Hongcheng Liu's research while affiliated with PPD - Pharmaceutical Product Development and other places

Since the first approval of the anti-CD3 recombinant monoclonal antibody (mAb), muromonab-CD3, a mouse antibody for the prevention of transplant rejection, by the US Food and Drug Administration (FDA) in 1986, mAb therapeutics have become increasingly important to medical care. A wealth of information about mAbs regarding their structure, stability, post-translation modifications, and the relationship between modification and function has been reported. Yet, substantial resources are still required throughout development and commercialization to have appropriate control strategies to maintain consistent product quality, safety, and efficacy. A typical feature of mAbs is charge heterogeneity, which stems from a variety of modifications, including modifications that are common to many mAbs or unique to a specific molecule or process. Charge heterogeneity is highly sensitive to process changes and thus a good indicator of a robust process. It is a high-risk quality attribute that could potentially fail the specification and comparability required for batch disposition. Failure to meet product specifications or comparability can substantially affect clinical development timelines. To mitigate these risks, the general rule is to maintain a comparable charge profile when process changes are inevitably introduced during development and even after commercialization. Otherwise, new peaks or varied levels of acidic and basic species must be justified based on scientific knowledge and clinical experience for a specific molecule. Here, we summarize the current understanding of mAb charge variants and outline risk-based control strategies to support process development and ultimately commercialization.

A battery of analytical methods is used to analyze recombinant monoclonal antibodies for lot release to ensure consistent product quality, safety, and efficacy. Additionally, state-of-the-art analytical methods have been used to thoroughly characterize various post-translational modifications and degradation pathways of those molecules. Scientifically sound and robust analytical methods are essential to providing reliable results for defining control strategy, including setting phase-appropriate specifications. Analytical artifacts can substantially impact analytical method performance, causing either overestimation or underestimation of the impacted attributes. However, these artifacts are often overlooked due to lack of the fundamental understanding of analytical methods. This review discusses several regularly encountered artifacts and provides a guidance on assessment and prevention of these artifacts. Understanding and preventing artifacts can help establish scientifically sound and robust methods with reliable performance throughout the method life cycle.

Therapeutic recombinant monoclonal antibodies are subject to various modifications during cell culture, and to a lesser degree, during purification. These modifications are expected to remain relatively constant during storage with the protection of appropriate formulations. However, after administration to patients, the levels of modifications may vary over time in circulation, where the recombinant monoclonal antibodies are exposed to the physiological conditions. Scientific understanding of those in vivo modifications can help drug candidate selection to choose the most stable molecules and set appropriate specifications for product release, which ultimately ensures safety and efficacy.

Recombinant monoclonal antibodies (mAbs) intended for therapeutic usage are required to be thoroughly characterized, which has promoted an extensive effort towards the understanding of the structures and heterogeneity of this major class of molecules. Batch consistency and comparability are highly relevant to the successful pharmaceutical development of mAbs and related products. Small structural modifications that contribute to molecule variants (or proteoforms) differing in size, charge or hydrophobicity have been identified. These modifications may impact (or not) the stability, pharmacokinetics, and efficacy of mAbs. The presence of the same type of modifications as found in endogenous immunoglobulin G (IgG) can substantially lower the safety risks of mAbs. The knowledge of modifications is also critical to the ranking of critical quality attributes (CQAs) of the drug and define the Quality Target Product Profile (QTPP). This review provides a summary of the current understanding of post-translational and physico-chemical modifications identified in recombinant mAbs and endogenous IgGs at physiological conditions.

Increasing attention has been paid to developability assessment with the understanding that thorough evaluation of monoclonal antibody lead candidates at an early stage can avoid delays during late-stage development. The concept of developability is based on the knowledge gained from the successful development of approximately 80 marketed antibody and Fc-fusion protein drug products and from the lessons learned from many failed development programs over the last three decades. Here, we reviewed antibody quality attributes that are critical to development and traditional and state-of-the-art analytical methods to monitor those attributes. Based on our collective experiences, a practical workflow is proposed as a best practice for developability assessment including in silico evaluation, extended characterization and forced degradation using appropriate analytical methods that allow characterization with limited material consumption and fast turnaround time.

Recombinant monoclonal antibodies have been routinely characterized at intact, subunit and peptide levels by LC-MS. Papain and pepsin have been the enzymes commonly used to cleave IgG into various fragments to facilitate in-depth characterization. However, non- specific cleavage for both papain and pepsin and the need for a reducing reagent for papain has limited their usage. In contrast, IdeS has gained popularity due to its specificity and independence of reducing reagent. Results presented in the current study demonstrated that the readily available endoprotease Lys-C can cleave IgG2 at a specific peptide bond in CH2 domain to generate a homogeneous F(ab')2 fragment, and the Fc regions was digested to peptides under limited digestion condition. The generated F(ab)2 fragment is suitable for further analysis because of its homogeneity. The posttranslational modifications located in the Fc region including glycosylation, deamidation and C-terminal heterogeneity can be rapidly analyzed by LC-MS.

Asparagine deamidation in the complementarity determining regions of recombinant monoclonal antibodies has been extensively studied and shown to have a negative impact on antigen binding. Those asparagine residues are typically exposed and thus have a higher tendency towards deamidation, depending also on local structure and environmental factors such as temperature and pH. Deamidation rates and products of a susceptible asparagine residue in the complementarity determining regions of a recombinant monoclonal antibody free in solution or in antibody-antigen complex were studied. The results demonstrated that incubation of the antibody or its antigen complex generated the same amount of aspartate. The expected amount of isoaspartate product was detected in free antibody, but it was completely lacking in the antibody-antigen complex.

In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen.

Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle.

Forced degradation studies have become integral to the development of recombinant monoclonal antibody therapeutics by serving a variety of objectives from early stage manufacturability evaluation to supporting comparability assessments both pre- and post- marketing approval. This review summarizes the regulatory guidance scattered throughout different documents to highlight the expectations from various agencies such as the Food and Drug Administration and European Medicines Agency. The various purposes for forced degradation studies, commonly used conditions and the major degradation pathways under each condition are also discussed.