DESARROLLO DEL CONTROL DE CONVENCIONALIDAD EN EL PERÚ

The expression of conventional monoclonal antibodies in culture can be analysed by immunoassays which use the generic binding proteins, protein A and protein G, or 'tracer antibodies' raised against the Fc region. Since none of these reagents interact with Fv, new binding reagents will be required.

A popular approach to tackling this problem is to tag the antibody fragment with short peptide epitopes for which tracer antibodies are readily available (Ward , 1989; Power et al, 1992). However, a potential disadvantage of this technique is that the presence of tags may be expected to have unpredictable effects on the expression, function, or toxicity of Fv fragments (Sassenfeld, 1990). Furthermore, these tags are thought to be susceptible to proteolytic cleavage (Ward et al, 1989) and so it may not be possible to design a quantitative analytical immunoassay with the tag system.

A novel approach would be to identify conserved motifs on Fv fragments - such as framework sequences - which could be used to raise generic tracer antibodies. An objective of this project was to take advantage of a side effect of the polymerase chain reaction (PCR). It was reasoned that Fv fragments whose genes have been cloned using 'generic' PCR oligonucleotide primers will carry an identical peptide 'm o tif at their termini. (This concept is the subject of a patent application, Berry 1994, a copy of which is bound in at the back of this thesis). This project set out to test the feasibility of this approach, by raising rabbit antibodies against the C-terminal motif of the chain and evaluating them for their utility as reagents for analysing Fv expression in Escherichia coli cultures.

2.2.2 Analvsis svstems which are compatible with culture supernatants.

Analysis systems need to function in culture supernatants, often with high biomass. This can be a problem when the analysis comprises an immunoassay since cell debris and other material can interfere with the specificity of binding at each stage of the assay - thus leading to inaccurate data. Therefore, a variety of different feedstocks needs to be analysed in the immunoassay system under investigation to determine the effect of non-specific biomass on the assay signal. The most thorough way of achieving this is to determine the signal that is generated in the immunoassay by a set of Fv standards made up in saline and then to repeat the exercise using the same set of standards but made up in a 'negative' culture supernatant.

This raises the question as to what a 'negative' culture should comprise. Initially, an obvious choice would be to use a supernatant which has been derived from the usual E.coli strain (JM109) and possessing the usual plasmid (puC19), but containing no Fv genes. However, biochemical analysis found such a supernatant to be an inappropriate 'negative' - due to it containing very few E.coli proteins. In contrast, E. coli cultures which are actively producing Fvs contain much more protein - and therefore potential contaminants for interfering with immunoassays. This extra protein is due to cell lysis resulting from Fv accumulating in the periplasm. (Somerville et al., 1994).

What is required is either 'positive' supernatants which have then been totally depleted of Fv by antigen affinity chromatography or 'positive' supernatants containing Fv of a different specificity to the one under test - and therefore should not produce a signal in the assay.

It is inevitable that 'negative' supernatants will interfere with immunoassays to a certain extent. However, for assays to be quantitative, this interference should be minimal and preferably less than 10% of specific signal. Therefore, an important objective of this project was to estimate the extent to which analysis systems were susceptible to interference from non-specific biomass. Immunoassays which showed little or no interference could then be taken forward for analysing Fv in E.coli cultures. This validation of analysis systems has not been reported by other researchers in the field.

2.2.3 Analvsis svstems which measure Fv protein and Fv activitv independents. Most analysis systems which have been previously described measure binding activity units (Ward et al., 1989; Schmidt and Skerra 1993). Whilst this is probably the most critical parameter to measure - as it is the property required for applications - there are other parameters which should also be measured. The most important of these is Fv protein - whether active or not. If these two parameters are measured in parallel it gives the fermentation technologist a measurement of the % activity of his product:- there are many problems which could arise during fermentation, leading to the production of inactive Fv. Examples include: an over production of one of the polypeptide chains (Vh and V J , poor association of Vh and Vl, incorrect folding of

assembled Fv, or a deleterious mutation. It is possible - even likely - that different Fvs and different culturing conditions will lead to different specific activity or ' immunoreactivity ' of the Fv that is produced in culture. Therefore, to optimise fully the culturing conditions, an analysis based on binding activity units alone is not sufficient. What is required is an independent measurement of Fv protein.

Fv protein (regardless of activity) has been determined in culture by immunoblotting techniques (Breitling et al, 1991). However, these investigators did not quantify their

Fv by cross-reference to a set of standards (see subsection 2.2.4) or compare Fv protein with active Fv. In contrast, this project set out to design an analysis system capable of measuring Fv protein and active Fv in parallel and in quantitative terms.

2.2.4 A method for producing a set of standards

A key objective of this project was to be able to establish the authenticity of an Fv preparation, in quantitative terms. For analysis systems to achieve this, signals generated by test samples need to be read against signal generated by a set of thoroughly characterised and quantified standards. Such Fv standards have not been reported by other researchers in the field. This project aimed to prepare and evaluate a set of standards according to the strategy given below

The first requirement is to purify a stock preparation of Fv to homogeneity and then to quantify it for different parameters. Total protein can be conveniently measured by the absorbance at 280nm (A2 8 0). This is a robust, reproducible, precise

measurement. Furthermore, the extinction coefficient can be readily calculated from the sequence of the target Fvs - absorbance at 280nm is defined by the relative quantity of tryptophans, tyrosines, and phenylalanines (Wetlaufer 1962). Since genes encoding Fvs will have been sequenced as a matter of course, this is a straightforward objective. Once the stock preparation of Fv has been characterised in this way, it can be diluted to make a set of standards against which analytical procedures determining Fv protein can be cross-referenced.

The specific activity of an Fv preparation, or "immunoreactivity", can be measured and in contrast to enzymes, antibody molecules appear to be either completely active or completely inactive. This opinion has been strengthened by recent research using analytical biosensors: - it was found that an antibody preparation of high concentration but low specific activity behaves exactly as though it were a preparation of fully active antibody but at a low concentration. (Gill and Hoare 1994). Therefore, immunoreactivities can be conveniently determined by passing a test sample down an antigen affinity column and determining the proportion which binds and is active - results can be expressed as a percentage. Once the stock preparation has been characterised in this way, it can be diluted to make a set of standards against which

analytical procedures determining Fv activity can be cross-referenced. Since the same stock preparation can also be characterised for Fv protein content, as described above, it should be possible to assign values to test samples in units of milligrams of active Fv.

The problem of standardising analytical procedures has been largely overlooked by other researchers, thereby making it difficult or impossible to compare the expression levels achieved in different laboratories. There is an urgent requirement for collaborating scientists to use thoroughly prepared and characterised standards so that these comparisons can be made. This project aimed to evaluate the above strategy as a generally-applicable approach for preparing such standards.

2.2.5 Elucidation of storage conditions for standards.

It is critically important that standards - once prepared - are stored in conditions which protect the properties against which they are measured. This is particularly relevant for activity standards. The timespans permissible for storage need to be determined -and fresh standards need to be prepared when these are exceeded. If this is not achieved, fermentation technologists will be generating progressively (and artificially) higher yields as the standards deteriorate in activity relative to Fv in the (fresh) culture. This project aimed to investigate the effect of different storage conditions on Fv activity and to make recommendations on which conditions to use for storing standards.

2.3 DESIGN AND EVALUATION OF A NEW RECOVERY SYSTEM.