June 1, 2018 (Vol. 38, No. 11)
Ken Hoffman Founder Cygnus Technologies
Determining Whether to Take a Generic or a Process-Specific Approach
The terms “process-specific” and “generic” are commonly used in scientific publications and regulatory guidelines in reference to host cell protein (HCP) analysis of recombinant biopharmaceuticals. The terminology distinction is based on theoretical concerns that generic assays could potentially fail to detect HCPs specific to a given culture and purification process. This concern has led to an arguable belief among many in the biopharmaceutical industry that a process-specific assay should be developed.
In the absence of a comprehensive definition of what constitutes a process-specific assay and a detailed procedure for generating a process-specific antibody, any presumption that a process-specific assay must be better than a generic assay is specious.
Given the variables in antigen selection and methods employed in antibody generation and purification, one cannot make an assumption that antibody response to a particular strain or growth process will be superior to antibodies generated to an essentially identical strain and process.
Generation of antibodies to the relevant downstream HCPs must incorporate multiple elements beyond just antigen selection. A truly process-specific assay cannot be assured by simply injecting a few rabbits with an arbitrary choice of upstream antigen.
Generic assays have often been comprehensively qualified and validated by critical analytical parameters and supported by orthogonal methods to demonstrate acceptable coverage to HCPs that co-purify with the drug substance.
Regulatory agencies have accepted generic assays as lot-release tests for approved biopharmaceuticals. Likewise, published reports of some so-called process-specific assays have shown poor coverage and accuracy for downstream HCPs.
The distinction in terminology is more semantic than scientific as evidenced by the fact that the objective scientific criteria used to qualify any analytical method are the same for both generic and process-specific assays.
This article provides clearer definition to the terminology while proposing how any assay, regardless of the terminology used to describe it, should be comprehensively validated to show it is fit for purpose of HCP detection in final drug substance.
Generic is used in the biological sense whereby the antibody is intended to detect downstream HCPs independent of the growth and purification process. The presumption for generic assays is that the proteins in cell lines such Chinese hamster ovary (CHO) cell lines are highly conserved among different strains. This presumption has been supported by published genomics and proteomics studies for both CHO and prokaryotic expression systems like Escherichia coli.
Generic assays, like process-specific assays, typically use antigen derived from upstream, null, or mock-transfected cells. While culture processes and conditions may up- or downregulate certain HCPs, qualitatively most of the HCPs are conserved among strains and processes. With comprehensive qualification using orthogonal methods of antibody analysis, regulatory agencies will accept generic assays as fit for product release and process monitoring without requiring development of a redundant process-specific assay.
The term “process specific” is poorly defined in the literature. Different sources of antigen have been used to generate so-called process-specific antibodies. Important antigen selection issues include the degree to which antigen is found upstream or downstream in the purification process, as well as the use of null cells, mock-transfected, or product-expressing cells.
To add to the confusion, papers claiming a process-specific antibody have been published that admit to including non-process-specific material such as lysates or cell debris in their antigens.
This leads to an obvious counterpoint. How can an antibody generated from non-process-specific antigen be labeled as a process-specific antigen? Process specific most often means using very upstream HCPs from null cells of the same strain and the growth process used to express the drug. Antigen derived from such a process is fundamentally the same as what is used for generic assays.
A process-specific antigen is only distinguishable from a generic antigen if the HCP content among very highly conserved strains and between similar culture processes may be assumed to consist of a few unique HCPs that co-purify with drug substance.
In the absence of a process-specific assay, companies should use available generic assays for process development and early clinical trials. A qualified generic assay is a valuable purification process development tool to better assure safety and efficacy in the clinic. The suitability of a generic or the need to develop of a process-specific assay as an approved drug-release test can be determined as the manufacturing and purification process is finalized.
It must be understood that the ability of any assay to accurately and quantitatively detect HCP in final drug substance is influenced by arbitrary choices and method limitations that are more significant than just broad coverage to an upstream antigen. Taken together, all these points require that any assay, regardless of what it is termed, must be subjected to comprehensive qualification to demonstrate it meets the accuracy, specificity, and sensitivity requirements to serve as a process control and QC release test.
The conventional procedure was to use 2D Western blot of upstream antigen to demonstrate that the antibody is broadly reactive. Due to poor sensitivity and specificity, 2D Western blot has no predictive value in determining how that antibody, when used in an ELISA format, will quantitatively detect the most important HCPs—those that co-purify with product. We now have new orthogonal technologies that can address the theoretical concern about what an HCP assay might be missing, and that can answer the question of what it actually detects.
Cygnus Technologies developed a method, termed antibody affinity extraction (AAE), that is now being widely used to show coverage to both upstream and, more importantly, downstream HCPs. AAE is over 100-fold more sensitive than 2D Western blot and, as such, can show reactivity to HCPs that co-purify with drug substance.
Figure 1 shows a 2D PAGE fractionation of an upstream sample prior to AAE. Cy3 staining detected 976 HCP spots (blue circles). After AAE, the 2D PAGE staining with Cy5 detected 896 of the same spots for a coverage of 92%. The 80 spots not detectable by AAE are represented with red circles. By contrast, 2D Western blot gave a coverage of only 55%.
Advances in mass spectrometry (MS) now allow for specific identification of individual downstream HCPs. AAE can be used as the LC step in LC–MS/MS methods, reducing the cost and complexity of MS analysis. AAE and MS, when used in conjunction with critical assay qualification criteria for accuracy and specificity by analysis of dilution linearity and spike data on downstream sample, will demonstrate objectively if any assay is fit for purpose as a routine lot release test.
Figure 2 shows the most effective methods to develop and qualify HCP assays. Utilization of the techniques implied in the red boxes will allow for a broadly reactive assay. The green circles are orthogonal methods that can detect individual downstream HCPs. Use of these methods for comprehensive qualification of the assay may obviate the costly and time-consuming development of a redundant process-specific assay when an acceptable generic exists.