography column and at least some of the target antibody is eluted from the column. 

Half Antibodies:

Birck-Wilson (US 2004/0092719) discloses methods for separation immunoglobulin half antibodies from whole antibodies by reducing the pH of the sample such that the half antibodies dissociate from one another and applying the resulting solution to a column that differentially retards mobility of the half antibodies and whole antibodies. In some embodiments the column is a cation exchange column, a size exclusion colum a hydrophobic interaction column or an affintiy column. In prefered embodiments that column binds to the half antibodies whereas in other embodimetns the column binds the whole antibodies present in the resulting solution. 

High Molecular Weight (HMW) impurities: (see outline, “aggregates”)

HMW impurities include mAb dimers and trimers. (Wang, US 16/259095, published as US 2019/0234959)

Intermediate HMW Species (HMW):

HMW include monomer with extra light chain (H2L3 and H2L4 species) (see outline), monomer plus Fab fragment complexes, Fab2-Fab2, Fc-Fc and Fab2-Fc. (Wang, US 16/259095, published as US 2019/0234959).

Free Fc:

Delvaille (US 2010/0249381) discloses purification of Fc fusion proteins via blue dye affinity chromatography for the reduction of free Fc moieties. The therapeutic moeity of the Fc fusion protein may be dervied from a receptor, preferably from the extracellular domain of a receptor. Examples include CD2, CD3, VEGF recetpor. The method results in elimination of free Fc moeities to less than 1%. Free Fc may contain dimers of the IgG hinge, CH2 and CH3 domains which are not linked to significant poritions of the therapeutic moiety. 

Eon-Duval (US 2010/0267932; see also (WO 2008/025748) discloses a process for the purification of an Fc-fusion protein which includes Protein A/G affinity chromatography, CEX, AEX and hydroxyapatite chromatogrpahy. The process was suitable for removal of free Fc, i.e., immunoglobulin H domains which are not fused to a complete therapeutic moeity such as a ligand binding extracellular porition of a member of the TNFR family. The two TNF receptors p55 (TNFR1) and p75 (TNFR (TNFR2) are examples of such members of the TNFR superfmaily. Ethanercept is an Fc fuion protein contianing the soluble part of the p75 TNR. OX40 is also a member of the TNFR superfamily. In particular, a ligand binding extracellular portion of a member of the tumor necrsos factor recetpor (TNFR) superfamily includes less than 0.1% of free Fc protein. The term “free Fc” encompasses any part of the Fc fusion protein which is dervied form the immunoglobulin part of the Fc fusion protein and does not contain a significant porition of the therapeutic moeity of the Fc-fusion protein. Thus, free Fc may contain dimers of the IgG hinge, CH2 and cH3 domains, which are not linked or boud to significant poritions of a therapeutic moeity. 

Nti-gyabaah (WO 2013/009526) discloses methods for purifying an Fc-fusion protein such as TNFR-Fc (etanercept) produced in eukaryotic systems using Protein A affinity chromatography and two ion exchange chromatography steps which are operated in the bind and elute mode resulting in highly purifed TNFR-Fc with an overall reduction of misfoled Fc fusion protein to less than 5% and reduction of fragments (including free Fc levels) to less tan 5%. Etanercept is a therapetuic recombinant fusion protein comprised of the extracellular ligand binding portion of the human 75 kDa (referred to as p75) human TNFalpha receptor linked to the constant region of human IgG1 (Fc region). 

Host Cell Proteins

Whether mammalina, bacterial or yeast derived, process related impurities can be host cell derived, including a myriad of proteins with various physicochemical characteristics, referred to as host cell proteins (HCPs). Originating from the production cell line, their level in the harvested cell culture fluid (HCCF) depends on both the cell culture process as well as harvest conditions. The potential that HCP has for eliciting immune reactions in patients has drawn significant interest, as the said adverse patient events can delay candidates and/or reduct drug efficacy. For this reason, it is not only important but also required to that porduct and process related impuriteties be removed to an acceptably low level during the manufacturing process. HCP levels can vary significantly for different mAbs. HCPs are one of the most challenging to remove impurities because of their varied physiochemical properties. The analytical method of choice used to monitor HCPs during the recovery process is a multiproduct immunoasaay. (Nogal Biotechnol. Prog. 28(2): 454-458 (2012). 

Chinese hamster ovary (CHO) cells are integral to the 125 billion biopharmaceutical market, which includes mAbs. Typically, therapeutic proteins are secreted into the extracellular media along with hundreds of endogenous host cell protoein (HCP) impurities, comprising both secreted proteins and intracellular proteins released during cell death. Putification processes for these extracellular CHO HCPs from the product becasue even low levels of HCP impurities have the potential to cause adverse patient reactions. A subset of these HCPs are difficult to remove during downstream purification becasue they exhibit product assocation with mAbs and have similar retention to mAbs on chromatographic media. (Valente, Biotechnology and Bioengineering 112(6): 1232-1242 (2015). 

Aboulaich “A novel approach to monitor clearance of host cell proteins associated with onoclonal antibodies”, Biotechnol Prog. 30(5) 1114-1124) discloses a method to identify HCPs (lists m HCPs accoiated with several antibodies) that specifically assocaite with mAb by immobilizing purified mAb onto chromatography resin via cross-linking, followed by incubated with HCPs obtained from supernatant of non-mAb producer cells that are representative of the expression systems used in mAb manufacturing. 

–-PLBL2: cathepsin L

Falkenstein (US 15/900, 449, published as US 20180186866) discloses that HCP, particularly PLBL2, can be reduced if the conductivity of the aqueous solution used in the wash step is low (below 0.5 mS/cm). Advantageously futher process steps can be obviated before loading hte eluate to the next chromatographi material if a low conductivity aqueous solution wash step is ued in the preceeding affintiy chromatography step.

Beigie (US 16/228,291, published as US 2019/0233468) discloes that using 0.5 M sodium benzoate pH 7.0 and benzyl alcohol in an intermeiate wash was very effective at removing HCPs such as PLBL2 during Portien A purification.

Dumetz (US 16/330,588, published as US 2021/0284686) discloses a method of purifying an antibody from HCPs such as Phospholipase B-like 2 protien and cathepsin L using a superantigen chromatography solid support such as Protein A with a wash buffer that includes about 50 mM caprylate and more than about 0.5 M arginine. 

Chromatin

Gagnon (US 14/769,098, published as US 2016/0009762) discloses adding allantoin to clarify chromatin from a cell culture harvest, then methylene blue or ethacridine or cetyl trimethyl ammonium bromide or chlorhexidine is added. Then particles bearing an electropositive metal affinity ligand tris (2-aminoethyl)amine (trEN) is added. In a separte embodiment allantoin is added to a cell culture and then heptanoic acid  or octanoic acid or pelargonic (nonanoic) or capric acid is added. The mixture is then stirred for 2 hours and the electropositive metal affinity ligand tris(2-aminoethyl)amine (TREN) are added. 

DNA and Endotoxins:

Protein A:

Morgan (J of Biotechnology, 4 (1986) 189-204) disclsoes purification of a mAb with a protein A-Sepharose column. The antibody absorbed to the colume at pH 8.4, followed by extensive washing with phosphate-buffered saline, pH 8.4 to remove endotoxins and other protein contaminants. The antibody was desorbed at pH 4.5. A 60-140 fold purificaiton of antibody with a 100-1000 fold reduction of endotoxins was acheived. 

Ion Exchange:

Since DNA and endotoxins are negatively charged over a wide pH interval, a CEX chromatography step at a pH below the isoelectric point of the antibody will bind the target protein and allow the negatively charged molecuels to wash through the colunm. Consequently, if AEX is used as the initial capture step, these contaminants will be removed at an early stage in purification. (GE Antibody Purificaton Handbook, (December 2007). 

Amino-terminal leader extension variants: 

Amino-terminal leader extension variants refers to a mian species antibody with one or more amino acid residues of the amino terminal leader sequence at the amino-terminus of any one or more heavy or light chains of the main species. (Harris, US2009/0202546)

Leachables/Extractables

Skudas (US 15/524824, published as US 2017/033741) teaches that activated carbon is usefuful in the removal of leacables/extractables which results from the use of disposible equipment in the purification of antibodies. 

Polyelectrolytes

Polyelectrolyte precipitation may require subsequent removal of the polyelectrolyte. Bozzano (J Membrane Science, volume 55, issues 1-2, pp. 181-198, 1991) discloses using ultrafiltraiton membranes to recover proteins in the permeate and polyelectroyte in the retentate. 

Proteases

Proteases can be subdivided into serine, cystein, aspartyle, and metalloprotease families, all of which can diminish protein drug performance. Proteolytic enzymes such as pepsin and papain often target unstructured hinge regions of proteins and antibodies. Becasue upstream and downstream processes generate myriad proteases, selection of suitalbe protease inhibitors is a common practice in the biopharmaceutical industry. Such inhibiotrs are classified by seze: e.g., small molecules (pheynylmethylsufonly fluoride), peptides (E64) and proteins (aprotinin). (perez, “Opporunities in the field of host-cell proteins” Bioprocess Technical, 2020). 

Clipped Masking Moieties in Cleavable moiety conjugates:

A “masking moeity” (MM) refers to a peptide that when positioned near an antigen binding domain (AB), interferes with binding of the AB to the biological target. The term “cleavable moeity” (CM) refers to a peptide that includes a substrate for at least one protease. Activated antibodies are designed to be selectively activated in dieased tissue by incorporated within the CM a substrate for a protease that is more prevalently found in the disease tissue. A “clipped impurity” or “clipped varaint” refers to a molecule that resutls after protease mediated clevage of an intact antibody. It contains the AB of the activated antibody but lacks all or a portion of the MM. It includes both “single-armed clipped” and “fully clipped” species. Removing clipped impurity from intact activatable antibody conompositions is challenging due to the relatively small differences between desired product and clipped impurity. Masking moeities are relatively short peptides such as less than 50 amino acids. Single-arm clipped variants are hte predominant clipped variant such that the entire difference between an intact activatable antibody and a clipped variant is the absence of a portion othat lacks a MM and a protion of a CM. Patrick (US Patent Application No: 17/227,029, published as US 20210317188)

By Hydrophobic Chromatography:

Patrick (US Patent Application No: 17/227,029, published as US 20210317188) discloses a process for purifying intact activatable antibody that includes a MM, a CM and a AB from clipped variant as determined by SDS-cGE which includes loading an aqueous feedstock that includes water, the intact activatable antibody and the variant and a first salt onto a support matrix that inclues hydrophobic substituents and eluting with water and a second salt. Exemplary hydrophobic substituents include hydrophobic itneraciton chromatogrpahy (HIC) and mixed mode chromotography (MM).  Salts for use as a first salt in the aqueous feedstock may be any salt that promotes binding of intact activatable antibody and clipped impurity to the column. Illustrative first salts include arignine chloride, arginine hydrochloride, NACl and the like. The salt may be introduced into the aqueous composition prior to the hydorphobic chromatography step or it may be introduced in connection with a process step upstream of the hydorphobic chromatogrpahy process step. When the column includes a HIC stationary pahse, the first salt typically exhibits kismotropic (salting out) behavior. Such salts can be identified for example form the hofmeister series of ions. In the case of HIC, the eluent is generally less porlar than the aqueous feedstock. Patrick further discloses that amounts of an activatable antibody and a clipped masking moiety can also be determine by subjecting a sample composition containing activatable antibody and clipped variants to a gel capillary electrophoresis. 

By Protein A chromatography:

Acoba (WO2020247574) discloses purifying masked antibody with protein A chromatography under conditions suitable for binding the masked antibody, washing the column with the bound masked antibody once with an acidic wash buffer and eluteing at pH 2.5-4 to form an eluate that includes the masked antibody. 

Biosimilars

A biosimilar product can have minor difference in clinically inactive components, provided that there are no clinically meaninful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. 42 USC 262(i)(1). 

Different Molecular Forms (i.e., monomeric, dimeric, tetrameric forms)

Aoyama (J. Immunological Methods, 162 (1993) 201-210) discloses HPLC on spherical beads of hydroxyapatite (HA) to separate different molecualr forms of mouse IgG and IgM mAbs. Monomeric, dimeric, trimeric and tetrameric forms of an IgA mAb were eluted from the column separately with appreciable differences in retention volume by a 20 ml gradient of pohsphate buffer (pH 6.8) of concentraiton from 10-400mM. IN addition, a monomeric form was resolved from a pentameric form of IgM mAbs under the same conditions.