Characterization of the co-elution of host cell proteins with monoclonal antibodies during protein A purification.

Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co-purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D-LC-HDMS(E) approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (∼10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co-purification with individual Fc and (Fab')2 antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708-717, 2016.

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry.

An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms.

Assessment of naturally occurring covalent and total dimer levels in human IgG1 and IgG2.

Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally.

Profiling the effects of process changes on residual host cell proteins in biotherapeutics by mass spectrometry.

An advanced liquid chromatography/mass spectrometry (MS) platform was used to identify and quantify residual Escherichia coli host cell proteins (HCPs) in the drug substance (DS) of several peptibodies (Pbs). Significantly different HCP impurity profiles were observed among different biotherapeutic Pbs as well as one Pb purified via multiple processes. The results can be rationally interpreted in terms of differences among the purification processes, and demonstrate the power of this technique to sensitively monitor both the quantity and composition of residual HCPs in DS, where these may represent a safety risk to patients. The breadth of information obtained using MS is compared to traditional multiproduct enzyme-linked immunosorbent assay (ELISA) values for total HCP in the same samples and shows that, in this case, the ELISA failed to detect multiple HCPs. The HCP composition of two upstream samples was also analyzed and used to demonstrate that HCPs that carry through purification processes to be detectable in DS are not always among those that are the most abundant upstream. Compared to ELISA, we demonstrate that MS can provide a more comprehensive, and accurate, characterization of DS HCPs, thereby facilitating process development as well as more rationally assessing potential safety risks posed by individual, identified HCPs.

IgG2 disulfide isoform conversion kinetics.

Human IgG2 antibodies contain three types of disulfide isoforms, classified by the number of Fab arms having disulfide links to the heavy chain hinge region. In the IgG2-B form, both Fab arms have interchain disulfide bonds to the hinge region, and in IgG2-A, neither Fab arm are disulfide linked to the hinge. The IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide linked to the hinge. Changes in the relative levels of these forms over time are observed while IgG2 circulates in humans, suggesting IgG2-A→IgG2-A/B→IgG2-B conversion. Using a flow-through dialysis system, we studied the conversion kinetics of these forms in vitro under physiological conditions. For two IgG2κ antibodies, in vivo results closely matched the kinetics observed in vitro, indicating that the changes observed in vivo were solely conversions between isoforms, not differential clearance of specific forms. Moreover, the combined results validate the accuracy of the physiological model for the study of blood redox reactions. Further exploration of the conversion kinetics using material enriched in the IgG2-A forms revealed that the IgG2-A→IgG2-A/B rate was similar between IgG2κ and IgG2λ antibodies. In IgG2κ antibodies, conversion of IgG2-A/B→IgG2-B was slower than the IgG2-A→IgG2-A/B reaction. However, in IgG2λ antibodies, little IgG2-A/B→IgG2-B conversion was detected under physiological conditions. Thus, small differences in the C-terminus of the light chain sequences affect the disulfide conversion kinetics and impact the IgG2 disulfide isoforms produced in vivo.

Identification and quantification of host cell protein impurities in biotherapeutics using mass spectrometry.

Residual host cell proteins (HCPs) in biotherapeutics can present potential safety risks to patients or compromise product stability. As such, their levels are typically monitored using a multicomponent HCP enzyme-linked immunosorbent assay (ELISA) to ensure adequate removal. However, it is not possible to guarantee ELISA coverage of every possible HCP impurity, and the specific HCPs remaining following purification are rarely identified. In the current study, we characterized the ability of an advanced two-dimensional liquid chromatography/mass spectrometry platform (2D-LC/MS(E)) to identify and quantify known low-level spiked protein impurities in a therapeutic peptide Fc fusion protein. The label-free quantification procedure based on the "top 3" intensity tryptic peptides per protein was applied and improved on for this application. Limits of detection for unknown HCPs were approximated from the spiked protein data along with estimates for the quantitative accuracy of the method. In all, we established that most protein impurities present at 13±4ppm can be identified with a quantitative error of less than 2-fold using the more sensitive of two tested method formats. To conclude the study, we characterized all detectable Escherichia coli proteins present in this Fc fusion protein drug substance and discuss future applications of the method.

Rates and impact of human antibody glycation in vivo.

Glycation of immunoglobulin G (IgG) can result from incubation with a reducing sugar in vitro or during circulation in vivo. Upon injection of a recombinantly produced human therapeutic IgG into humans, changes in the glycation levels could be observed as a function of circulation time. Mass changes on the individual IgG polypeptide chains as the results of glycation were determined using reversed-phase liquid chromatography/mass spectrometry. Changes to the light and heavy chains were low but easily detectable at 0.00092 and 0.0021 glucose (Glc) additions per chain per day, respectively. Levels of glycation found on the Fc portion of IgG isolated from healthy subjects, using a similar analytical approach, were on average 0.045 Glc molecules per fragment. In vivo glycation rates could be approximated in vitro by modeling the physiological glycation reaction with a simplified incubation containing physiological Glc concentrations, pH and temperature but with a high concentration of a single purified IgG. To test the impact of glycation on IgG function, highly glycated IgG1 and IgG2 were prepared containing on average 42-49 Glc molecules per IgG. Binding to FcγIIIa receptors, neonatal Fc receptor or protein A was similar or identical to the non-glycated IgG controls. Although the modifications were well distributed throughout the protein sequence, and at high enough levels to affect the elution position by size-exclusion chromatography, no changes in the tested Fc functions were observed.

Rapid LC-MS screening for IgG Fc modifications and allelic variants in blood.

A new method for simultaneously screening allelic variants and certain Fc modifications on endogenous human IgG1 and IgG2 directly from blood samples is described. The IdeS endoproteinase was used to cleave IgG in serum to generate Fc, which, after denaturation, was analyzed directly as monomeric Fc (Fc/2) by LC-MS to identify the haplotype(s) present in each individual. The relative levels of IgG isotype and haplotype ratios were generated along with the profile of the major Fc glycans and several other modifications associated with each IgG1 or IgG2 haplotype. Since only minute quantities (5 µL) of blood are required and analysis can be highly automated, this approach lends itself to screening large populations. We demonstrate its utility in examining possible correlations between Fc properties and allelic variants. IgG1 core fucosylation, which significantly impacts antibody dependent cellular cytotoxicity (ADCC), showed an asymmetric distribution, with a small number of individuals showing unexpectedly high core afucosylation levels. In these individuals, IgG2 afucosylation levels were normal. Finally, a new IgG1 allotype, previously not characterized, was identified using this analytical methodology.

High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans.

Glycan structures attached to the C(H)2 domain of the Fc region of immunoglobulin G (IgG) are essential for specific effector functions but their role in modulating clearance is less clear. Clearance is of obvious importance for therapeutic monoclonal antibodies (Mabs) as it directly impacts efficacy. Here, we study the impact of Fc glycan structure on the clearance of four therapeutic human IgGs (one IgG1 and three IgG2s) in humans. The therapeutic IgGs were affinity purified from serum samples from human pharmacokinetic studies, and changes to the glycan profile over time were determined by peptide mapping employing high-resolution mass spectrometry. Relative levels of high-mannose 5 (M5) glycan decreased as a function of circulation time, whereas other glycans remained constant. These results demonstrate that therapeutic IgGs containing Fc high-mannose glycans are cleared more rapidly in humans than other glycan forms. The quantitative effect of this on pharmacokinetic area under the curve was calculated and shown to be relatively minor for three of the four molecules studied, but, depending on the dosing regimen and the relative level of the high-mannose glycan, this can also have significant impact. High-mannose content of therapeutic Mabs should be considered an important product quality attribute which may affect pharmacokinetic properties of therapeutic antibodies.

N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies.

Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.

Assessing monoclonal antibody product quality attribute criticality through clinical studies.

Recombinant therapeutic proteins, including antibodies, contain a variety of chemical and physical modifications. Great effort is expended during process and formulation development in controlling and minimizing this heterogeneity, which may not affect safety or efficacy, and, therefore, may not need to be controlled. Many of the chemical conversions also occur in vivo, and knowledge about the alterations can be applied to assessment of the potential impact on characteristics and the biological activity of therapeutic proteins. Other attributes may affect the drug clearance and thereby alter drug efficacy. In this review article, we describe attribute studies conducted using clinical samples and how information gleaned from them is applied to attribute criticality assessment. In general, how fast attributes change in vivo compared to the rate of mAb elimination is the key parameter used in these evaluations. An attribute with more rapidly changing levels may have greater potential to affect safety or efficacy and thereby reach the status of a Critical Quality Attribute (CQA) that should be controlled during production and storage, but the effect will depend on whether compositional changes are due to chemical conversion or differential clearance.